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Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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Abstract |
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 Top Abstract I. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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ERKO phenotype and oncogene-induced tumorigenesis: Wnt-1/ERKO mice and aromatase deficiency |
I. Introduction—A Historical Perspective |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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At the time of the above statement, the laboratories of Elwood Jensen and Jack Gorski had spent 10 yr providing experimental evidence to support the concept of an intracellular "receptor" protein for steroid hormones. Their combined work had even led to a proposed model by which the interactions of the receptor and the steroid were involved in mediating the cellular effects of the hormone (1). The first of these receptors to be characterized was for the female sex hormone, 17ß-estradiol (E2) (2, 3). Since this time, similar receptors for testosterone, progesterone, glucocorticoids, thyroid hormone, vitamin-D3, and retinoids have been discovered and now form a portion of a large family of nuclear hormone receptors (4). Although significant strides have been made since, Jensen’s introductory statement in a review published more than 25 yr ago is still contemporary with the current research goals toward understanding the growing family of nuclear receptors. This is not to say that little progress has been made, which is quite to the contrary, but rather to state that although advances in technology and molecular biology have allowed for extensive insight, there is still a great deal to be learned as we move into the next century.
Our current understanding of the various roles of the steroid, thyroid, and retinoid hormones in development and normal physiology and the mechanisms by which these actions are mediated is due, in large part, to the generation of a series of reagents and tools over the past 40 yr. The first of these was the synthesis and use of tritium-labeled E2 with high specific activity, allowing for the first reports of the detection and simple characterization of an estrogen-binding component, or "estrophilin" (1, 3, 5). This protein exhibited a binding affinity for estradiol that was several fold higher compared with the other gonadal steroids and was found only in tissues previously shown to respond to estradiol in terms of growth and increased RNA synthesis (1, 3). These studies described the uptake and concentration of radiolabeled estradiol by a specific protein unique to the cells of the uterus, vagina, and pituitary and thereby disputed the current thought that estrogen action required enzymatic metabolism of the hormone (2, 3, 6). Thus, the concept of a hormone receptor protein in target tissues was initiated. Autoradiographic studies with radiolabeled ligand further demonstrated the strong association of estradiol with the nuclei of cells lining the rat uterus within 4 h after injection (7). These studies were significantly advanced by pharmacological approaches with early nonsteroidal estrogen antagonists, the first being the triphenylethylene, MER-25 (2), followed soon after by a series of similar compounds, e.g., clomiphene, nafoxidine, CI-628, and tamoxifen (1, 8). These reagents were previously known to inhibit the uterotropic effects of estradiol in a dose-dependent manner but, more importantly, were later shown to block estradiol uptake and binding in target tissues. Still, at this time the exact role that the hormone-receptor complex played in the final manifestation of the hormonal effects remained unclear. It was suggested that the receptor may simply fulfill a "transport" role to move the steroid hormone from the cytoplasm to the nucleus of the cell (1). Nonetheless, from these early studies, the definition of an estrogen target tissue now included not only the exhibition of a measurable response to the natural hormone (E2) but also one that possessed detectable levels of the estrogen receptor (ER).
Much of the early characterization of the ER relied on the use of sucrose gradient analysis of nuclear and cytoplasmic cell fractions under varied salt concentrations (3). This procedure, along with gel electrophoresis and filtration using radiolabeled estradiol, allowed for the generation of semipure fractions of the estradiol-binding protein. These preparations led to the generation of antisera specific to the ER protein, another notable step in receptor research (9). The later development of monoclonal antibodies to the ER and the immunohistochemical techniques that soon followed provided evidence of the predominantly nuclear localization of the receptor protein in target cells (10, 11). The late 1970s brought numerous reports of the tissue distribution and localization of the ER in humans and laboratory animals, confirming much of the findings of earlier steroid autoradiography studies (7, 11). The antibodies were also used to further purify large amounts of the ER from target tissues, allowing for more detailed studies of the receptor structure and function.
The 1980s witnessed the cloning and sequencing of the cDNAs for several of the steroid hormone receptors, which proved to be a seminal step toward understanding their mechanisms of action. The first cDNA to be cloned for a member of the nuclear receptor family was that for the human glucocorticoid receptor (12), followed soon after with the description of the human ER cDNA (13, 14). Since this time, the cDNAs encoding several other members of the nuclear receptor family have been described (4). The current list of isolated ER cDNAs includes those for the chicken (15), mouse (16), rat (17), Xenopus laevis (18), and rainbow trout (19). Sequence analysis of the various receptor cDNAs demonstrated a high degree of similarity and led to their inclusion in a superfamily of nuclear receptors possessing a defined motif of functional domains (4).
A vital contribution from the cloning of the nuclear receptor cDNAs was the ability to express the receptor proteins in in vitro mammalian (20) or yeast (21) cell systems. These techniques, as well as cell-free in vitro transcription (22), allowed detailed characterizations of the different functional domains of the receptor. Recombinant DNA methodologies provided for the construction of receptor cDNAs possessing precise truncations, deletions, point mutations, or additional sequences. These powerful techniques led to the in vitro expression of chimeric and mutant receptors and great advances in the dissection and mapping of the specific domains and distinct residues critical to receptor function (21, 22, 23, 24, 25). The late 1980s witnessed multiple descriptions of naturally occurring variants and mutations of several of the nuclear receptor transcripts (26), including the ER (27, 28, 29). Their identification and functional characterization have led to further insight into the mechanisms of action of specific domains of the receptor proteins as well as the receptors as a whole. Furthermore, the detection of these nuclear receptor transcript variants in vivo has allowed speculation concerning their possible roles in alternate or abnormal hormonal signaling in normal and neoplastic tissues (27).
By the 1990s, it was evident that the members of the steroid/thyroid hormone superfamily of receptors were intracellular proteins that functioned in the nucleus to regulate transcription of target genes. This growing family of receptors now includes those for the sex and adrenal steroids, thyroid hormones, retinoids, vitamin D3, and eicosinoids (reviewed in Refs. 4, 30). The inclusion of nuclear receptor-like proteins with no known ligand, termed orphan receptors, such as those for the chicken ovalbumin upstream promoter (COUP), and steroidogenic factor-1 (SF-1), has expanded the family to now include approximately 150 distinct proteins (30).
Our knowledge of the expression patterns and mechanism of action of the
nuclear receptors has led to a greater appreciation of their
involvement in normal physiology and disease. The current decade has
witnessed several advances in our understanding of the molecular
biology and overall physiological role of these proteins. Especially
significant to these efforts has been the discovery and/or development
of three distinct aspects. The first of these was the discovery of
coregulators, a second group of nuclear proteins that further modulate
the actions of unoccupied as well as ligand (agonist or
antagonist)-bound receptors (reviewed in Ref. 31). The continued
characterization of the coregulator proteins has provided some insight
toward the long sought explanations for the cell- and tissue-specific
mixed agonist/antagonist activity of certain ligands (31). The second
of these advances was the generation of crystal structures for the
ligand-binding domains of the retinoic acid receptor- (32), retinoic
X receptor- (33), thyroid receptor- (34), and ER (35, 36).
These new data continue to allow for the description of the long
speculated conformational changes that result in the receptor after
binding of the natural ligand, and how these changes may differ from
those induced by natural or synthetic agonists and antagonists.
The third development of great impact in this decade has been the use of gene-targeting technology and transgenic techniques to disrupt the genes encoding several members of the steroid/thyroid hormone receptor superfamily. This methodology has allowed for the generation of transgenic mice that lack a functional gene for a specific receptor, as well as germline passage of this mutation. Hormone resistance due to naturally occurring mutations in the genes encoding the receptors for androgen (37, 38), glucocorticoids (39), and thyroid hormones (40) had already been described in humans and laboratory animals and thereby provided great insight into the role of these hormones in development and normal physiology. However, similar mutations had not yet been reported for other members of the superfamily of nuclear receptors. The impact of the gene-targeting technique is evident in the generation of several "knockout" mice for the nuclear receptors, including mice lacking multiple forms of the retinoic acid receptors (41, 42, 43), the progesterone receptor (44), the vitamin D3 receptor (45), the ERs (46, 47), and the coregulator protein, steroid receptor coactivator-1 (48). If nothing else, the naturally occurring mutants combined with the generation of the knockout models, have confirmed the basic hypotheses put forth almost 30 yr ago, i.e., that the receptor proteins found to tightly and specifically bind a hormone within a tissue are indeed critical for mediating the biological effect of the hormone within the target cell (1).
This review will discuss one such advance originating from the
gene-targeting boom that occurred in the 1990s, the ER knockout mice.
The broad content of this review is a reflection of the current state
of research in the expanding field of estrogen action, as illustrated
by the discovery of a second ER, the ERß. A large extent of the
discussion will focus on the mice lacking the classical ER, the ER
knockout mice (ERKO). The reasons for this are 2-fold: 1) the
ERKO mouse has been available for more than 6 yr, compared with less
than 1 yr for the ßERKO, and therefore has been more thoroughly
studied and characterized; 2) the phenotypes of the ERKO mice appear
to be more broad in nature compared with those of the less well studied
ßERKO mice, although continued research may lessen this disparity.
The extent to which the ERKO models have been used in various fields of
biological research is illustrated in Table 1
. This review will discuss the
phenotypes of the ERKO mice and the multiple ways in which these models
continue to influence the field of estrogen research. Foremost, several
of the hypotheses of estrogen and ER action put forth from the efforts
of numerous investigators over the years were confirmed by our
observations in the ERKO. Furthermore, certain phenotypes have
introduced unforeseen critical roles of estrogen in some physiological
systems, such as in male fertility. Later years have witnessed the use
of the ERKO as a research tool to investigate specific biochemical
pathways and neoplasia in the absence of estrogen action. Where
applicable, we will contrast the phenotypes of the two ERKO models, as
well as compare the relevant phenotypes of knockout models for other
hormone signaling systems. And finally, we will discuss how these
models compare with the few cases of insufficient estrogen synthesis
and the single reported case of estrogen insensitivity in humans.
Before we continue, however, we wish to take this opportunity to
establish consistency in the abbreviations used to refer to the
ER-knockout (ERKO) mice in the literature. Although the terms ERKO
and ERßKO have appeared in previous reports from our own as well as
other laboratories, we propose that the abbreviations used above and
throughout the remainder of this review, i.e., ERKO and
ßERKO, be the consensus abbreviations used hereafter to refer to the
two models.
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II. Estrogen Receptors |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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The two receptors are not isoforms of each other, but rather distinct
proteins encoded by separate genes located on different chromosomes. As
a result of this discovery, we now know that the ER shares a phenomenon
of multiple forms previously described for other members of the nuclear
hormone superfamily, including the receptors for thyroid hormone,
retinoids, mineralocorticoids, and progesterone (26, 30). A detailed
description of the structure and mechanism of action of the ERs is
beyond the scope of this review, and therefore only a brief discussion
of the relevant points will appear here. For more detailed descriptions
of the mechanism of steroid receptor-regulated gene
transcription, readers are encouraged to seek several recent reviews
(52, 53, 54, 55).
Transcription of the mouse ER gene in vivo
predominantly results in a single transcript of approximately 6.3 kb
transcribed from 9 exons. This transcript encodes a protein of 599
amino acids with an approximate molecular mass of 66 kDa (16).
The human ER is slightly shorter at 595 amino acids but exhibits a
similar molecular mass (13, 14). Whereas the human ER gene has been
mapped to chromosome 6 (56), the mouse ER gene is located on
chromosome 10 (57). The existence of multiple promoter and regulatory
regions in the 5'-untranslated sequences of the human and rat ER
have been described, but only a single open reading frame appears to
exist (58, 59, 60). Numerous reports have described the discovery and
characterization of naturally occurring variants and mutations of the
ER mRNA in normal as well as neoplastic tissues of several species
(reviewed in Refs. 27, 28, 61). Although the existence of true
protein products of these ER mRNA variants in vivo
remains controversial, their transactivational activities in in
vitro cell culture systems have been intensely described and have
furthered our understanding of the functional domains of the receptor
(27, 61).
Initial studies indicated that the rodent ERß was composed of 485
amino acids and an estimated molecular mass of 54 kDa and therefore was
slightly smaller than the ER (49, 50, 62). The majority of this
difference in size between the two ERs was due to a significantly
shorter N' terminus in the deduced ERß protein. Unlike the ER
gene, Northern blot analyses of ovarian RNA from both the rat and mouse
indicates the presence of multiple ERß transcripts (50, 63, 64).
Furthermore, open reading frames initiating up-stream from those
originally described have now been discovered in the mouse, rat,
bovine, and human ERß mRNA (65, 66, 67, 68, 69). These studies have also
provided evidence to support that the upstream start codons are the
likely initiation sites of translation and therefore suggest the
possibility of an ERß protein of 527–530 amino acids and a
calculated molecular mass of approximately 60 kDa (65, 66, 67, 69).
However, convincing Western blots from tissue extracts to indicate the
true in vivo molecular mass of the ERß have remained
difficult to produce with the antibody preparations currently
available. Similar to ER, a number of variants of the ERß mRNA
have already been described. These include a conserved insertion of 18
amino acids in a C'-terminal region of the ERß in the rat (70, 71),
human, and mouse (72), the deletion of one or more exons in these same
species (70, 72, 73), and various isoforms in the extreme C'-terminus
of the human ERß (68).
B. Mechanism of ER action
The ERs are classified as class I members of the
superfamily of nuclear hormone receptors, defined as a ligand-inducible
transcription factor (30). Early studies indicated the ER was
cytoplasmic and became localized to the nucleus only upon ligand
binding, providing the basis of the initial "two-step mechanism" of
hormone action (1, 3). However, it is now accepted that the ER is a
predominantly nuclear protein regardless of whether
or not it is complexed with ligand (74). The inactive ER exists in a
complex consisting of several heat-shock and other proteins that appear
to disassociate upon ligand binding, resulting in a
"transformation" of the receptor to an active state (75). With
continued research, the two-step mechanism model has evolved to state
that upon binding of estradiol, or an estrogenic ligand, the
transformed receptors form dimers that tightly associate with specific
consensus DNA sequences, consisting of 15-bp inverted palindromes in
the regulatory regions of target genes (52, 74, 75, 76). This complex then
interacts with basal transcription factors, coregulator proteins, and
other transcription factors to ultimately regulate transcription of the
target gene (52, 55, 76). However, in recent years, pathways of gene
activation by the steroid receptors that deviate from this classical
model have been described. These include gene activation by
ligand-bound steroid receptors without evidence of direct DNA
binding, but rather via interaction with other DNA-bound transcription
factors, such as an AP-1 complex (77, 78). In addition,
ligand-independent activation of the receptor through pathways that
alter the activity of cellular kinases and phosphatases has been
demonstrated both in vitro and in vivo (reviewed
in Ref. 55). The discovery of these pathways strongly supports the
great importance of the ER and its ability to possibly provide diverse
physiological functions even in the absence of ligand.
The ER and ERß proteins are composed of six functional domains,
labeled A–F, a signature characteristic of members of the superfamily
of steroid/thyroid hormone nuclear receptors (Fig. 1). The N'-terminal A/B domain is the
least conserved among all members and demonstrates only 17% identity
between the human ER and ERß (64). In contrast, the C domain is
the most highly conserved among the different members of the family. It
possesses two zinc fingers forming a helix-loop-helix motif and
primarily functions in tightly binding the receptor to the DNA hormone
response elements. The sequences encoding the two zinc fingers possess
97% homology between the ER and ERß genes and are located in
separate exons (exons 3 and 4) in each (50, 64, 79). The E domain, or
ligand-binding domain, confers ligand specificity to the receptor and
is moderately conserved among the members of the superfamily. The ER
and ERß proteins possess 60% conservation of the residues in the E
domain; however, each binds estradiol with nearly equal affinity and
exhibits a very similar binding profile for a large number of natural
and synthetic ligands (80). The D domain possesses signals for nuclear
localization of the receptor and exhibits approximately 30% identity
between the two human forms of ER (64). The C'-terminal F domain is
unique to the ER among the nuclear receptors for the gonadal and
adrenal hormones (6) but is not well conserved among the ERs of
different species nor between the ER and ERß, which share
approximately 18% homology (64). Studies using forms of the ER
missing the C' terminus have indicated a role for the F domain in
modulating transactivational activity of the ER when complexed with
mixed agonist/antagonist ligands, possibly via influencing coregulatory
function and/or dimerization of the receptor (81, 82).
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(85).
The discovery of the ERß has introduced a new level of complexity to
the current model as well. To date, there exist no data indicating a
physiological response solely mediated by ERß. In contrast, the
ERKO mouse has confirmed the requirement for ER in mediating
several actions of estradiol, as will be discussed in this review.
Nevertheless, in vitro experiments from several laboratories
have indicated the possibility of cooperative activity between the two
receptors, acting in the form of heterodimers (50, 62, 65, 86). These
studies generally report a tendency of ER to form homodimers whereas
ERß prefers to heterodimerize with ER. However, Giguere et
al. report (87) that the heterodimer is the preferred state when
both mouse ERs are present. The transactivational activity of the
heterodimer when assayed in in vitro mammalian cell
transfection assays appears to lie between that of the more active
ER homodimer and the less active ERß homodimer (50, 62, 86). A
major consideration when evaluating the possible physiological
functions of an ER/ERß heterodimer is evidence of coexpression of
the two receptors in the same cell, which has not yet been definitively
reported (88). To this end, studies and reagents are only now becoming
available to directly assess this question.
Several functional characteristics of the two ERs are similar. The
residues critical to function of the AF-2 domain appear to be identical
in the mouse ER and ERß (87). Tremblay et al. (50, 89)
demonstrated that a tyrosine residue critical to the function of the
AF-2 domain was conserved in both the ER and ERß and that mutation
of this amino acid resulted in similar constitutive, ligand-independent
transactivational activity in both receptors. In contrast, the
N'-terminal AF-1 domain shows no significant regions of similarity
between the two ERs (87). However, a potential activation site of the
mitogen-activated protein (MAP)-kinase pathway previously shown for
ER is present and active in the ERß (50, 89). Additionally, when
acting on a basal promoter linked to a consensus estrogen response
element, both ER and ERß were able to recruit the coactivator
SRC-1 and were equally susceptible to inhibition by the antiestrogens
raloxifene, ICI 164,384, and EM-800 (50).
However, as studies continue, distinct differences at the molecular
level and in the transactivational capacities between ER and ERß
have been described. Two separate studies have demonstrated the
specificity of the agonist activity of 4-hydroxytamoxifen to be unique
to ER, although this appears to be highly dependent on the cell and
promoter context as well as experimental design (50, 90). Furthermore,
Paech et al. (91) reported that when interacting with
DNA-bound AP-1 transcription factors, the in vitro
transactivational activity of estrogen agonists and antagonists was
quite different depending on which form of ER was present. Whereas
antagonists, such as raloxifene, tamoxifen, and ICI 164,384, were able
to block the stimulatory activity of the ER/AP-1 complex, these same
compounds acted as potent agonists when bound to an ERß/AP-1 complex
(91). Further experimental support for the existence of distinct
structural and functional differences between ER and ERß was
recently provided by Sun et al., who showed that certain
nonsteroidal ligands were receptor selective in their binding and
agonist/antagonist activities (92).
Perhaps the most significant disparity lies in the tissue distribution
of the two receptors. Studies employing the techniques of RT-PCR and/or
ribonuclease protection assay (RPA) have indicated that ER mRNA is
predominant in the uterus, mammary gland, testis, pituitary, liver,
kidney, heart, and skeletal muscle, whereas ERß transcripts are
significantly expressed in the ovary and prostate (Fig. 2) (63, 80, 93, 94). These same studies
have indicated relatively equal levels of mRNA for the two receptors in
the epididymis, thyroid, adrenals, bone, and various regions of the
brain (80, 93, 95, 96, 97). However, as more studies are reported, several
discrepancies in the expression patterns of ER and ERß among
different species are becoming apparent (80, 93, 96). For example,
whereas ERß mRNA is easily detectable in the pituitary of the rat
(70, 98, 99), human (100), and rhesus monkey (96), levels in the
pituitary of the mouse appear low to undetectable (93). A similar
difference in expression is apparent in the mammary gland, in which
normal and neoplastic human tissue and cell lines express detectable
ERß mRNA (64, 68, 73, 101, 102), although the mammary gland of the
mouse appears to predominantly express ER (93). Furthermore, even in
those tissues expressing both ERs, there is often a distinct expression
pattern within the heterogeneous cell types composing the tissue. In
the ovary, ERß is apparently localized to the granulosa cells of
maturing follicles, whereas ER is detectable in the surrounding
thecal cells (69, 103, 104). In the prostate of the rat, expression of
ER and ERß is detectable in the stroma and epithelium,
respectively, but does not appear to be colocalized in any portion of
the tissue (49). However, through the combined use of
immunohistochemistry and in situ hybridization, Shughrue
et al. (88) have demonstrated colocalization of ER and
ERß to select regions of the rat forebrain.
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The rationale for generating mice that possess no functional ER is multifaceted, but in its most simple terms, was founded on the classical ablation experiments of the early part of this century. In 1900, Knauer (105) described the ability of ovarian grafts to prevent uterine atrophy in the castrated guinea pig. Five years later, Marshall and Jolly (106) described the capacity of ovarian extracts to induce estrus when administered to ovariectomized dogs. Similar protocols were later elegantly employed by Jost (107) to substantiate the endocrine function of the testis and the importance of testosterone in sex determination. These studies were critical to establishing the following basic criteria required to verify an endocrine role for a particular organ or tissue: 1) removal or destruction of the synthesizing organ should result in predictable symptoms presumed to be related to the absence of the hormone; 2) administration of material prepared from the removed organ should relieve these symptoms; and 3) the hormone should be present in and extractable from both the organ and blood (108). In the spirit of these earlier studies, the later part of this century has witnessed the marriage of two relatively new methodologies to introduce a modern version of the ablation experiment. The combination of in vitro culture of mouse embryonic stem cells and targeted homologous recombination has generated a tool that allows for the precise disruption or knockout of a particular gene of study and the passage of this mutation to offspring. Although one can debate whether this new technique is more or less invasive than the previous surgical methods, it is obvious that the classic "ablation" experiment has been elevated to a molecular level. The function of a specific hormone can now be studied rather than the function of a whole endocrine organ that may produce multiple secretions. Furthermore, this new technology allows for the study of a particular cellular component, such as a receptor, that is intrinsic to one or more tissues. Such studies were previously impossible or relied on the use of chemical antagonists, which introduced their own inherent limitations. Additionally, gene targeting provides for in vivo methods to study the roles of a particular receptor throughout the life of the animal, including the early development stages. The current tools that allow for the generation of transgenic mice have already made significant contributions to our knowledge of particular genes, especially those involved in development and reproduction (reviewed in Refs. 109, 110).
At the time the ERKO mouse was envisioned, the ER was the only form
of ER known to exist. Furthermore, there were no reports of ER
mutations in normal tissue that resulted in estrogen insensitivity in
humans or laboratory animals. This was in contrast to the descriptions
of syndromes of receptor-based insensitivity to androgens (37, 38) and
thyroid hormones (40). Therefore, investigators were inclined to
conclude that mutations that resulted in insufficient estrogen
synthesis or resistance to the hormone at the level of the target organ
were lethal at the earliest developmental stages (111, 112). This view
was strengthened by reports of the detection of ER mRNA in both
human (113) and mouse (114) oocytes as well as in mouse blastocysts
(115). Accordingly, the concept of generating a mouse devoid of ER
was initially met with skepticism but was pursued as a collaborative
effort between our laboratory and that of Dr. Oliver Smithies of the
University of North Carolina at Chapel Hill. If disruption of the mouse
ER gene did prove lethal, a model to study the precise time and
locations of critical ER-mediated actions during early development
would be available. However, providing the animal was viable, an
in vivo model of estrogen insensitivity would now be
accessible for continued study. Six years after the initial description
of the ERKO mouse, we now take for granted that disruption of the
ER gene proved not to be lethal, but rather the animal develops
normally and exhibits a life span comparable to its wild-type litter
mates (46). However, as will be elaborated in detail in this review,
the adult ERKO mice exhibit several abnormalities and deficiencies,
most notable of which are the phenotypic syndromes that result in
infertility in both sexes. Since this time, additional discoveries have
been made to enhance the utility of the ERKO model. Soon after the
generation of the ERKO mouse, Smith et al. (116) reported
the first and only known case of clinical estrogen insensitivity due to
an inactivating mutation of the ER gene in a human patient. Later
came multiple descriptions of aromatase and subsequent estradiol
deficiency in humans (117, 118, 119, 120). And finally, a second collaborative
effort resulted in the successful generation of the ßERKO mice, which
also survive to adulthood and exhibit phenotypes unique from those of
the ERKO (47). However, splicing variants of the disrupted genes
that may encode for receptors with decreased functional activity have
been detected in small amounts in each of the respective ERKO models
(as discussed below) and therefore complicate the interpretation of the
nonlethality of the gene targeting. Nonetheless, we now know that a
loss of full function of any one of the two ERs is neither lethal nor
detrimental to embryonic and fetal development in both mice and humans.
Perhaps the survival of the ERKO mice, the aromatase-deficient humans,
and the ER-deficient male will prompt a renewed effort among
clinicians to suspect and investigate the possibility of estrogen
insufficiency or resistance in patients not responding to conventional
therapies.
The ERKO mice provide broad and multiple advantages to the research
efforts toward understanding the function and mechanism of estogen
action. Much of what is known about estrogen action was inferred from
in vivo studies involving castration or the administration
of ER antagonists or inhibitors of estradiol synthesis. These findings
have been complemented by the vast knowledge gained from in
vitro cell culture studies, employing chimeric and mutant versions
of the ER, varied cell types, multiple combinations of
promoter-reporter gene constructs, as well as synthetic agonists and
antagonists. However, there are distinct disadvantages to these
experimental schemes. Studies using aromatase inhibitors and/or
estrogen antagonists are complicated by several factors, including
variability of the compound to block the action of the natural hormone
or enzyme. The effectiveness of various antagonists is highly dependent
upon the animal model, the tissue or cell of study, the bioavailability
of the compound at different target tissues, and the class of
antiestrogen used (8). This dilemma is further complicated by the
discovery of the ERß, since no known ER-selective agonists or
antagonists have been characterized at an in vivo level. The
limitations of in vitro cell culture experimental approaches
are obvious and mostly based on their finite application to the whole
animal. Therefore, the ERKO models provide a unique tool to investigate
the role of the ER in the context of the whole animal, and equally
important, during the complete life span of the animal. At their most
fundamental level, the ERKO mice address the role of the ER in the
development and normal physiology of all organ systems, as well as in
carcinogenesis, toxicity, and aging. Furthermore, unlike the
"castrate" model in which several hormones are removed from the
system, the ERKO mice retain the capability to synthesize the gonadal
steroids, including the natural ER ligand, estradiol. Therefore, the
biochemical functions of estradiol and the ER can be investigated in
the presence of presumably intact pathways for the other gonadal
hormones. The presence of estrogens in the absence of ER also provides
for the possibility of discovering pathways of estrogen action that are
independent of nuclear ER, or mediated via previously unknown forms of
the receptor. Additionally, although the majority of in
vitro studies indicate that ER and ERß may have redundant
functions, their differences in tissue distribution and response to
certain ligands indicate the presence of distinct roles fulfilled by
each. The fact that the ERKO mice exhibit an unaltered pattern of
ERß mRNA expression strengthens the usefulness of this model to
dissect these potential ERß-mediated actions (93, 121). Finally,
consistent with those criteria discussed earlier for establishing an
endocrine function to an organ, the ERKO animals now provide a null
background available for transgenic reintroduction of the ER of other
species, mutated ERs, or targeted ER expression to a specific tissue or
cell type.
A detailed description of the targeting scheme employed to disrupt the
mouse ER gene can be found in the initial description of the ERKO
mice (46). As shown in Fig. 1
, a 1.8-kb insert possessing the gene for
neomycin (neo) resistance under the control of the
phosphoglycerate kinase (PGK) promoter and including a
PGK-polyadenylation signal was inserted into a NotI site in
exon 2 of an ER gene fragment subcloned from a genomic library of
129/J mouse DNA. The targeting insert was placed in a replacement 
type targeting vector (122) with the appropriate ER gene-flanking
sequences. Upon successful targeting in mouse embryonic stem cells
(129/J), the neo insert is placed approximately 270 bp
downstream of the ER translation start site and thereby inhibits
proper expression of the ER gene. Since this was the current state
of the technology, no portion of the ER gene was removed during the
targeting event. Standard protocols of clone selection and blastocyst
(C57BL/6J) injection were used to generate chimeric mice possessing the
disrupted gene, some of which demonstrated germ-line transmission of
the mutation when bred with wild-type mates (122). Southern blot and
PCR analysis of genomic DNA from mice of all three genotypes indicated
the correct targeting of the ER gene and the absence of any
heterologous recombination in other regions of the genome. Inbreeding
of mice heterozygous for the ER disruption resulted in a Mendelian
distribution of all three genotypes as well as a balanced sex ratio,
indicating that the ER is not critical to sex determination at the
level of the external genitalia (46).
The generation of mice homozygous for a disruption of the ERß gene
was similar to that described above for the ERKO and can be found in
detail in the initial description (47). A genomic clone that spanned a
15-kb region possessing the first three exons of the mouse ERß gene
was selected from a 129/SvJ mouse library. A replacement type
targeting construct was generated to include 5' and 3' homologous
sequences of 1.3 and 7.4 kb, respectively (Fig. 1). A PGK
promoter-regulated neo gene was inserted in the reverse
orientation into a PstI site in exon 3 of the ERß clone.
Therefore, correct targeting resulted in disruption of the sequences
coding for the first zinc finger of the ERß protein, a domain
critical to normal function of the receptor. Chimeric and heterozygous
offspring were generated as described above for the ERKO mice. Once
again, mice possessing the targeted disruption of the ERß gene were
identified by diagnostic Southern blotting and PCR of genomic DNA. As
with the ERKO, inbreeding of mice heterozygous for the disruption
yielded a Mendelian distribution of all three genotypes as well as a
balanced sex ratio (47).
In both knockout models, RT-PCR on RNA from target tissues indicated
the presence of multiple splicing variants of the respective ER
transcripts (47, 123). In neither case has wild-type-like mRNA
transcribed from the disrupted receptor gene been detected. The greater
proportion of the variants detected in each ERKO model possessed frame
shifts that would result in a severely truncated or mutated ER if
translated. However, in the ERKO, a single-splice variant capable of
encoding a mutant ER protein with significantly decreased
transactivational capacity in vitro was detected at very low
levels (123). A similar ERß splice variant, in which the reading
frame was preserved although coding sequences were removed, was
detected in ovaries of the ßERKO mice. This single variant, if
translated, would encode a mutant ERß lacking the first zinc finger
and therefore would be unlikely to transactivate due to an inability to
tightly associate with the chromatin structure within the regulatory
regions of target genes.
This is not the first report of targeted insertions resulting in
aberrant splicing of a disrupted gene. Mice possessing a targeted
disruption of the transforming growth factor- gene produce a
transcript in which the entire exon possessing the disrupting insert is
accurately removed via the conventional donor and acceptor splice
sites, preserving the normal reading frame of the gene (124). Studies
in three different human genes have demonstrated that point mutations
resulting in a premature stop codon can lead to complete excision of
the exon possessing the mutation (125, 126). Furthermore, Reed and
Maniatis (127) used artificial deletions and insertions within exons of
genes that normally display alternative splicing to demonstrate that
the proximity of the acceptor and donor splicing sequences to one
another plays a role in splicing mechanisms. It is possible that
insertion of sequences as large as those used in the ERKO mice may
disrupt the spatial requirements necessary for proper mRNA splicing.
Therefore, the above studies, along with our experiences, are relevant
to the practice of targeting genes by insertion of a large disrupting
sequence possessing internal stop codons.
1. Interpretation of phenotypes in receptor null mice. The use of methodologies to target and disrupt individual genes has created numerous models available for study (reviewed in Refs. 109, 110, 128). Furthermore, this impact has been felt in several facets of the biological sciences. Although it may be initially thought that a particular gene plays no role in the physiology of certain animal systems, such as reproduction or behavior, disruption of the gene and the subsequent phenotypes often prove otherwise. Therefore, transgenic and knockout technologies have spawned a number of collaborative efforts between investigators of varied disciplines that may have never occurred.
An issue that has become apparent from the numerous gene disruption studies and interdisciplinary collaborative efforts is a collection of caveats to be considered when evaluating phenotypical data from a knockout model. These have arisen mostly from the behavioral sciences (reviewed in Refs. 129, 130), but have expanding application to all areas of study provided by transgenic animals. The first of these caveats is one that may be most relevant to the steroid receptor mutant models, i.e., when studying the target tissue of an adult receptor-knockout mouse, one must realize the tissue passed through all the stages of development and "organization" in the absence of the respective receptor. Therefore, this tissue, and in essence the whole animal, cannot be assumed to be truly identical to the wild type in all aspects except for the absence of the targeted gene product. Any genetic redundancies or compensatory mechanisms that took place during development cannot be readily detected or accounted for during most experiments. Therefore, the lack of a phenotype does not necessarily discount the function of the disrupted gene in the physiology being studied. Additionally, it is difficult to distinguish between an organizational vs. activational basis for an observed deficit in a particular physiology when studying the adult mutant. For example, observed resistance to a hormone due to alterations downstream of the function of the disrupted gene may be apparent during adulthood, but may have been imprinted during development.
Other caveats of interpreting data from receptor null mice are founded in the methods used to generate and maintain a line of knockout mice. The standard protocol for generating a knockout mouse involves the incorporation of embryonic stem cells of a 129 strain of mice that carry the desired mutation into the blastocyst of a C57BL/6 strain. The resulting chimeric animals are then often back-crossed to the C57BL/6 strain once again until mice homozygous for the disruption are acquired. Therefore, early generations of knockout mice are composed of a somewhat chimeric genome, especially in the chromosomal regions closest to the targeted locus. This is of special importance in behavioral studies in light of the known variations in the sexual behavior of different strains of mice (131). However, most relevant to the ERKO, significant variations in estrogen responsiveness of the female reproductive tract among the different strains of mice are also known to exist (132, 133). A recent report by Roper et al. (134) has further defined the genetic basis for the variations that exist in the effects of estradiol on classical uterine parameters in mice. These limitations can be overcome to some degree by increasing experimental sample sizes and including parental strains as a control group in all experiments (135). In addition, the various models of hormone and steroid receptor deficiency that are now available no longer make necessary the complete interpretation of data from any one model. Therefore the data from these models, when interpreted as a whole, should prove invaluable to elucidating the roles of the different sex steroid receptors in both development and adult physiology.
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III. Reproductive Tract Phenotypes of the Female |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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ERKO and ßERKO female mice exhibit a properly
differentiated female reproductive tract possessing the constituent
structures (46, 47). However, estrogen insensitivity has severely
disrupted sexual maturation of the whole reproductive tract in the
ERKO female and ovarian function in the ßERKO female. The
consequences of ER gene disruption on the individual components of the
female reproductive tract is the topic of this portion of the review.
Before we continue, we believe it is necessary to briefly reiterate
those studies carried out to verify successful targeting of the ER
gene in the ERKO. This discussion is appropriate for this portion of
the review because the majority of these experiments were performed on
uterine tissue. To determine the effectiveness of the gene targeting,
Western blots of adult ERKO uterine nuclear and cytosolic extracts
were probed with the H222 antibodies, a rat monoclonal antibody
specific to the ligand-binding domain of the human ER (10). Our
studies, as well as those of others, have demonstrated that this
antibody possesses high cross-reactivity to the mouse ER (139, 140, 141).
These assays detected no wild-type ER or any other immunoreactive
fragments unique to the ERKO uterus. Similar results were obtained
when blots were probed with the rabbit antiserum ER-21, directed toward
the 21 amino-terminal residues of the rat ER (141). However, binding
assays using 3H-E2 on ERKO uterine extracts
indicated the presence of high-affinity binding of the hormone at
levels approximately 3–9% of the wild type (123). In agreement with
these data, sucrose gradient analysis with
3H-E2 on low-salt cytosol extracts from ERKO
uteri indicated a binding factor with an 8S sedimentation value,
similar to that of the wild-type ER (123). The discovery of the
ERß, reported approximately 3 yr after the generation of the ERKO,
prompted a renewed assessment of this ERKO estrogen-binding data in
several publications. Unfortunately, in a number of these reports, the
original datum discussed above is not evaluated in full, and the
authors elude to ERß as the likely binding source in the ERKO
uteri. Certainly at the time of the initial characterization, concern
over the residual level of binding in the ERKO uteri was often mixed
with the wonder of possibly discovering an unknown ER. However, during
these studies we also demonstrated that when the H222 antibodies were
included in the sucrose gradient assays, the estradiol binding peak in
the ERKO uterine extract was shifted accordingly (123). The H222
antibodies have been shown by us, as well as by other laboratories, to
be ER specific and unable to recognize ERß by Western blot
analysis or immunohistochemistry (142). As described earlier in this
review, our RT-PCR analysis on mRNA from ERKO uteri demonstrated the
presence of a splicing variant of the disrupted ER gene that could
encode a mutant ER possessing both the ability to bind estradiol as
well the H222 epitope (123). Furthermore, we have recently shown that
ERß mRNA is undetectable in the uteri of adult wild-type as well as
ERKO mice when assayed by ribonuclease protection assay (93).
Therefore, we believe that relatively conclusive data have been
generated to indicate that the estradiol-binding factor present in
ERKO uteri is most likely not ERß.
A. Uterus
1. Uterine phenotype and estrogen insensitivity. The ER has
been detected by steroid autoradiography and immunohistochemical
methods in the ductal structures of the rodent female reproductive
tract during several stages of development, including the late fetal
and neonatal stages through puberty and adulthood (reviewed in Refs.
112, 143). Several reports describe the initial appearance of ER
immunoreactivity in the developing uterus as early as fetal day 15
(112, 143). ER immunoreactivity was first detectable in mesenchymal
cells, whereas induction in the epithelial cells occurs during the late
fetal stages and increases significantly during the neonatal period
(112, 143). The fully developed uterus is composed of many
heterogeneous cell types comprising three major anatomical
compartments, the outer myometrium, endometrial stroma, and
luminal/glandular epithelium. In the immature CD-1 mouse, ER
immunoreactivity is easily detectable in the stroma on day 1 and
continues to rise to a maximal level on day 10, whereas the appearance
of epithelial ER is delayed and reaches a peak around day 16 (144).
Other reports indicate variations in the exact timing of the appearance
of ER among different strains and species, most likely reflecting
temporal deviations in development (112, 143).
The presence of an intact estrogen-signaling system appears to coincide
with the appearance of ER. In several species, estrogen treatment of
fetal and neonatal females results in the stimulation of increased
uterine levels of nucleic acid (136, 145), protein synthesis (146),
ornithine decarboxylase (147), progesterone receptor (148), and
cellular proliferation (145, 149, 150). However, a full biological
response to estradiol in terms of maximum increases in uterine weight
is not possible in the neonatal uterus, and can be observed only after
the animal approaches weaning age (146). Furthermore, significant
differences in the uterine response to estradiol between the neonate
and sexually mature rodent are known (151). For example, estrogen
stimulates cellular proliferation in all tissues of the immature
uterus, whereas this response becomes limited to the epithelial
compartment during adulthood (151, 152). Therefore, sexual maturation
of the uterus is not simply marked by the presence of ER, but rather
the acquisition of the capacity to undergo the correct synchronized
phases of proliferation and differentiation elicited by the
ovary-derived sex steroids.
As shown in Fig. 3, the uteri of both
adult ERKO and ßERKO females possess all three definitive uterine
compartments, the myometrium, endometrial stroma, and epithelium.
However, in the ERKO, each is hypoplastic and results in whole
uterine weights that are approximately half that recorded for wild-type
littermates (46). In contrast, the uteri of adult ßERKO females
appear normal and able to undergo the cyclic changes associated with
the ovarian steroid hormones (47). Therefore, perinatal development of
the female reproductive tract in the mouse appears to be independent of
ER and ERß actions. However, estrogen responsiveness and
subsequent sexual maturity in the uterus has been ablated by disruption
of the ER gene. The ERKO endometrial stoma is characterized by a
less organized structure and hypotrophy, with a sparse distribution of
uterine glands compared with that of the wild type (153). Luminal and
glandular epithelial cells in the ERKO uterus most often appear
healthy, but are consistently cuboidal and lack the normal
"estrogenized" morphology of a tall columnar shape and basally
located nucleus (Fig. 3). This phenotype is interesting in light of the
increased levels of estradiol found in the serum of adult ERKO
females (Table 2). However, Lindzey
et al. (154) demonstrated that the concurrently elevated
ovary-derived androgens in the ERKO female (Table 2) do provide for
some maintenance of uterine weight, which can be further reduced upon
ovariectomy. We recently reported that ERß mRNA is barely detectable
in the adult mouse uterus, including those from ERKO mice (93),
making it unlikely that ERß could provide a compensatory role in the
ERKO uterus. Numerous immunohistochemical studies for ER and the
apparent loss of estrogen sensitivity in the ERKO uterus indicate
that the classical ER is the predominant form responsible for
mediating estrogen actions in the mouse uterus.
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ERKO females
fail to exhibit components of both phases after estrogen treatment,
providing strong evidence for the requirement of ER in the full
response (46, 123). In brief, when treated with 40 µg E2
or diethylstilbestrol (DES) per kg body weight for three consecutive
days, wild-type mice exhibited the expected 3- to 4-fold increase in
uterine wet weight, whereas no such response was observed in the uteri
of ERKO mice (46, 157). It should be noted that this pharmacological
dose of estrogen is well beyond that required to achieve a maximum
response in the wild-type rodent. Nonetheless, estrogen-treated ERKO
uteri exhibited no apparent components of the initial phase of estrogen
effects, including water imbibition and hyperemia. Histological
analysis and [3H]thymidine incorporation assays indicated
a lack of significant cellular proliferation and DNA synthesis in uteri
from the estrogen-treated ERKO mice (123, 153). Interestingly,
although the heterozygous females possess approximately one-half the
normal complement of ER, their uterine response to estrogens is
equal to that of the wild-type females. In a similar study, wild-type
and ERKO mice treated with hydroxy-tamoxifen (1 mg/kg) produced
comparable results (157), eliciting the expected estrogenic response in
the wild-type and having no effect on the ERKO uterus. These studies
thereby confirm that the estrogen agonist activity of
hydroxy-tamoxifen, which is somewhat unique to the mouse uterus (8), is
mediated via the ER pathway.
The mitogenic and stimulatory action of estradiol in the uterus is a
complex process involving increased RNA polymerase and ribosomal
activity (158), resulting in the regulation of a plethora of genes. It
is well accepted that the ligand-bound ER complex is not directly
involved in the mediation of all responses elicited by estrogens in the
uterus, but rather serves as a stimulus for a cascade of signaling
pathways that act to amplify the estrogen action. However, certain
genes appear to be directly regulated by the ER-estradiol complex
and possess functional estrogen-responsive elements within their
regulatory regions. Two such examples are the genes encoding the
progesterone receptor (PR) (159, 160) and the secretory protein,
lactoferrin (161). In fact, the regulation of the uterine PR and
lactoferrin genes have often been used as assays for the estrogenic
activity of experimental compounds. Therefore, with a similar intent,
we used these estrogen markers to attest for estrogen insensitivity in
the uteri of the ERKO mouse. A single dose of estradiol, known to be
effective in inducing the PR and lactoferrin genes within 24 h in
uteri of wild-type mice, produced no such up-regulation in the uteri of
the ERKO mice, confirming the need for a direct action of the ER
in this mechanism (123). Interestingly, a recent report by Tibbetts
et al. (162) demonstrated that the estrogen-stimulated
increases in PR are localized to the stromal and myometrial
compartments, whereas the increases in lactoferrin are isolated to the
luminal and glandular epithelium in the mouse uterus. Therefore,
disruption of the ER gene has resulted in estrogen insensitivity in
all three anatomical compartments of the uterus. However, it must be
noted that constitutive levels of PR and lactoferrin mRNA are present
in the ERKO uteri, suggesting that these genes are also under the
influence of pathways independent of ER. A testimony to the
complexity of estrogen action in the uterus is the finding that while
estradiol up-regulates PR expression in the myometrium and stroma, it
simultaneously abolishes PR levels in the luminal epithelium (162).
This would indicate an inhibitory role of the estradiol-ER complex
on PR expression in this portion of the uterus. Speculating that this
pathway may therefore be lacking in the ERKO uterus, an
investigation as to the source of the PR mRNA in the ERKO uteri is
warranted.
2. Changes in growth factor functions. A component of the
cascade of events that lead to the obvious changes in the physiology of
the adult uterus after estrogen exposure are the auto- and paracrine
actions of polypeptide growth factors. Several members of the epidermal
growth factor family have been suggested as possible mediators of
estrogen-induced mitogenesis in the uterus. This hypothesis is based on
experiments demonstrating that estradiol up-regulates the uterine
levels of epidermal growth factor and its receptor (EGF, EGF-R) (163, 164), transforming growth factor- (165), and insulin-like growth
factor-I (IGF-1) (166). Furthermore, mice homozygous for a targeted
disruption of the EGF-R gene exhibit a hypoplastic uterus that is
significantly reduced in size (167), similar to that of the ERKO.
Experimental data indicate that treatment of ovariectomized wild-type
mice with EGF mimics the early effects of estradiol and DES in terms of
inducing modified cell morphology and increases in the levels of ER,
DNA synthesis, phosphatidylinositol turnover, PR, and lactoferrin in
the uterus (168, 169, 170). Further studies have illustrated that
cotreatment with anti-EGF antibodies was able to attenuate the uterine
response to estradiol, presumably due to inactivation of the
EGF-signaling pathway (168). In turn, cotreatment with the estrogen
antagonist ICI-164,384 was able to reduce the uterine response to EGF
(169). These in vivo studies suggest a cross-talk mechanism
between the EGF and ligand-independent ER- signaling pathways. The
results of the animal studies have been supported by numerous in
vitro experiments demonstrating ligand-independent activation of
the nuclear signaling pathway of the ER, possibly via altering the
phosphorylation pattern of the ER (reviewed in Ref. 55). The
culmination of these and several other studies has led to the proposed
model in which the mitogenic actions of estradiol in the rodent uterus
appear to be at least partially mediated by EGF; however, in turn the
mitogenic effects of EGF require the presence of ER.
Therefore, the ERKO female provides an excellent in vivo
model to study this cross-talk between the ER- and EGF-signaling
systems in the uterus. The uteri of ERKO females possess wild-type
levels of functional EGF and EGF-R (170). Nonetheless, the mitogenic
actions and induction of estrogen-responsive genes elicited by EGF in
the wild-type uterus have been ablated in the ERKO, confirming the
interaction of these two signaling systems (170). However, not all EGF
responses are lacking in the uteri of ERKO females, as this same
study demonstrated that the mechanisms for EGF-mediated up-regulation
of the c-fos gene remained intact (170). These studies have
thereby confirmed the need for functional ER for the mitogenic
actions of EGF in the uterus.
Cunha et al. has extended the use of the ERKO mouse to
investigate the intersecting roles of ER-mediated estrogen
stimulation and growth factors in the uterus through a series of tissue
recombination experiments. The observation of estrogenic effects in
wild-type uterine epithelial cells that are apparently lacking ER
has prompted numerous investigations to illustrate a role for paracrine
factors secreted by the underlying ER-positive stromal compartment,
and thereby mediating the epithelial response (143). These studies have
been advanced by methods that provide for the delicate construction of
tissue recombinants, in which uterine stoma and epithelium are
enzymatically disassociated and recombined with similar tissue from
uteri from animals of different treatments or models to ultimately
regenerate a chimeric stromal-epithelial unit (reviewed in Ref. 143).
These tissue recombinants are implanted under the kidney capsule of
ovariectomized nude mice, which are then acutely treated with estrogen
agonists or antagonists. Later removal of the recombinant grafts allows
for the evaluation of certain end points of estrogen action in each
portion of the recombinant. Cooke et al. (171) described
experiments in which wild-type (ER+) uterine stroma were recombined
with ERKO (ER-) uterine epithelium and vice versa.
The results of these studies illustrate that proliferation of the
epithelial portion of the recombinant was possible only when ER+
stroma were present and did not require ER in the epithelium (171).
Interestingly, similar recombinant experiments using tissue from the
EGF-R knockout mice illustrated that the estrogen-signaling pathways
required for stimulation of the stroma and subsequent induction of
epithelial growth are intact in the absence of EGF-R (167). Aside from
the proliferative effects of estradiol, previous studies suggested that
estrogen stimulation of secretory products from uterine epithelium,
e.g., lactoferrin, is directly mediated by the epithelial
ER (140, 162). However, Buchanan et al. (172) recently
employed tissue recombinants similar to those described to demonstrate
that both stomal and epithelial ER are required for
estrogen induction of the uterine epithelial secretory products,
lactoferrin and complement component C3. Therefore, estrogen-induced
proliferation of the uterine epithelium requires the presence of ER
in the stromal compartment only, whereas induction of certain
epithelial secretory products is dependent on the presence of ER in
both uterine compartments.
3. Maintenance of selective estrogen actions in the ERKO
uterus. A distinct advantage of null receptor models, whether
naturally existing or experimentally generated via molecular
methodologies, is their use as an in vivo tool for
discerning alternate pathways of hormone action. Recent studies by the
Lubahn laboratory have indicated the preservation of a distinct
estrogen-signaling pathway in the ERKO uterus (173, 174). Das
et al. (173) reported that two consecutive treatments (over
a period of 12 h) with the catecholestrogen, 4-hydroxyestradiol
(4-OH-E2) at 10 µg/kg body weight resulted in significant
increases in water imbibition in the uteri of ovariectomized ERKO
mice. Induction of lactoferrin mRNA in the uterine epithelium of
wild-type mice was 97- and 85-fold after treatment with 10 µg/kg body
weight estradiol or 4-OH-E2, respectively (173). In
contrast, only the 4-OH-E2 was active in the ERKO
uterus, resulting in a 60-fold increase in lactoferrin mRNA levels
compared with a 1.4-fold induction by estradiol (173). A similar, yet
more modest, response was reported in the wild-type and ERKO uteri
after treatment with the xenoestrogens, kepone (15 mg/kg body weight)
(173) and methoxychlor (15 mg/kg body weight) (174).
Most interesting was the lack of inhibition of this response by the
pure estrogen antagonist, ICI-182,780, in both the wild-type and
ERKO mice, indicating the possibility of a non-ER-mediated
signaling pathway for certain compounds exhibiting estrogenic activity.
Additionally, the nature of the timing and type of lactoferrin response
elicited by the 4-OH-E2 and xenoestrogens in the ERKO is
quite distinct from that of the original descriptions by Teng et
al. (175) concerning estrogen regulation of this gene in the mouse
uterus. Given the knowledge of the low-to-absent expression of ERß in
the uterus and the ability of the ICI compounds to antagonize ERß
signaling in vitro, it is not likely that this receptor is
involved in this phenomenon. The catecholestrogens are naturally
synthesized and proposed to play a role in steroid regulation of the
hypothalamus and pituitary (176, 177), ovarian function (178), and
embryo implantation (179). Furthermore, the discovery of local
synthesis of these estrogens in mammary tissue has led to implications
of their involvement in breast cancer (180). Therefore, further
investigation into the alternate mechanisms by which these compounds
may activate nuclear processes is needed.
4. Maintenance of progesterone action in the ERKO uterus.
Like estradiol, ovarian derived progesterone, is an integral steroid
hormone in the physiology and function of the uterus. The PR has been
localized to cells composing all three anatomical compartments of the
uterus and exhibits varied levels in each during the stages of the
estrous cycle (reviewed in Ref. 181). The PR also exists in two forms,
PRA and PRB, which differ only in the length of
the N' terminus. In contrast to the ER, PRA and
PRB are encoded by a single gene but transcribed from two
distinct promoters in both the rat (160) and human (159).
As previously discussed, the PR gene is strongly regulated by the
estradiol-ER complex. This is evidenced by in vitro and
in vivo studies showing inhibition of estrogen stimulation
of the PR gene with antiestrogens as well as the presence of a single
imperfect estrogen-response element in the proximal region of the PR
gene promoter (160). However, in vitro assays indicate that
both the proximal as well as a distal promoter appear to be at least
partially ER dependent and may also involve ligand-independent
pathways of ER action for full expression (160, 182). The lack of
estradiol-induced increases in PR mRNA levels in the ERKO uterus
confirms a regulatory dependence of the PR gene on ER action (123).
Therefore, it was hypothesized that disruption of the ER gene may
subsequently result in abnormally low levels of PR in the ERKO
uterus, and thereby render this tissue refractory to progesterone as
well. However, Northern blot and ribonuclease protection assays have
indicated basal levels of PR mRNA in the ERKO uterus that are equal
to those in wild-type, although estrogen-stimulated increases in these
levels are absent (123). Binding assays with a radiolabeled progestin
indicate levels of PR protein in the ERKO uteri are present but
reduced to approximately 60% of that in wild type (183). A notable
finding was the greater proportion of PR found to be tightly associated
with the nuclei of cells from the ERKO uteri (
25%), compared
with the wild type (5%) (183). It is possible that a lack of ER
has resulted in a more plentiful pool of available coregulator
proteins, allowing the PR present in the ERKO uterus to maintain a
more "active" state. Western blots indicate no difference in the
relative levels of PRA and PRB between the two
genotypes, with PRA consistently present in greater amounts
in both (183). Therefore, a loss of ER action in the uterus has led
neither to a complete loss of PR nor to altered and preferential
transcription from one of the two PR gene promoters.
Curtis et al. (183) carried out a series of studies
demonstrating the preservation of PR-mediated progesterone actions in
the ERKO uteri. Stimulation of the genes encoding amphiregulin (184)
and calcitonin (185) in the rodent uterus has been shown to be an early
and late response to progesterone, respectively. A treatment regimen of
progesterone shown to be effective in inducing these two genes in the
ovariectomized wild-type uterus was equally effective in the uteri of
ovariectomized ERKO females (183). Furthermore, the documented
ability of estradiol to inhibit the progesterone induction of the
amphiregulin gene was absent in the ERKO (183). These studies
indicate that a sufficient level of PR and an active progesterone
signaling pathway are present in the uteri of ERKO mice.
A well known physiological role for progesterone is the preparation of the uterine endometrium for the forthcoming pregnancy. Implantation of the embryo is a complex process that requires a high degree of synchronicity between the blastocyst and the hormone-induced changes of the uterine endometrium (reviewed in Ref. 186). In general, ovarian synthesis of estradiol, which peaks at ovulation, serves to "prime" the uterus by eliciting increased differentiation and proliferation of the luminal and glandular epithelium, and the induction of significant increases in PR in the endometrial stroma and myometrium (162, 186). Subsequent increases in progesterone released from the ovarian corpora lutea then cause decidualization, a complex process involving massive proliferation and differentiation of the endometrial stroma along with localized increases in vascular permeability and edema (186). This process involves the synthesis of specific hormones, such as PRL, cytokines, prostaglandins, extracellular matrix components, and various enzymes within the uterine tissues (186). The result is a remarkable swelling of the uterine stroma thought to be necessary for implantation by forcing the uterus to close down on the blastocyst (186). The final stage of apposition in the mouse is the grasping of the blastocyst and ultimate attachment to the uterine wall, a process thought to be dependent on the secondary rises in ovarian derived estradiol (186). Therefore, preparation of the uterus for blastocyst implantation is dependent on the multifunctional and sometimes opposing effects of estradiol and progesterone.
A laboratory model for the decidualization process has been generated
for several species and generally involves exogenous steroidal
treatments designed to mimic those of the ovarian cycle, coupled with a
mechanical stimulus or trauma of the uterine lining to simulate
implantation (186, 187). In brief, ovariectomized mice are treated with
estradiol (100 ng) for 3 consecutive days, followed by 2 days of no
treatment. The animals are then administered progesterone (1 mg) and a
reduced dose of estradiol (7 ng) for 8 consecutive days, with
mechanical stimulation of the uterus taking place on the third day.
Using this treatment scheme and the progesterone receptor-knockout
(PRKO) model, Lydon et al. (44) demonstrated the absolute
dependence of the decidualization process on progesterone action and
the PR by reporting the complete lack of decidualization in the PRKO
mice. However, Curtis et al. (183) demonstrated that a
progesterone-induced decidual response is possible in the ERKO
uterus, despite its inability to respond to the estradiol priming meant
to mimic estrus. Most interesting were investigations indicating that
although the uterine decidualization in the wild type was dependent on
estradiol, as evidenced by its inhibition with ICI-182,780,
progesterone alone was sufficient to induce decidualization in the
ERKO uterus (183).
The reasons for the apparent loss of estrogen dependence for successful
decidualization in the ERKO uterus can only be speculated upon at
this time. One role of estrogen in uterine decidualization is thought
to be the induction of significant increases in PR in the endometrial
stroma (186). This is supported by recent immunohistochemical studies
by Tibbetts et al. (162) demonstrating strong up-regulation
of stromal PR and simultaneous down-regulation of epithelial PR in the
mouse uterus after 4 days of estradiol treatment. In light of the
puzzling results indicating the PR in the ERKO uterus is strongly
associated with the nucleus, and therefore possibly in a more
"active" state, the ER-independent decidualization observed in
the ERKO uterus may be due to an enhanced ability to respond to
progesterone. However, a release of suprabasal levels of estradiol
during the luteal phase of the ovarian cycle is synchronized with the
time of implantation and thought to be critical to enhancing and
maximizing the uterine decidualization process (186). Nonetheless, the
deciduomas induced in the ERKO females are neither reduced in size
nor appear less complex or differentiated than those observed in the
wild type (183). It is also possible that a lack of ER during
development as well as adulthood has resulted in a uterus with a
heightened tendency toward decidualization, caused by to the altered
expression of other gene products. The complexity of the decidual
process is illustrated by the several models that lack the ability to
exhibit uterine decidualization, such as mice lacking
leukemia-inhibitory factor (188), prostaglandin synthase-2 (189), and
Hoxa-10 (190). Furthermore, a process thought to be critical to
implantation is the acquired ability of portions of the uterine
epithelium to self-destruct and become detached from the uterine wall,
possibly clearing a route by which the underlying swelling endometrium
can breach and provide a site for implantation (186, 191). Histological
analysis of ERKO uteri indicates a uterine epithelium that may be
less healthy and more often exhibits sloughing compared with wild-type
uteri. It is therefore possible that the inherently impaired luminal
epithelium of the ERKO female has resulted in a lowering of the
threshold required to induce decidualization.
B. Vagina
The fully developed adult vagina serves as both a copulatory
receptacle and a birth canal in the female and may be divided into two
distinct sections, the upper vagina and lower vagina. The cranial end
of the upper vagina is attached to the cervix and is derived from the
Müllerian ducts during differentiation of the female tract (143).
The lower vagina, which connects the tract to the vulva and external
genitalia, is differentiated from the urogenital sinus (143). As shown
in Fig. 3, the wild-type vagina is a highly sensitive estrogen target
tissue, composed of an inner mucosal layer of stroma and overlying
epithelia, a middle layer of muscularis, and an outer sheath of
connective tissue. Detectable levels of ER are present in both the
stromal and epithelial compartments making up the mucosa of the duct
(7). Estradiol exposure during the ovarian cycle induces a series of
effects in the vaginal mucosa that are often used to estimate serum
gonadal hormone levels and approximate the current stage in the estrous
cycle (107). These changes in the mucosa include cytodifferentiation of
the stromal cells and a rapid proliferation and differentiation of the
epithelial cells, resulting in a stratified and cornified epithelial
layer closest to the lumen (192). This process also involves the
estrogen stimulation of a series of keratins (193, 194). As shown in
Fig. 3, despite the chronically elevated levels of estradiol in the
serum of ERKO females, histological analysis consistently indicates
a complete lack of vaginal estrogenization. A similar effect on the
vaginal mucosa has been produced in mice (195, 196) and rats (197)
after prolonged ovariectomy or treatments with the antiestrogens,
ZM-189,154, EM-800, and tamoxifen. Administration of exogenous
estradiol, DES, or hydroxytamoxifen to ERKO mice results in no
discernible vaginal response (153). In contrast, the vaginal mucosa of
the ßERKO female appears to undergo the normal cyclic changes
associated with ovarian steroidogenesis (Fig. 3) (47), strongly
indicating that this is an ER-mediated process.
Buchanan et al. (198) have used the stromal-epithelial
tissue recombinant scheme described above for uterine tissue to dissect
the contributions of the different tissue compartments in the estradiol
response of vaginal tissue as well. As in the uterus, these studies
demonstrated that stromal ER, but not epithelial ER, are required
for estradiol-induced epithelial proliferation in the mouse vagina
(198). Similar to the observations in the uterine recombinants, both
vaginal stromal and epithelial ER were required for
estradiol-induced stratification and cornification of the epithelium,
including the induction of the gene for the secretory protein
cytokeratin 10 (198). All recombinations involving tissue from the
ERKO vagina became atrophied, even in the presence of estradiol
(198).
C. Oviduct
The mouse oviduct is a coiled tubular organ connecting the uterus
to the ovarian bursa and derived from the Müllerian duct during
fetal development of the female reproductive tract (143). It functions
as a route for passage of sperm to the ovulated oocyte and the
subsequently fertilized blastocyst to the uterus. In the CD-1 mouse,
ER immunoreactivity is easily detectable in both the stroma and the
epithelium of the oviduct as early as day 2 of life (144). The levels
of ER immunoreactivity in the epithelium continue to rise and
plateau at approximately day 15 and remain high throughout adulthood
(144). Furthermore, Newbold et al. (199, 200) described the
high degree of sensitivity of the fetal and neonatal oviduct to the
detrimental effects of developmental DES exposure. During adulthood,
the levels of ER in the oviductal epithelium fluctuate during the
ovarian cycle, reaching peak levels during the proliferative phase
(201). The ovarian sex hormones found in high concentrations in the
oviductal fluid are thought to play a role in ovum and zygote transport
through the oviduct (201). However, studies using ovariectomized
laboratory animals have produced conflicting results in terms of what
this role may be, depending on the animal model and hormone dosing
regimen used (202). Despite the apparent ontogeny of ER in the
developing and adult mouse oviduct, no gross phenotypes in the oviduct
of ERKO females have been observed. Similar to the uterus, the
epithelium of the ERKO oviduct usually appears healthy yet
unstimulated, despite the chronically high levels of serum estradiol.
Due to the inability of the ERKO to ovulate, possible defects in the
transport functions of the oviduct are not easily studied. Assays for
ERß mRNA in the mouse oviduct detect little if any ERß transcripts.
Accordingly, in ßERKO females there appears to be no obvious defects
in the structure and function of the oviduct that impede fertility.
D. Ovary
In most mammals, differentiation of the bipotential fetal gonad to
an ovary in the genotypic female occurs later in gestation than
differentiation of the testis in the male (111). The factors involved
in development and differentiation of the ovary are not well
understood, although the process does not appear to be dependent on the
presence of primordial germ cells (111). The appearance of follicles
and the onset of estrogen synthesis in the fetal gonad may be the first
indication of differentiation to an ovary, although the secreted
estrogens do not appear to be critical to development of the ductal
structures of the female reproductive tract. Still, fetal ovarian
estradiol may play a role in development of the ovary itself, as
evidenced by the complete lack of ovaries in SF-1 knockout mice (137, 138). Furthermore, a recent study has demonstrated immunohistochemical
detection of ER and ERß in the neonatal rat ovary (103).
Interestingly, a lack of ER or ERß during development appears to
have no gross effect on ovarian differentiation, since individual
knockouts of both respective receptors possess normal ovaries at birth
and during prenatal development (47, 142). A study of ovarian
development in a double-knockout lacking both forms of ER will
therefore prove interesting in the future. Still, distinct ovarian
phenotypes become apparent in the adult ERKO and ßERKO females,
resulting in infertility and subfertility in each, respectively (46, 47).
1. Review of the physiology and function of the ovary. A brief description of ovarian morphology is necessary for a discussion of phenotypes that result from a lack of ER. The ovary may be conveniently divided into three broad functional units: the follicles, corpora lutea, and interstitial/stromal compartment (203, 204). All three possess the capacity to synthesize hormonal factors, especially steroids, in response to gonadotropins secreted from the anterior pituitary. The maturing follicle is a relatively ellipsoidal unit that can be further divided into three main cell types: the outermost thecal cells, which surround a single or multiple layers of granulosa cells, and together act to encase the germ cell (oocyte) at the approximate core. The overall size of the follicle and the number of cells composing the thecal and granulosa cell compartments are dependent on the stage of maturation (204). The corpora lutea are clearly defined and vascularized structures formed from the terminally differentiated thecal and granulosa cells that remain after ovulation (203). And finally, the interstitial and stromal tissue is composed of undifferentiated cells that may eventually be recruited for the thecal or granulosa units as well as dedifferentiated thecal and granulosa cells from past atretic follicles or regressed corpora lutea. This compartment also functions as the matrix within which the follicles are suspended (203).
Ovarian function is often divided into two separate phases. The follicular phase refers to the period of follicle maturation and increased estradiol synthesis that leads up to and terminates with ovulation of the ovum. Ovulation marks the beginning of the luteal stage in which the developing corpora lutea secrete large amounts of progesterone as well as estradiol to allow for successful implantation of the blastocysts in the uterus. During the follicular stage of the ovarian cycle, the follicles may be categorized based on size, responsiveness to gonadotropins, and steroidogenic capabilities. These stages are most commonly referred to as the primordial, primary, secondary, tertiary or antral, atretic, and mature Graafian follicle (204). The primordial, or nongrowing follicles, are the most prevalent stage in the ovary at any one time and provide the pool from which follicles will be selected for maturation. These follicles consist of an oocyte arrested at the diplotene stage of the first meiotic division, surrounded by a single layer of cuboidal granulosa cells (204). Commencement of the follicular phase of the ovarian cycle involves the recruitment of primordial follicles to form the assembly of primary growing follicles to be prepared for ultimate ovulation. The factors required for this recruitment are not well understood. Henceforth, each stage is characterized by dramatic changes in the structure and functional capabilities of the follicle, which have been well characterized in several reviews (204, 205, 206, 207, 208). As the follicle progresses toward the secondary stage, rapid proliferation of the granulosa cells results in the formation of several concentric layers surrounding the oocyte (204). By this stage, stromal cells have differentiated to produce a defined stratum of thecal cells that encapsulate the granulosa cell/oocyte unit. A basement membrane acts to separate the heavily vascularized thecal layers from the granulosa cells and ovum. Since capillaries do not penetrate the basement membrane, the granulosa/oocyte compartment depends on the passive movement of hormonal factors through this extracellular structure (204). Oocyte and follicular growth are linear up to the tertiary stage, at which time the ooctye appears to reach a maximum size, while the follicle as a unit continues to enlarge. The tertiary follicle is characterized by a hypertrophied thecal layer, multiple layers of granulosa cells, and the appearance of an antrum, a space that separates the granulosa cells from the ooctye/cumulus complex. The process of follicular selection for ovulation, although not well understood, appears to occur at this stage of folliculogenesis, when several secondary-tertiary follicles will divert toward a pathway of atresia. Still under the influence of gonadotropins, the "selected" follicles will continue to enlarge, mostly due to increases in antrum size, to eventually reach the Graafian, or ovulatory stage. Follicular rupture and ovulation occur in response to a surge in gonadotropin levels, at which time cellular proliferation is ceased, and the remaining cells of the follicle terminally differentiate to form the corpus luteum (209).
In consonance with its gametogenic function, the ovary fulfills a
critical role as an endocrine organ, serving as the principal source of
sex steroids in the female. Therefore, a normal functioning ovary is an
essential prerequisite to the function and maintenance of the
reproductive tract, mammary gland, and behavior of the female. The
research efforts of several investigators during the past decades have
generated the well accepted "two-cell, two-gonadotropin" model of
ovarian estradiol synthesis. This model and the investigations leading
to its description have been discussed in great detail in several
recent reviews and therefore will be only summarized here (204, 206, 210). The two steroid-producing components of the maturing follicle are
the thecal and granulosa cells, which predominantly produce androgens
and estrogens, respectively (210). Ample evidence exists to indicate
that thecal cells possess the full complement of steroidogenic enzymes
necessary for estradiol synthesis. In contrast, estradiol synthesis by
the granulosa cells is dependent on thecal-derived androgens as
substrates for aromatization. The amount and activity of the expressed
steroidogenic enzymes within the two cell types vary depending on the
follicular stage, and thereby determine the predominant steroid being
produced. The cell-specific and temporal actions of the gonadotropins,
LH and FSH, regulate the type and activity of the steroidogenic enzymes
expressed within the granulosa and the thecal cells. The model states
that LH acting via the constituitively expressed LH receptor on the
cell surface of thecal cells stimulates the synthesis of androgens
(androstenedione) in the growing follicle. This requires the initial
conversion of cholesterol stores to pregnenolone by the
cholesterol side-chain cleavage enzyme (P450scc) and is
thought to be a rate-limiting step in thecal cell steroidogenesis
(204). Still within the thecum, pregnenolone is converted to
progesterone and then to androstenedione via the enzymatic actions of
3ß-hydroxysteroid dehydrogenase and
17-hydroxylase/C17–20 lyase (P45017),
respectively (204, 206, 210). Regulation by LH has been shown to occur
at both the transcriptional and translational levels for the
P450scc and P45017 genes (206). Granulosa
cells lack expression of the P45017 enzymes required to
produce androgens, the precursor of estradiol, and therefore are
dependent on the passage of the thecal-derived androgens through the
basement membrane and into the granulosa compartment. This cellular
cooperation provides the basis of the two-cell portion of the model.
The second gonadotropin, FSH, acts solely upon the granulosa cells to
stimulate the enzymatic conversion of the androstenedione and
testosterone to estrone and estradiol, via P450-aromatase
(P450arom), and 17ß-hydroxysteroid dehydrogenase,
respectively (204, 206, 210). The estradiol is then released into the
follicular fluid, whereupon the bulk passes back through the basement
membrane and enters the circulation. Upon ovulation, the luteal phase
begins with luteinization of the follicle and differentiation of the
remaining granulosa and thecal cells to form the corpus luteum. The
relative amounts and activities of the steroidogenic enzymes are
altered once again and shift toward synthesis of large amounts of
progesterone.
Recent data have challenged the "two-cell" model to incorporate the descriptions of a role for the oocyte in regulating granulosa cell steroidogenesis. Elegant in vitro experiments involving the surgical removal of the oocyte from isolated growing follicles have demonstrated the existence of an ooctye-secreted factor that is able to inhibit granulosa cell estradiol and progesterone synthesis (211, 212, 213).
2. Review of intraovarian estrogen actions. In 1940, both Pencharz (214) and Williams (215) independently reported a direct and specific ability of estradiol or DES to induce significant increases in ovarian weight in the hypophysectomized rat. These same seminal studies also described the synergistic effect of estradiol on gonadotropin-stimulated increases in ovarian weight (214, 215). Since then, numerous intraovarian effects of large amounts of locally synthesized estrogens have been described and postulated to be essential to normal follicular development and ovarian function. In granulosa cells of the growing follicle, estrogen has been reported to increase the levels of its own receptor (216), as well as induce DNA synthesis and proliferation (205, 217, 218, 219, 220), increase the number and size of intercellular gap junctions (221), stimulate synthesis of IGF-I (222), and attenuate apoptosis and follicular atresia (223, 224). Estradiol is also known to augment the actions of FSH on granulosa cells, resulting in the maintenance of FSH-receptor levels (218, 225, 226, 227) and the acquisition of LH-receptor (218, 228, 229, 230, 231), an event critical to successful ovulation.
Ultimately, the actions of estradiol act to enhance follicular responsiveness to gonadotropins and thereby result in increased aromatase activity and further estrogen synthesis (231, 232). Therefore, normal ovarian function appears to be dependent on a multitude of auto- and paracrine actions of estradiol that act in concert with the gonadotropins secreted from the anterior pituitary to provide for successful folliculogenesis and steroid production. Nonetheless, immunohistochemical detection and characterization of ER in the different ovarian compartments have proven difficult, although studies employing binding assays with radiolabeled ligands report the presence of ER in ovarian granulosa cells of the rat (216, 233, 234, 235), mouse (236), rabbit (236), and pig (235).
The discovery of the ERß and its reportedly high mRNA levels in the
ovary (49, 63, 93) reinforces the need for thorough immunohistochemical
studies for the two distinct ERs in the ovary. Reports of localization
of ER and ERß transcripts in the rat ovary by in situ
hybridization indicate the presence of low levels of ER mRNA with no
specific pattern (63), whereas ERß mRNA is easily detectable and
predominantly localized to the granulosa cells of growing follicles
(49, 63). Sar and Welsch (103) recently described immunohistochemical
studies with ER- and ERß-specific antibodies, reporting that ERß
immunoreactivity is indeed highly expressed in and localized to the
granulosa cells of growing follicles, whereas ER staining appears
limited to the interstitial/thecal cells in the rat ovary. Similar
findings of immunohistochemical localization of the ERß to the
ovarian granulosa cells were reported in the rat by Hiroi et
al. (104) and in the cow by Rosenfeld et al. (69).
Brandenberger et al. (94) reported the RT-PCR detection of
ER and ERß transcripts in normal and neoplastic human ovary and
ovarian cell lines. This study further described the presence of easily
detectable levels of ERß mRNA and very low levels of ER mRNA in
the granulosa cells, whereas the opposite was found in a cell-line
derived from the ovarian outer surface epithelium (94). Misao et
al. (237) also used RT-PCR to detect ER and ERß transcripts
in human corpus luteum. Both Misao et al. (237) and Byers
et al. (63) demonstrated a down-regulation of ERß mRNA
during luteinization of the follicle and the differentiation of the
corpus luteum in the human and rat, respectively. Interestingly, a
report by Iwai et al., before the knowledge of ERß,
described the detection of ER immunoreactivity in the granulosa
cells of the rabbit ovary (238), possibly illustrating another
variation in the expression pattern of the two ERs among different
species. Nonetheless, ER and ERß are present in the adult rodent
ovary. Therefore, disruption of the genes encoding these receptors may
be expected to result in distinct ovarian phenotypes. In addition, the
dissimilar expression patterns for ER and ERß among the functional
units of the follicle suggest that compensatory mechanisms fulfilled by
the remaining functional gene in each respective ERKO may not be
possible in the ovary.
3. ERKO ovary. The ovary of the neonatal and prepubertal
ERKO female does not exhibit any gross differences when compared
with those of wild-type littermates (142). The mature ERKO ovary
possesses a normal complement of primordial follicles, indicating no
defects in germ cell generation or migration to the gonad during fetal
development. However, at the commencement of sexual maturity, it
becomes apparent that the ERKO female is anovulatory and exhibits a
distinct ovarian phenotype of enlarged, hemorrhagic, and cystic
follicles as shown in Fig. 4. These
cystic structures often accumulate in the ovary, making the gonad
appear grossly as a bundle of dark grapes, a signature phenotype of the
ERKO female. However, growing follicles in the tertiary and pre- to
small antral stage are present in the mature ERKO ovary, indicating
that ER is not required for the recruitment of primordial follicles
and the initial stages of the follicular phase. This is in contrast to
the described phenotypes of mice possessing a mutation of the steel
(Sl/Slt) locus (239), or a targeted disruption of the genes
encoding growth differentiation factor-9 (240), or the vitamin D
receptor (45), all of which exhibit phenotypes of follicular arrest at
very early stages. The growing and cystic follicles in the ERKO are
often characterized by a hypertrophied thecum, indicating stimulation
by LH. Induction of thecal cell function by the chronically elevated
serum LH is illustrated by the similarly elevated levels of serum
androgens exhibited in the adult ERKO female (Table 2). Histological
analysis of ovaries from numerous ERKO females has revealed no
corpora lutea, indicating an inability of the follicles to
spontaneously ovulate and differentiate. Anovulation in the adult
ERKO could not be rescued even after appropriate stimulation with
exogenous gonadotropins (142), strongly indicating that infertility in
the ERKO female is due in large degree to alterations in the
hormonal milieu and ovarian responsiveness.
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ERKO female mice possess serum FSH
levels within the normal range (Table 2) and actually show elevated
levels of FSH-receptor mRNA in the ovary (142). Furthermore, since FSH
signaling has been proposed to be a critical factor in estradiol
synthesis (243, 244), the extremely elevated levels of serum estradiol
in the ERKO female indicate that this pathway is intact in the
granulosa cells. Interestingly, neither report of mice lacking FSH
action mention any significant differences in ovarian estradiol
synthesis but describe a much smaller and thinner uterus in the female
(241, 242). It is possible that the estradiol present in these models
lacking FSH action is synthesized by the ovarian thecum. Alternatively,
the presence of compensatory mechanisms that have allowed for granulosa
cell aromatase activity that is independent of FSH stimulation must
also be considered. Progression of the follicle to the more mature
antral stage is also arrested in mice after targeted disruption of the
gene for IGF-I (245), cyclin-D2 (246), connexin-37 (247), activin type
II receptor (248), and superoxide dismutase 1 (249), although low serum
gonadotropin levels are thought to be the cause of this phenotype in
the latter two models.
Therefore, a definitive cause of the ERKO ovarian phenotype, in
addition to the loss of ER action, was not obvious. In fact, some
facets of ovarian physiology thought to be dependent on estrogen
action, such as the attenuation of apoptosis and the induction of LH
receptors in granulosa cells of antral follicles, were apparently
preserved in the follicles of the ERKO ovary. Follicular atresia in
the ovary is a hormonally controlled process that is critical to oocyte
selection. Although the factors that trigger atresia are not well
understood, it is characterized by apoptosis of the granulosa cells of
the follicle (reviewed in Refs. 223, 224). Estradiol has been shown
to be one of the many factors reported to protect the follicle from
becoming atretic (250). Furthermore, androgens reportedly accelerate
the process, and it may be an altered estrogen/androgen synthesis ratio
in the follicle that leads to atresia (250). However, despite elevated
androgen production, the presence of androgen receptor (AR) mRNA, and a
lack of ER action in the mature ERKO ovary, an inordinate amount
of apoptosis is not observed in the follicles (142). Estradiol has also
been shown to facilitate the FSH induction of LH receptors in the
granulosa cells of the mature ovulatory follicle in both in
vivo (225, 231, 252) and in vitro experiments (229, 230). Nonetheless, the granulosa cells of the growing follicles, in
addition to the enlarged cysts in the ERKO ovary, possess
significant levels of LH receptor mRNA when assayed by in
situ hybridization (142). The most plausible explanation for these
observations is that these estrogen actions are mediated by ERß,
which has been shown to be expressed in a normal pattern in the
granulosa cells of the ERKO ovary, and concomitant with the
expression of LH receptor (93, 142).
Therefore, with data suggesting that disruption of the ER gene
did not result in an ovary completely refractory to estrogen, other
aspects of ovarian physiology must be considered as possible factors in
the etiology of the ERKO ovarian phenotype. As previously discussed,
ovarian function is tightly controlled by pituitary gonadotropins (see
Section III.D.1). In turn, gonadotropin synthesis and
secretion from the anterior pituitary are at least partially regulated
by gonadal steroids acting via classical feedback mechanisms in the
hypothalamic-pituitary axis (reviewed in Ref. 251). Indeed, disruption
of the ER gene has resulted in significant phenotypes in the
hypothalamic-pituitary axis of the ERKO female (see Section
VI.A). Most notable is the increased and chronic secretion of LH
in the ERKO female that results in levels that are 4–7 times that
found in wild-type females (Table 2) (252). As discussed above,
synchronized increases in serum FSH and LH levels are critical to
follicular maturation and ultimate ovulation in the ovary. However, it
has been proposed that the follicular requirements for LH are finite,
and the presence of abnormally high levels may force maturing follicles
to either prematurely luteinize or become atretic (253).
Therefore, the ERKO ovarian phenotype is likely caused by the
chronic exposure to abnormally high levels of LH. Support for this
hypothesis can be drawn from a number of studies. Investigations
involving prolonged treatment with antiestrogens over a period of at
least 28 days have produced an ovarian phenotype in both mice (195, 196) and rats (197) that is similar to that of the ERKO. Of course,
interpretation of these studies is complicated by the ability of the
antiestrogens to inhibit both ERs as well as estrogen action in both
the ovary and hypothalamic-pituitary axis. However, these studies
reported that the "ERKO" phenotype of enlarged cystic follicles
was produced only after prolonged treatments with those antiestrogens
that possessed the ability to cross the blood-brain barrier and
concurrently produce serum LH levels that were several fold higher than
controls. Therefore, whereas the estrogen antagonists ZM-189,154 (197)
and EM-800 (195, 196) produced chronically elevated LH levels and an
ovarian phenotype strikingly similar to that of the ERKO, tamoxifen
did neither (195, 196, 197). More definitively, Risma et al.
(254, 255) report that targeted transgenic overexpression of the
LHß-subunit in the mouse that results in a 15-fold increase in serum
LH levels produces females that are anovulatory and exhibit an ovarian
phenotype almost indistinguishable from that of the adult ERKO
female.
Therefore, the similarity in the ovarian phenotypes described in the
above studies, in which the models presumably possessed normal levels
of ovarian ER, combined with our observations in the ERKO,
strongly indicate that the ER is not directly involved in the
development of ovarian cysts due to hypergonadotropism. However, there
are descriptions of at least two models in which serum LH is
chronically elevated, yet do not manifest an ovarian phenotype similar
to the ERKO or those induced by antiestrogens or transgenics as
described above. Female mice that are homozygous for a targeted
disruption of the FSHß-subunit gene exhibit an approximate 5-fold
increase in serum LH but do not show indications of enlarged cystic
follicles in the ovary (241), indicating a role for FSH in this process
as well. Bogovich (256) has provided supporting evidence by
demonstrating that FSH is required along with prolonged exposure to LH
(in the form of human CG) to induce follicular cysts in the rat. A
likely role for FSH is the induction of LH receptor in the granulosa
cells of the maturing follicles, thereby rendering the follicle
responsive to the increased levels of LH. Another contrasting knockout
model is that of the P450arom gene (ArKO), in which the
homozygous females possess significantly elevated serum LH and FSH
levels but lack the capacity to synthesize estradiol (257). Although
folliculogenesis is arrested at an antral stage in the ArKO ovary, no
ERKO-like cystic structures are reported (257). Therefore, assuming
that a lack of estradiol synthesis would disrupt ligand-dependent
activity of the ERß in the granulosa cells, the lack of ovarian cysts
in the ArKO indicate an intraovarian role for estradiol in this
phenotype. Therefore, although the ERKO phenoytpe may be triggered
by hyperstimulation of the follicles by LH, it is likely influenced by
both FSH and ERß actions in the granulosa cells as well. Current
studies utilizing prolonged treatment of ERKO females with a GnRH
antagonist to reduce serum gonadotropins are being carried out to
further define the etiology of the ovarian phenotype (J. F. Couse,
D. O. Bunch, J. Lindzey, D. W. Schomberg, and K. S. Korach, manuscript
in preparation).
Since the ERKO ovarian phenotype develops and worsens only after
sexual maturity, we have began studies to characterize ovarian function
in the immature ERKO female (J. F. Couse, D. O. Bunch, J. Lindzey,
D. W. Schomberg, and K. S. Korach, manuscript in preparation). Although
superovulation with exogenous gonadotropins was not successful in
eliciting ovulation in the older ERKO females, immature (28 days)
females do respond and produce viable oocytes that can be collected
from the oviduct. However, the average number of oocytes collected from
superovulated ERKO females is significantly less than that yielded
from age-matched superovulated wild-type and heterozygous females.
Therefore, intraovarian ER action does not appear to be
essential to ovulation when stimulated with exogenous
gonadotropins.
4. ßERKO ovary. As discussed above, the ERKO has provided
a number of indications to suggest that intraovarian ER action is
not critical to ovarian function. Furthermore, several presumed
functions of estradiol in ovarian granulosa cells appear to be
preserved in the ERKO, such as the attenuation of apoptosis and the
induction of LH receptors. Based on the reported localization of ERß
mRNA (49, 50, 63) and protein (69, 103, 104) to the granulosa cells of
growing follicles, as well as the maintenance of a normal expression
pattern for ERß in the ERKO ovary (93, 142), it is likely that the
newly discovered ER is the predominant mediator of estrogen action in
the follicle. In the rat ovary, ERß is easily detectable by
immunohistochemistry and exhibits an almost ubiquitous expression
pattern within the granulosa cells of follicles ranging from the
primary up to the ovulatory or Graafian stage (69, 103, 104). Byers
et al. (63) demonstrated that ERß mRNA levels remain
relatively constant during the follicular stage of the ovarian cycle
but are rapidly decreased after the gonadotropin surge-induced
luteinization of the granulosa cells. Interestingly, in 1975, Richards
(216) reported a similar profile for high-affinity
[3H]-E2 binding in the granulosa cells of the
rat ovary, showing increased binding levels as follicles matured
followed by marked decrease after luteinization. Because the affinities
of the two ERs for estradiol do not significantly differ, it was not
possible at that time to realize that the receptor being detected in
the ovary was distinctly different (i.e., ERß) from that
which was being concurrently described in the uterus (i.e.,
ER).
As stated previously, female mice homozygous for the targeted
disruption of the ERß gene possess no gross aberrant phenotypes as
neonates or during adulthood (47). However, during a continuous mating
study of 8 weeks in which sexually mature wild-type or ßERKO females
were housed with a known fertile wild-type male, a significant deficit
in fertility in the ßERKO became obvious. As shown in Table 3, ßERKO females produced substantially
fewer litters as well as significantly less numbers of pups per litter
when compared with their wild-type littermates (47). Whereas the
average litter size among wild-type females was 8.8 (±2.5) pups per
litter, this number was reduced to 3.1 (±1.8) in ßERKO females
(Table 3) (47). Furthermore, two of the tested ßERKO females yielded
no litters, despite the observation of seminal plugs on multiple
occasions, suggesting that abnormal sexual behavior was not a cause for
the infertility. Gross analysis of the uteri from the ßERKO females
used in this study demonstrated no indication of embryo resorption
during gestation. Therefore, the observed subfertility in the ßERKO
females did not appear to be due to uterine dysfunction that results in
premature termination of pregnancy. However, a possible defect in
embryo implantation due to a lack of ERß could not be assessed in
these studies.
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, histological analysis of
ovaries from sexually mature ßERKO females illustrated the presence
of a relatively normal interstitial compartment and follicles at
various stages of the follicular cycle, ranging from primordial and
primary to those with a clearly defined antrum, all possessing the
expected thecal shell. Therefore, as demonstrated for ER by the
ERKO, ERß does not appear to be essential for the establishment of
germ cell number or ovarian development. Several of the speculated
intraovarian roles of estrogen were discussed previously and include a
proposed critical role in proliferation of the granulosa cells in the
maturing follicle (217, 218, 219, 220, 231). However, the multiple large
follicles present in the ßERKO ovaries indicate no marked differences
in granulosa cell number. Furthermore, serum estradiol levels in adult
ßERKO females do not appear to differ from age-matched wild-type
females, at 24.2 (±3.3) and 30.5 (±2.8) pg/ml, respectively.
Biological evidence of estradiol synthesis in the ßERKO ovary is also
illustrated by the apparently normal uterus and vagina, which exhibit
the proper cyclic changes in morphology of a sexually mature wild-type
mouse. Nonetheless, there were indications of an increased number of
early atretic follicles and a sparse presence of corpora lutea in the
ßERKO ovary when compared with the wild-type (47), suggesting that
the observed subfertility in the ßERKO may be due to a reduction in
completed folliculogenesis.
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To gain further insight into the ovarian phenotype of the ßERKO
female, immature knockout and wild-type mice were stimulated to ovulate
by administration of superphysiological levels of gonadotropins (PMSG
and hCG) (47). After several independent trials, it became obvious that
the ovulatory capacity of the ßERKO female was dramatically reduced,
yielding an average of 6 (±1.5) oocytes per female vs. 33
(±4.8) and 52 (±5.7) oocytes per female in the wild-type and
heterozygous animals, respectively (Table 3) (47). Additionally, the
cumulus mass that surrounded the ovulated follicles from the ßERKO
females was consistently composed of a decreased number of cells and a
lessened integrity when compared with ova yielded from wild-type
controls. Most interesting was the histology of the ovaries from the
superovulated ßERKO females, which indicated the presence of numerous
preovulatory but unruptured follicles (Fig. 5). It therefore appeared
that the follicles of the ßERKO ovary were able to respond to the
proliferative effects of PMSG in terms of increased size and antrum
formation. However, a severe deficit in the response to the
gonadotropin surge (hCG), required to induce luteinization and rupture
of the follicle, was obvious in the ßERKO. A small number of the
selected follicles were able to be expelled, as evidenced by the
presence of ova in the oviduct and of corpora lutea in the
corresponding ßERKO ovary. Therefore, a lack of ERß resulted in a
drastic reduction in ovulatory capacity, yet with incomplete
penetrance. Until ERKO females are treated and tested in a similar
manner to the ßERKO studies described, it will be difficult to
determine the precise role for each ER in the ovary.
The observation of numerous unruptured Graafian follicles in the ovaries of superovulated ßERKO females is strikingly similar to the phenotype reported for mice possessing a targeted disruption of the cyclin-D2 gene (246). Cyclin-D2 is a positive regulator of cell cycle progression that is highly expressed in granulosa cells (reviewed in Ref. 258). Robker and Richards (259) demonstrated strong up-regulation of the cyclin-D2 gene by estradiol in rat granulosa cells and suggested that this protein may be a downstream mediator of the synergistic actions of FSH and estradiol that result in increased granulosa cell numbers in the maturing follicle. The dramatic increases in cyclin-D2 induced by estradiol are evident in vivo at both the mRNA and protein levels in the rat. Furthermore, assays on primary granulosa cell cultures from the rat indicate that estradiol stimulation of the cyclin-D2 gene can be inhibited by the estrogen antagonist, ICI-164,384, strongly suggesting that it is an ER-mediated process (259). Therefore, given that ERß is the predominant form of ER in the granulosa cells, disruption of the ERß gene may likely result in significant deficits in cyclin-D2 expression in the granulosa cells of the growing follicles in the ßERKO female. However, FSH is also able to stimulate increases in cyclin-D2, although the temporal pattern of regulation by FSH is distinct from that elicited by estradiol (259). Nonetheless, it is possible that FSH action in the follicles of the ßERKO ovary have provided for some degree of cyclin-D2 expression and thereby may explain the incomplete penetrance of the ßERKO ovarian phenotype.
The ßERKO ovarian phenotype that becomes apparent after superovulation is also similar to that reported for knockout models of the genes for the PR (PRKO) (44) and prostaglandin synthase-2 (189). The dramatic increases in both PR (260) and prostaglandin synthase-2 (261) in the granulosa cells of the ovulatory follicle shortly after the gonadotropin surge have been well documented. Furthermore, the lack of follicular rupture in the respective knockout models supports a critical role for each of these components in ovulation (reviewed in Ref. 262). Lydon et al. (44) reported infertility in the PRKO and a consistent inability of superphysiological doses of hCG to induce follicular rupture. Although regulation of the PR gene is strongly influenced by estradiol in the uterus (see Section III.A), sufficient evidence exists to indicate that this may not be the case in the ovary. For example, although the wild-type ovary possesses extremely high intraovarian levels of estradiol and the presence of ERß, levels of PR mRNA and protein remain at a modest basal level in granulosa cells of the pre- and antral follicle (260). However, within 4–6 h after the gonadotropin surge, transcription of the PR gene has peaked at levels several fold that before the surge, only to return to near-basal levels within 20 h (260). The mechanism of this strong and transient induction of the PR gene by LH is known to include significant increases in intracellular cAMP, but may also involve phosphorylation of ERß and/or a coactivator, which then combine to act in a synergistic nature (262). Therefore, the elevation in PR levels in the granulosa cells of the ovulatory follicle that is critical to follicular rupture may be attenuated in the ßERKO ovary.
Another possibility for a lack of spontaneous ovulation in the ßERKO
female may be not intaovarian in nature, but rather due to altered
gonadotropin synthesis and secretion from the hypothalamic-pituitary
axis. The sex steroids play an important role as a positive regulator
of the preovulatory surge (reviewed in Refs. 263, 264). Although the
exact mechanism of action by which estrogens may be involved is not
well defined, studies have shown that estradiol can induce GnRH release
from the hypothalamus as well as cause increases in the level of GnRH
receptors in the anterior pituitary (263). The ER may be the
predominant form of ER in the pituitary of the adult female mouse (93);
however, both ER and ERß have been detected in various regions of
the hypothalamus (88, 97, 265). Preliminary data in the ßERKO female
indicate that tonic levels of serum LH are within the normal range.
However, a lack of hypothalamic ERß may have reduced the potential
for positive regulation by estradiol in the hypothalamic-pituitary axis
and thereby may result in a reduction in the frequency and/or amplitude
of the preovulatory gonadotropin surge. Nonetheless, the results of the
superovulation studies described above, in which an artificial bolus of
gonadotropin is administered to induce ovulation, indicate a severe
phenotype that can be localized to the ovary of the ßERKO female.
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IV. Mammary Gland |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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-dihydrotestosterone, or PRL has been reported to
result in an apparent increased sensitivity of the gland to
mammotrophic hormones during adulthood, leading to varied degrees of
excessive ductal growth and differentiation (267, 269). The later four
stages of mammary gland development occur after birth and terminate in
a gland capable of milk production. These stages are strongly regulated
by the endogenous ovarian steroid hormones and are characterized by
massive growth of the glandular ducts that emanate from the nipple
until they have progressed through the fat pad composing the bulk of
the breast. Upon pregnancy and the onset of lactation, the gland
undergoes dramatic differentiation to produce milk- secreting
structures, termed alveoli, throughout the ductal network. Therefore,
since the majority of mammary gland growth and differentiation occurs
after birth, this tissue provides a unique tool within which the
interactions of gonadal and peptide hormone systems may be studied.
A. ERKO phenotype
The mammary gland of an adult wild-type female mouse consists of a
network of epithelial ducts originating from the nipple and forming a
tree-like structure. The growth of the epithelial ducts begins during
prepubertal development and continues during puberty until the branches
of the gland have reached the limits of the fat pad (266). This ductal
elongation is via cap cell proliferation at the terminal end buds of
the individual ducts, as they maintain close contact with the stromal
fat pad through which they are progressing. Studies using
ovariectomized models have indicated that estradiol and GH, locally
mediated as IGF-I, are required for the development of this ductal
structure (266, 268). The mammary glands of adult ERKO female mice
exhibit a phenotype similar to the glands of a newborn female,
confirming the need for ER-mediated estradiol actions for ductal
growth (269). However, the ERKO gland does possess the component
structures necessary for mammary gland development, i.e.,
the epithelial and stromal portions, connective tissue, and a small
rudimentary ductal tree. Therefore, embryonic and fetal development of
the mammary gland in the mouse occurs independently of ER actions.
However, this may be strain dependent, since studies in the C57BL mice,
the background strain of the ERKO, have also reported little effect
of neonatal steroid exposure on the mammary gland (267). Nonetheless,
despite apparently unaltered GH levels and the existence of
significantly elevated levels of serum estradiol, the mammary gland of
the ERKO female has lost the capacity to commence the pre- and
postpubertal stages of growth.
Estradiol has been shown to directly stimulate the formation of
terminal end buds and stimulate cellular proliferation of the mammary
ductal epithelium (270). Furthermore, this physiological effect can be
inhibited by antiestrogens (271), indicating a receptor-mediated
pathway of estrogen action. Although ER has been detected in both the
ductal epithelial cells as well as the stromal tissue of the mammary
gland (270, 272), the mechanism by which estrogens directly regulate
growth remain unclear. The lack of ER in the outermost proliferating
cap cells of the terminal end buds suggests that the effects of
estradiol may be indirect and may involve the regulation of cell cycle
genes and/or paracrine-acting peptide growth factors and their cognate
receptors (270). Both EGF and transforming growth factor- are
thought to be critical to proper mammary growth and development and are
at least partially regulated by estradiol via the ER (273, 274, 275).
Support for a role of estradiol-induced growth factor activity is
provided by the study of Xie et al. (276), in which
transgenic mice overexpressing a dominant-negative form of the EGF-R
exhibited an attenuated response to EGF action in the mammary gland
in vivo. The virgin transgenic mice of this study exhibited
severe deficits in ductal growth and branching in the mammary gland,
which were overcome only with pregnancy, when ovarian steroids
increased endogenous EGF expression several fold that found in virgin
females (276). Further evidence comes from Ankrapp et al.,
who recently reported that estradiol-releasing pellets implanted into
the mammary fat pad of ovariectomized mice result in significantly
increased levels of EGF and PR and induce terminal end bud formation in
the gland (277). However, implantation of EGF-releasing pellets induced
similar increases in PR and terminal end bud formation, suggesting that
EGF may mediate the mitogenic actions of estradiol (277). Therefore,
this study demonstrated a scenario of ER-EGF cross-talk similar to
that previously described in the uterus (see Section
III.A.2). In brief, treatment of mice with pellets releasing both
EGF and the antiestrogen ICI-182,780 exhibited inhibition of the
mitogenic effects of the growth factor; in turn, an anti-EGF antibody
was able to neutralize the growth effects induced by an implanted
estradiol-releasing pellet (277). Therefore, as was confirmed in the
uterus through the use of the ERKO mice, EGF is also able to mimic
the mitogenic effects of estradiol in the mammary gland, but once again
appears to require the presence of functional ER to complete this
mechanism.
It is well known that the mammary fat pad serves not only as a matrix,
but also provides biochemical signals and/or factors necessary for
normal ductal growth (reviewed in Ref. 266). In the study by Ankrapp
et al. (277) described above, it was found that the greatest
induction of EGF by estradiol occurred in the stromal compartment of
the gland. Confirmation of a requirement for ER in the glandular
stroma and the likely involvement of paracrine growth factors in
estrogen regulation of mammary gland growth has been demonstrated by
Cunha et al. using the tissue recombinant methodology
described previously (see Section III.A.2). Tissue
recombinations of mammary fat pad and epithelium from wild-type and
ERKO females were constructed and grafted under the kidney capsule
of athymic nude mice for 4 weeks (278). Extensive ductal growth was
observed only in recombinants involving wild-type (i.e.,
ER+) stroma, including those composed of ERKO ductal epithelium
(278). However, ERKO (ER-) stroma was unable to induce growth in
the overlying ductal epithelium from either genotype (278). The authors
therefore concluded that stromal ER is essential to the mitogenic
actions of estradiol in the mammary epithelium, a conclusion also
reached from similar studies in the uterine and vaginal tissue. In
light of these data produced by Ankrapp et al. (277) and
Cunha et al. (278), current studies to determine whether the
underdeveloped ERKO mammary gland may be rescued by exogenous
treatment with growth factors will prove interesting.
Complete maturation of the mammary gland during pregnancy involves further ductal growth, extensive branching, and differentiation of the lobuloalveolar structures along the ducts, the hallmark of the lactating gland (266). These alveoli provide for milk production and will eventually fill the remaining spaces of the fat pad. This stage of mammary gland development is thought to require the combined actions of estrogen, progesterone, and PRL (266). The need for progesterone action in lobuloalveolar development was confirmed by studies of the PRKO mouse, which exhibited a normal pubertal ductal structure but was completely refractory to the induction of lobuloalveolar development after progesterone treatment (44). A similar phenotype is observed in the female PRL-receptor knockout (PRLR-/-) mice, which possess a normal virgin mammary gland as an adult, but a severe deficit in lobuloalveolar development and lactation after pregnancy (279). Interestingly, because the homozygous PRL-receptor (PRLR-/-) knockout females like others are infertile, this mammary gland phenotype was first detected in the heterozygous (PRLR+/-) females (279). Furthermore, the defects in lactation in the PRLR+/- mice were lessened after multiple pregnancies, due to the compensatory actions of other pregnancy-associated mammotrophic hormones that accumulated with each pregnancy (279).
Due to the documented ability of the estradiol-ER complex to
increase PR expression in various tissues, including the mammary gland
(280), a loss of ER action may also result in a loss of PR-mediated
progesterone functions. Although progesterone actions in the mammary
gland were once thought to be strictly differentiative and may actually
oppose the proliferative effects of estrogens, recent work has
suggested that progesterone may also act as a mitogen in the breast
(reviewed in Ref. 181). This is best illustrated by the less extensive
ductal arborization of the mammary gland, as well as the lack of
alveolar structures, in the PRKO mice (44). Furthermore, in
vitro studies have indicated that PR-mediated progesterone actions
can regulate the transcription of genes for cell cycle-related
proteins, growth factors, and growth factor receptors (181). Therefore,
the lack of ductal growth and differentiation in the ERKO mammary
gland may be largely due to a lack of physiologically sufficient PR and
subsequent progesterone action. As shown in Table 2, the average serum
progesterone levels in adult female ERKOs are 4.0 (±1.1) ng/ml
vs. 2.3 (±0.6) ng/ml in wild-type and are therefore within
the normal range of cycling wild-type females (123). Preliminary
analysis by ribonuclease protection assay indicates that the mammary
glands of adult virgin ERKO females possess slightly detectable
levels of PR mRNA, but exhibit no increased regulation when treated
with estrogen. Furthermore, current studies in our laboratory indicate
that prolonged progesterone treatment of adult ERKO females results
in the formation of terminal end buds and differentiation in the
mammary gland, indicating that the lack of progesterone action caused
by disruption of the ER gene can be partially overcome with
treatment of superphysiological levels of exogenous progesterone.
A similar secondary effect of targeted disruption of the ER gene on
the mammary gland may involve a lack of PRL stimulation. PRL is
primarily secreted from the anterior pituitary, although local
synthesis in the mammary epithelium has also been described (279). This
peptide hormone plays a critical role in mammary gland physiology,
especially in the induction of lobuloalveolar structures and milk
production, as evidenced in the phenotype of the PRLR+/- mice
(reviewed in Ref. 279). However, the synthesis and secretion of PRL
from the anterior pituitary are strongly regulated by estradiol and
ER (281). Confirmation of the regulatory action of ER on the PRL
gene is provided by the ERKO females, which exhibit a 20-fold
decrease in PRL mRNA in the anterior pituitary (282) and a 5-fold
decrease in serum PRL levels (Table 2) (see Section VI.A.4).
Therefore, given the significant role of PRL in the differentiation to
a lactating mammary gland, it is likely that the phenotype described in
the ERKO female is also at least partly due to a lack of PRL
stimulation. Support for this hypothesis is provided by current studies
in which elevated serum PRL levels are achieved in the ERKO female
via wild-type pituitary transplants placed under the kidney capsule and
have resulted in significant ductal growth and differentiation in the
ERKO host mammary gland (W. P. Bocchinfuso, J. Lindzey, S. W.
Curtis, J. E. Clark, P. H. Myers, and K. S. Korach, in
preparation). However, preliminary analysis indicates that this partial
rescue of the ERKO mammary gland by the PRL secreting wild-type
pituitary graft does not occur in an ovariectomized ERKO host
female, indicating a requirement for ovarian derived factors as well.
Nonetheless, the ERKO mammary gland appears to possess the intrinsic
tissue components necessary for pubertal development and
pregnancy-induced maturation, but fails to develop because of the loss
of multiple stimuli that are downstream of ER action.
B. ßERKO phenotype
Unlike the dramatic underdevelopment observed in the mammary gland
of the ERKO, no such phenotype is observed in adult ßERKO females.
Virgin ßERKO females of 4–5 months of age exhibit mammary glands
that possess a normal ductal structure that fills the entire fat pad
and are indistinguishable from those of age-matched wild-type females.
This phenotype agrees with our description of minor amounts of ERß
mRNA in the adult mouse mammary gland, whereas ER transcripts are
easily detectable (Fig. 2) (93). Furthermore, mammary glands from
pregnant and nursing ßERKO females appear to have undergone normal
differentiation and exhibit the lobuloalveolar structures required for
lactation. Although litter sizes of the ßERKO females were reduced
during the mating study described above, no marked abnormalities in
nursing were observed. Therefore, the combined data from the ERKO
and ßERKO models indicate that ER is the predominant receptor
required to mediate the actions of estrogen in the mammary gland of the
mouse.
C. ER and oncogene-induced tumorigenesis: Wnt-1/ERKO mice
Several lines of evidence indicate that breast cancer in humans is
strongly correlated with the extent of lifetime exposure to estrogen.
Most notably, breast cancer almost exclusively occurs in females and is
never seen before puberty but rather only after several years into the
reproductive life span (283, 284). Furthermore, an increased length of
a woman’s reproductive years, i.e., early menarche and late
menopause, has been associated with an elevated risk of breast cancer
(284), whereas women who have experienced premature menopause due to
natural causes or castration appear to be at a much lower risk (283).
Furthermore, studies have indicated that women who circulate higher
levels of active estrogens may also be at greater risk of developing
breast cancer (283). In apparent contrast, early pregnancy tends to
provide a protective effect, although it is obviously associated with
an increased level of steroid hormone exposure. In addition, several
years of research have indicated little positive correlation with the
risk of breast cancer and prolonged use of contraceptive pills composed
of synthetic estrogens and progestins, although this issue remains
controversial (reviewed in Refs. 285, 286). Still, a large portion
of chemotherapeutics for breast cancer are aimed at either blocking
estrogen action or reducing estrogen levels (283). Therefore, although
an association between estrogen action and breast cancer is apparent,
it involves less understood yet critical mechanisms, including the
periodicity and cyclicity of hormone exposure as well as the
sensitivity of the end organ (283). This is further complicated by the
influence that environmental exposures, geography, diet, body weight,
and genetics also play in individual risk of developing breast cancer
(283).
Numerous studies have been carried out concerning the levels of ER and
PR in neoplastic breast tissue and the prognostic value that these
parameters may provide (reviewed in Refs. 287, 288, 289, 290, 291). Reports indicate
that more than 70% of primary breast tumors are ER-positive and
exhibit estrogen-dependent growth (291). However, the most malignant
mammary tumors are often ER-negative and exhibit
estrogen-independent aggressive growth, but are thought to progress
from a once ER-positive cell population (287). In addition, the role
of local aromatase activity and estrogen production in breast cancer is
receiving increased attention (reviewed in Ref. 292). Added complexity
is introduced by the detection and description of numerous variants of
the ER transcript in breast cancer tissues, although their possible
role in the etiology of the disease remains speculative (reviewed in
Refs. 28, 293). Recent reports also described the detection of ERß
transcripts in multiple immortalized human breast cancer cell lines,
normal human breast tissue, and human breast tumors (64, 73, 101, 102, 294). Vladusic et al. (73) also characterized an ERß mRNA
variant detected in normal as well as malignant human breast tissue
(73).
It is clear from the severely underdeveloped mammary gland of the
ERKO that estradiol acting via the ER is a potent mitogen in the
breast. To gain insight into the potential role of ER in the
induction and promotion of mammary gland carcinogenesis, we crossed the
ERKO mice with the MMTV-Wnt-1 mice, a transgenic line that is highly
susceptible to mammary adenocarcinoma. The family of Wnt genes encode a
series of secretory glycoproteins that act in auto- and paracrine
pathways to stimulate cell proliferation and differentiation (reviewed
in Ref. 295). The MMTV-Wnt-1 mice possess a transgene designed for
targeted overexpression of the Wnt-1 protooncogene in the mammary gland
and exhibit a nearly 100% and 15% incidence of mammary hyperplasia
and lobuloalveolar adenocarcinoma by 1 yr of age in females and males,
respectively (295, 296). Therefore, breeding of the two transgenic
lines allowed for the generation of animals that possessed the Wnt-1
transgene on either a wild-type or ERKO background and thereby
allowed for the assessment of the role of ER in the initiation and
promotion of protooncogene-induced mammary tumors (297).
At 6 months of age, virgin wild-type Wnt-1 females exhibit extensive
hyperplasia of the ductal epithelium and aberrant lobuloalveolar
development that occupies the entire fat pad. This was the expected
phenotype based on that described previously in the original line of
Wnt-1 females derived from a different mouse strain (296).
Interestingly, a similar phenotype of lobuloalveolar hyperplasia was
observed in the rudimentary duct of the ERKO-Wnt-1 females, although
the extent of ductal growth was much reduced compared with that seen in
the wild-type (269). Nonetheless, the rudimentary ductal structure
previously described in the ERKO female was obviously induced to
proliferate by the presence of ectopic Wnt-1 expression. However,
comparison of mammary glands from a series of age-matched animals
indicated that the ductal growth observed in the mammary gland of a
6-month-old ERKO-Wnt-1 female remained confined to the nipple region
and approximated that seen in a 2.5-month-old wild-type-Wnt-1 female,
illustrating a significant delay in the proliferation of the ERKO
epithelium (269). Interestingly, the mammary hyperplasia in the
ERKO-Wnt-1 females did not markedly progress into the inguinal fat
pad, but rather remained confined to the area of the nipple. Therefore,
although the lobuloalveolar phenotype characteristic of Wnt-1
overexpression was evident, ductal elongation did not occur in the
ERKO-Wnt-1 female, indicating that the hyperplastic action of
ectopic Wnt-1 expression cannot substitute for ER-mediated terminal
end bud formation and ductal morphogenesis (297). In the males,
Wnt-1-induced epithelial hyperplasia was obvious in both the wild-type
and ERKO animals as well, but no distinct difference in growth rates
between the two genotypes was evident (269).
The incidence of lobuloalveolar carcinoma in the Wnt-1 mice mirrored
the observations described above for the epithelial hyperplasia.
Wild-type-Wnt-1 and heterozygous-ER/Wnt-1 females developed mammary
tumors at a rapid rate, reaching an incidence of 50% by 6 months of
age (297). Ectopic expression of the Wnt-1 gene was also able to induce
tumorigenesis in the ERKO females and therefore did not require the
presence of functional ER (297). However, a 50% incidence in tumors
in the ERKO-Wnt-1 females was not observed until 12 months of age,
twice the time required for the wild-type-Wnt-1 colony (269).
Ribonuclease protection assays indicated that the level of Wnt-1
transgene expression was relatively equal in the two ER genotypes.
Prepubertal ovariectomy had no effect on the overall incidence of
tumors in either genotype, although it resulted in a delayed incidence
of palpable tumors in both, i.e., wild-type-Wnt-1 at 50% at
10.5 momths and a further delay in the ERKO-Wnt-1 at 50% at 15
months (269). Therefore, ovarian factors clearly accelerated the growth
rate of the Wnt-1-induced adenocarcinoma in both the wild-type and
ERKO females. However, multiple pregnancies had no significant
effect on the time of tumor onset in the wild-type-Wnt-1 females (269).
These studies indicate that ER signaling is not involved in the
induction of hyperplasia and tumorigenesis in the mammary gland that
results from overexpression of the Wnt-1 protooncogene, but indeed
plays a promotional role in these phenotypes. Similar findings of a
promotional role for the ER in uterine carcinogenesis were reported
in transgenic mice that overexpressed an ER gene (298). A possible
explanation for the observed tumor latency may be drawn from an
inherent difference between the wild-type and ERKO mammary glands,
i.e., because the ERKO-Wnt-1 glands possess a reduced
amount of ductal morphogenesis and proliferating epithelia due to the
lack of ER, the number of cells most susceptible to neoplastic
transformation may be decreased (297). Interestingly, the growth rates
of the ductal hyperplasia and ultimate adenocarcinomas were also
significantly reduced when the animals were ovariectomized, even in
those lacking functional ER. A possible explanation for this finding
may be that ovarian estradiol is acting via non-ER-mediated
pathways, suggesting a possible role for ERß. However, in contrast to
reports in other species, ERß mRNA remains difficult to detect by
standard assays in the mouse mammary gland, including the glands of the
ERKO (93) and Wnt-1 females (297). Furthermore, ovariectomy also
results in the loss of other gonadal hormones in addition to estrogen.
Remarkably, overexpression of the Wnt-1 gene in the ERKO mammary
gland resulted in significant increases in PR mRNA levels compared with
the low basal levels detected in control ERKO glands, when assayed
by ribonuclease protection assay (297). Recently, Shyamala et
al. (299) showed that misregulation of the PR gene resulting in
increased levels of PRA through transgenic methodologies
may be tumorigenic in the mammary gland. Therefore, the ability of
Wnt-1 to compensate for the reduced PR levels generated by the loss of
ER action may provide a pathway by which the role of ER in tumor
induction and promotion may be overridden. In addition, if the PR
pathway was involved in the promotion of the Wnt-1 lobuloalveolar
carcinomas, ovariectomy would be expected to result in a delay in their
growth.
In summary, the ERKO/Wnt-1 mouse model has demonstrated that
artificially induced hyperplasia and tumorigenesis of the mammary gland
can take place in the absence of ER signaling. These findings have
potential importance in understanding the etiology of human breast
cancer by illustrating that oncogenic induction of mammary neoplasia
can occur in an ER-negative tissue. Extending these observations
further may indicate that hormone-independent breast tumors can
originate from a cell population lacking ER as well as those that
are ER positive. The characterization of mice produced from crosses
of other overexpressing transgenic models, e.g.,
neu, with the ERKO are currently being carried out in a
fashion similar to those studies described above.
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V. Reproductive Tract Phenotypes of the Male |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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-dihydrotestosterone (DHT) (143). The importance of
androgen action in the development of the male reproductive tract is
evident by the feminized phenotype that results in human males with
complete androgen insensitivity syndrome (cAIS), caused by a naturally
occurring mutation that results in the lack of functional AR. The
phenotypes of the cAIS human male and the analogous testicular
feminized male (Tfm) rodent are characterized by the
complete absence of either male or female internal reproductive
structures except for the presence of inguinal testes (37, 301).
External genitalia in the cAIS human and Tfm mouse are
indistinguishable from the normal wild-type female, but a short and
often blunt-ended vagina is present (37, 301). During puberty and the
later stages of sexual maturation in the male, virulization of the
external genitalia is dependent on the actions of DHT (143, 302). This
is illustrated in human males lacking sufficient 5-reductase
activity and DHT synthesis, who exhibit normal internal reproductive
structures and testosterone levels but severely undervirulized external
structures (reviewed in Ref. 37). Once again, the critical role of
androgens in the development of the male reproductive tract was
reiterated by the complete lack of gonadal development in mice
homozygous for a disruption of the gene encoding SF-1 (137, 138).
Therefore, androgen action is critical to the development and function
of the tissues of the male reproductive tract from fetal stages through
adulthood, whereas a defined role for estrogen remains unclear. Although there is no apparent role for estrogen action in the development of the male reproductive tract, studies have reported significant effects of early exposure to estrogen agonists and/or antagonists on the adult male reproductive system. However, it was generally concluded that such effects were caused by estrogen actions at the hypothalamic-pituitary level that ultimately resulted in decreased stimulation of the testis and reduced androgen production (303, 304). Still, a series of studies by McLachlan et al. demonstrated that perinatal exposure to the synthetic estrogen, DES, results in an assortment of apparently direct defects in the murine male reproductive tract, including undescended testes, epididymal cysts, aberrant expression of estrogen-inducible genes, adenocarcinoma, and sterility (305, 306, 307, 308). However, these studies indicated a toxic effect of developmental exposure to pharmacological levels of estrogens rather than a required physiological function of estrogen in the male reproductive tract. Investigators have reported the detection of ER by steroid autoradiography (309, 310) and immunohistochemistry (112) in the testes and accessory tissues of the male tract, even as early as day 16 of gestation in the mouse. In addition, the newly described ERß also appears in varied amounts in the testis, epididymis, and prostate of the rodent (93, 121, 311, 312, 313, 314) and rhesus monkey (96). Hence, these studies suggest the presence of a functional estrogen- signaling system in the male reproductive tract and further demonstrate the direct detrimental effects that may occur when this system is aberrantly activated.
A. Testicular function and spermatogenesis
A defined role of ER and estrogen action in the function and
maintenance of the male reproductive tissues remained elusive until the
generation of the ERKO mice. As expected in the presence of a
functional androgen-signaling system, the reproductive tract of the
ERKO male mouse undergoes apparently normal prenatal development to
produce internal and external structures that are indistinguishable
from wild-type littermates. However, at approximately 20 weeks of age,
significant decreases in the weight of the testis and epididymis/vas
deferens are observed, whereas the seminal vesicle/coagulating glands
and prostate appear normal in size (315, 316). As shown in Table 2,
circulating gonadotropins in mature ERKO males include levels of FSH
within the wild-type range, whereas LH is slightly elevated (315, 317).
In accordance with the increased LH secretion are observations of
Leydig cell hyperplasia in the testis (121) and serum testosterone
levels of approximately 2-fold in the ERKO male compared with
age-matched wild-type males (Table 2) (315, 317). Therefore, the
decreased weights reported for the accessory organs of the male
reproductive tract are not a secondary effect of any phenotype in the
hypothalamic-pituitary axis, but rather a direct result of the loss of
ER-mediated estrogen action within these tissues.
One of the most striking initial observations in the ERKO was
complete infertility in the males when tested in a continuous mating
study with wild-type, known-fertile females (315). No difference in
fertility was observed in the heterozygous ERKO males (315).
Although the etiology of such infertility was unclear, initial
experiments in which ERKO males were placed in a mating environment
with hormone-primed wild-type females resulted in significantly fewer
copulatory plugs, indicating a severe deficit in normal sexual behavior
(315, 318). Further investigations have demonstrated that infertility
in the ERKO male is due to pleiotropic effects resulting from
disruption of the ER gene. In addition to a lack of normal sexual
behavior, ERKO male mice exhibit significantly lower sperm counts
that further diminish with age compared with their wild-type and
heterozygous litter mates (315). This is compounded by defects in the
function of those sperm that are produced in the testis of the ERKO
male, including obvious deficits in motility and a complete inability
to fertilize wild-type oocytes in an in vitro assay (315).
The testes of the ERKO male are slightly smaller than wild-type but
do develop normally and possess the usual complement of seminferous
tubules surrounded by interstitial tissue and Leydig cells (Figs. 6 and 7).
Stimulation of the Leydig cells by LH and subsequent androgen synthesis
in the ERKO testes appears sufficient as mentioned earlier. However,
a report by Hutson et al. (316) indicated a greater
incidence of retraction of the testes into the abdomen and a smaller
yet more muscular cremaster sac in ERKO males compared with
wild-type counterparts. This anomaly was noticed only after excision
and fixation of the urogenital system and was not observable upon
external examination of the animals. Interestingly, this is one of the
few phenotypes of the ERKO that has also been observed in the
heterozygous littermates as well (316). This same laboratory had
earlier employed the Tfm mouse, a naturally occurring
androgen-resistant mutant, to produce strong evidence in support of a
biphasic model of testicular descent in which androgens are critical to
the second step of testis migration, i.e., from the internal
inguinal ring to the scrotum via action on the genito-inguinal ligament
(or gubernaculum) (319). Previous studies had shown that exposure of
fetal male mice to exogenous estrogens resulted in a failure in
testicular descent, although it was concluded that this was probably an
indirect effect due to inhibition of the hypothalamic-pituitary axis
and anti-Müllerian hormone (319). However, the significant
incidence of undescended or retracted testes in the ERKO strongly
suggests a previously unrecognized and possibly direct role for the
ER in development of the male reproductive tract.
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Estradiol has been thought to play only a minor role if any in sperm
production, although Sertoli cells lining the seminferous tubules
produce estradiol (325) and express detectable levels of ER (326).
Therefore, it was surprising to observe severe impairments in
spermatogenesis at multiple levels in the ERKO male. At 8 wk, the
earliest age studied, ERKO males possess epididymal sperm counts
that are approximately 55% of that found in wild-type littermates
(315). This value continues to decrease with age, with ERKO males
possessing approximately 13% of wild-type sperm counts at 16 wk of age
(315). Furthermore, the epididymal sperm collected from the ERKO
males are characterized by significantly decreased levels of motility
and increased incidence of sperm heads separated from the flagellum
(315). Even those sperm that possessed normal structure and motility
were unable to fertilize wild-type oocytes in an in vitro
fertilization assay (315). Therefore, despite levels of circulating
gonadotropins and androgen within the normal range, disruption of the
ER gene has resulted in severe impairments in both spermatogenesis
and sperm function.
As shown in Fig. 7, histological analysis of testes from sexually
mature ERKO males indicated significant atrophy of the seminiferous
epithelium and severe dilation of the tubule lumen. At 10–20 days of
age, no morphological difference in the testis was apparent when
comparing the ERKO with wild-type. However, a distinct morphological
phenotype becomes obvious by 40 days of age and progresses to produce a
completely atrophied testis by 150 days in the ERKO male (315).
Accordingly, sperm counts decrease as the testicular phenotype worsens,
although immunohistochemical detection of Hsp70–2, a germ
cell-specific protein, was possible even in the most severely disrupted
tubules (315).
Further characterization of testes from mature ERKO males indicated
a prominent rete testis that is dilated and protrudes into the interior
of the organ as well as severely dilated efferent ductules (Fig. 7)
(315, 327). The rete testes are composed of a network of
intercommunicating channels located in the posterior-cranial portion of
the testes and serve as a pathway by which suspended spermatozoa can
pass from the testis to the epididymis. Connecting the rete testis to
the epididymis are the efferent ducts, a series of multiple channels
thought to play a significant role in reabsorption of much of the
testicular fluid, and therefore act to also concentrate the sperm
(303). Steroid autoradiography has indicated that the efferent ducts of
the mouse possess the highest concentration of ER compared with any
other region of the excurrent duct system (309). This expression
pattern is evident in the mouse as early as neonatal day 3 (310).
Immunohistochemical and RNA analyses have shown that ER is the
predominant form of ER in the efferent ducts and the cranial portion of
the epididymis (311, 328), although ERß mRNA is also detectable (121, 311).
Previous studies employing surgical ligation of the efferent ductules
reported a severe dilation of the seminiferous tubules similar to that
observed in the ERKO male (329, 330). Based on the similarity of
phenotypes, it became clear that the testicular anomaly observed in the
mature ERKO male may be the result of a severe imbalance in the
fluid equilibrium. However, was it due to hypersecretion of fluid from
the testis or insufficient reabsorption of fluid by the epithelial
cells lining the efferent ducts, or possibly a combination of both?
Using surgical techniques to inhibit fluid transport at different
points in the excurrent duct system, Hess et al. (327)
demonstrated that the reabsorption abilities of the efferent ductules
in the ERKO male were lacking and, in fact, the secretory activity
is actually reduced in the ERKO testis. Further characterization
indicated a reduction or often a complete lack of endocytotic vesicles
and organelles common to fluid uptake in the epithelial cells lining
the ERKO efferent ducts (327). This study was the first report of a
direct ER-mediated estrogen function in the male reproductive tract.
Interestingly, however, was the inability of the pure antiestrogen,
ICI-182,780, to produce a similar phenotype in wild-type ductal
fragments in in vitro experiments (327). Although the
antagonist was able to cause some loss of fluid absorption in the
wild-type ductal fragment, the resulting phenotype was not nearly as
extreme as that observed in the ERKO tissue fragments (327). The
authors proposed that perhaps ERß was possibly mediating an agonistic
effect of the ICI-182,780 and thereby may explain the lack of full
corroboration with the in vivo ERKO phenotype (327). This
hypothesis was based on the work of Paech et al. (91), which
demonstrated that estrogen antagonists, including ICI-164,384, may
function as an agonist when interacting with AP-1 complexes in
vitro. We, as well as others, have since shown that ERß mRNA
expression in the ERKO male reproductive tract is not altered,
although its function remains unclear (93, 121). However, the
preservation of ERß expression in the ERKO strongly indicates that
the reabsorption functions of the efferent ducts are indeed dependent
on the presence of functional ER. This view is strengthened by the
lack of a similar testicular phenotype in ßERKO male mice observed at
ages as old as 14 months (47).
Interestingly, the luminal swelling, loss of germinal epithelium, and
atrophy in the seminiferous tubules of the ERKO testes appeared to
commence at the caudal portion of the organ and progress toward the
cranial region as the animal aged (Fig. 7) (315). This is thought to be
due to a gradual increases in testicular pressure leading to restricted
blood flow as the phenotype in the rete testis worsens and fluid
accumulates within the encapsulated organ (315). The result of such
decreased circulation is likely to become initially manifested in the
less vascularized caudal region of the testis and eventually advance to
affect the whole gonad.
Despite the severe testicular phenotype that occurs in the ERKO male
with age, younger males do produce viable sperm. However, the motility
and fertilization abilities of epididymal sperm collected from ERKO
males are severely compromised. ER (326) as well as
P450arom (331) have been reported in Sertoli cells and germ
cells of the testis, respectively. Therefore, a loss of ER-mediated
estrogen action in the Sertoli cell may alter sperm function. It is
also known that spermatozoa entering the epididymis are unable to
fertilize, and undergo a critically active maturation process as they
pass through the epididymal cords (303). Estradiol treatment of adult
male mice has been reported to increase the rate at which spermatozoa
pass through the epididymis (332). Furthermore, expression of ER in
the mouse epididymis appears to be highest in the caput epididymis,
where sperm first enter after exiting the testis (309). Reports of
ERß expression in the epididymis indicate an opposite distribution,
i.e. highest levels are found in the cauda epididymis, in
both the rat (311) and mouse (121). This pattern of epididymal ERß
expression is preserved in the ERKO male (121). Nonetheless, normal
fertility in the ßERKO male indicates that any actions of estrogen
required for sperm maturation and fertilization capacity appear
dependent on the presence of ER.
The varied phenotypes leading to infertility in the ERKO male have
provided great insight into the role that ER plays in the
development and function of the male reproductive tract. The importance
of functional ER is reiterated by the lack of phenotypes and
apparent full fertility in the ßERKO male mice. It is certainly
possible that there are overlapping functions of the two ERs and that
compensatory mechanisms have reduced the observed phenotypes compared
with those that might result if both ERs were lacking. The speculated
phenotype of a double ER knockout, i.e., ßERKO, might
be similar to that reported in males homozygous for a disruption of the
P450arom gene (ArKO), and therefore lacking the
capabilities to synthesize estradiol. However, intriguing
inconsistencies are apparent when comparing the phenotypes of the
ERKO male with those of the ArKO. For example, the male ArKO mice
exhibit no defects in fertility, at least at the younger ages examined
(257). The testicular weight in adult ArKO males is within the normal
range, and histology indicates normal testicular development with no
indication of a phenotype similar to that of the ERKO males (257).
The possible existence of currently unknown ligands able to stimulate
the ER pathways required for function of the male reproductive tract
and therefore altering the phenotype in the ArKO must be considered. It
is also possible that much of the actions of ER in the male
reproductive tract, as inferred from the ERKO, may be ligand
independent. Such a phenomenon may explain the findings of Hess
et al. (327), in which an estrogen antagonist was unable to
completely induce an ERKO phenotype in the ligated wild-type
efferent duct segments in vitro. Since the testicular
phenotype in the ERKO becomes more severe with age, it is possible
that a similar, yet even further, delay in the manifestation of this
anomaly may exist when the ligand is removed; however, there are
currently no reports on older ArKO males.
B. Accessory sex organs
Certain accessory organs of the male reproductive tract that have
not been already discussed, namely the prostate, bulbourethral glands,
coagulating gland, and seminal vesicles, warrant mention. These glands
have no known specific function other than to secrete components
necessary to the volume of seminal plasma. All four tissues are
dependent on androgen stimulation for growth and maintenance (reviewed
in Ref. 333). However, ER has been detected in each during various
stages of development in the rat (310). Furthermore, the prostate in
various species appears to express significant mRNA levels for ERß as
well as ER (93, 96, 311, 313). A series of studies by the Prins
laboratory have described the toxic effects of neonatal DES exposure on
the morphology and biochemistry of the rat prostate, including the
regulation of AR, ER, and ERß (314, 334, 335, 336). Nonetheless, no
obvious abnormalities in the development of these glands have been
observed in either the ERKO or ßERKO mice (47, 315). However,
categorical studies of these tissues have not been carried out to date
on older animals of either model. One observation in ERKO males is a
significant increase in weight of the seminal vesicle/coagulating gland
that becomes more apparent with age, as shown in Fig. 6. This phenotype
is most likely the result of continued stimulation of this tissue by
the elevated levels of serum androgen that exist in the ERKO (Table 2).
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VI. Neuroendocrine System |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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Several reviews over the past 20 yr have thoroughly documented the known actions of steroid hormones in the central nervous system (see Refs. 337, 343, 344, 345, 346, 347, 348). Sexual dimorphism has been described in multiple regions of the central nervous system (reviewed in Ref. 343). Analogous to the reproductive tract, the neuroendocrine system undergoes a process of sexual differentiation and maturation that is heavily influenced by the steroid receptor-signaling pathways. Briefly, sexual maturation of the neuroendocrine system may be defined as the acquisition of pituitary responsiveness to hypothalamic factors and ovarian steroids and the onset of steroid-induced sexual behavior. In turn, differentiation of the neuroendocrine system is demonstrated by the unique ability of the female hypothalamus to induce an LH surge in response to a rise in serum estradiol (346, 348). The "organizational" or differentiating effects of perinatal steroids are permanent and manifested as measurable structural changes or as subtle fixations in the system’s sensitivity to the "activational" effects of steroids during adulthood (343).
Studies have indicated that the imprinting mechanisms controlled by the
sex steroids in the brain are likely via multiple pathways, including
the regulation of cell death, neuronal growth, and synaptogenesis (343, 347, 349). These effects are generally considered to be genomic events
mediated via the nuclear receptor pathways (337). Indeed, the nuclear
receptors for estrogen, androgen, and progesterone all have been
detected in various amounts in the fetal, neonatal, and adult brain of
several species (88, 337, 343, 350, 351). Furthermore, the discovery of
the ERß has introduced a new level of complexity to the
neuroendocrine system as it relates to estrogen action. Recent studies
have demonstrated an expression pattern for the ERß in the rat brain
that is as broad as that for ER as reviewed in Refs. 351, 352 .
Studies in the primate have reported similar findings (96, 354). Of
particular relevance are the descriptions by Shughrue et al.
(88) of colocalization of ER immunoreactivity and ERß mRNA in
certain regions of the rat brain, including the medial nucleus of the
amygdala and the periventricular preoptic nucleus. Therefore, with
preliminary studies indicating distinct tissue localization of the two
ERs in the reproductive tract, the brain may be the ideal tissue for
the study of possible transactivational actions of ER/ERß
heterodimers. It is important to reiterate that studies have indicated
a normal expression pattern for the ERß gene in the hypothalamus of
the ERKO mouse (93, 352).
Aside from the receptor-mediated genomic actions of sex steroids that
have been so well characterized, the possibility of nongenomic effects
of gonadal hormones and their metabolites has also received increased
attention. A number of rapid responses to gonadal steroids in various
tissues have been reported and are believed to occur too soon after
steroid exposure to be mediated by the classical mechanism of hormone
nuclear receptors; therefore, they have been termed as being
"nongenomic" (reviewed in Refs. 347, 355, 356, 357, 358). These include the
rapid activation of membrane calcium channels by progesterone in the
maturing oocyte and spermatozoa, by estrogens in myometrial cells, and
by androgens in rat osteoblast cells (347, 355, 356). Descriptions of
similar nongenomic effects of steroids in the neuroendocrine system
include rapid increases in cAMP levels in neurons, modulation of the
GABA-GABAA receptor function, release of GnRH and dopmamine
from nerve terminals, modulation of oxytocin receptors, and the release
of PRL from GH3/B6 pituitary cells (343, 356, 357). Supportive
experimental findings indicate the presence of membrane steroid
receptors, including those for estradiol, in various cell types
(359, 360, 361). Evidence that a membrane ER is structurally similar to the
nuclear ER was provided by Pappas et al. (362) in which
multiple ER-specific antibodies were shown to detect and localize
ER immunoreactivity in the cell membrane. Furthermore, Blaustein
describes findings of extranuclear ER immunoreactivity in the
cytoplasm, dendritic processes, and axon terminals of neurons and
suggests an active role for these receptors in neurotransmitter release
(reviewed in Ref. 338). Recently, Razandi et al. (363)
reported the detection of membrane ER and ERß receptors in Chinese
hamster ovary (CHO) cells transfected with an expression vector of the
respective receptor cDNA, indicating that the membrane and nuclear
forms of each ER originate from the same transcript and exhibit similar
affinities for estradiol. These studies further demonstrated that the
membrane-bound ERs were G protein linked and able to elicit a variety
of signal transduction events, including the induction of cell
proliferation (363). In contrast, Gu et al. (364) recently
employed the ERKO mouse to illustrate that the documented rapid
action of estradiol on kainate-induced currents in the hippocampus
occurs in the absence of a functional ER gene, nor does ICI-182,780
have an inhibitory effect, suggesting that ERß is not involved as
well. Therefore, the putative membrane receptor involved in mediating
the neuronal effect of estradiol in the hippocampus described by Gu
et al. appears to be distinct from the intracellular nuclear
form of the ER as well as that described by Razandi et al.
(363). Regardless, the ultimate function of the nongenomic signaling
pathways of the gonadal steroids in the proper organization and
function of the mammalian brain remains unclear. Therefore, the ERKO
mutant mice provide an excellent model to not only study the role of
the nuclear receptors, but also further the investigations of steroid
hormone actions that may be nuclear receptor independent.
A comprehensive review of the neuroendocrine system is beyond the scope
of this discussion and has been reviewed in detail elsewhere (337, 347). However, as was expected, distinct phenotypes in the
neuroendocrine system have become evident in mice after disruption of
the ER gene. The ultimate consequences of the lack of ER action
in the neuroendocrine system are manifested in the ovary of the ERKO
female and as severe deficits in sexual and field behavior in both
sexes of the ERKO mice. Due to the relatively short time in which
the ßERKO model has been available for study, no detailed
characterizations of possible phenotypes in the hypothalamic-pituitary
axis of this model have been carried out. Therefore, this section of
the review will concentrate on what is currently known about the
ERKO, but will attempt to shed light on the possible distinct roles
of both ERs based on the limited observations of the ßERKO.
A. Hypothalamic-pituitary axis
The hypothalamus may be thought of as the interface between the
central nervous system and the endocrine system, i.e., the
pituitary. The anatomical location of the hypothalamus, forming the
base of the brain and residing just above the pituitary, is conducive
to a function of translating neuronal signals from the brain into
humoral factors that stimulate the appropriate actions in the anterior
pituitary (365). The two components are connected by the
hypothalamo-hypophyseal portal system, within which blood flows
predominantly from the hypothalamus to the anterior pituitary, carrying
the appropriate hormonal factors (365). These hormones act as releasing
or inhibiting factors to control the secretory activity of the
pituitary. In contrast, the posterior pituitary is connected directly
to the hypothalamus via neurons passing through the pituitary stalk and
functions as a storage organ for the hypothalamic hormones, oxytocin
and vasopressin.
The anterior pituitary is composed of at least five distinct cell
types, all derived from a common primordium, which have been
categorized by the particular peptide hormone they produce and secrete.
These cell types are as follows, with the secretory hormone in
parentheses: gonadotrophs (FSH and LH), corticotrophs (ACTH),
thyrotrophs (TSH), somatotrophs (GH), and lactotrophs (PRL). Early
studies employing steroid autoradiography demonstrated estrogen binding
to varied degrees throughout the different cells of the anterior
pituitary, although discrepancies among species are evident (reviewed
in Ref. 339). These studies have been followed by those using in
situ hybridization and immunohistochemistry, indicating that the
majority of estradiol binding in the anterior pituitary is due to the
expression of ER (366, 367). In most species described, gonadotrophs
and lactotrophs exhibit the greatest level of ER followed by lower
and varied levels of localization to the other cell types (339).
Furthermore, Shupnik et al. (29, 368) have described in the
rat pituitary a number of ER mRNA isoforms characterized by the
removal of single or multiple exons as well as 5'-sequences that
exhibit little homology to the full-length wild-type ER. Although a
distinct function for these ER isoforms has not yet been determined,
their transcription as well as that of the full-length wild-type ER
gene appears to be regulated by ovarian steroids (368, 369). Further
complexity has been introduced by several recent descriptions of the
presence of ERß mRNA in the anterior pituitary of the human (100),
monkey (96), and rat (70, 98, 99). Wilson et al. (98) report
that ERß mRNA levels were easily detectable in the pituitary of the
15-day-old rat, exceeding the levels of ER mRNA. These levels
decreased in the adult, whereupon a distinct sex difference became
evident in which the anterior pituitary of the female expressed much
higher levels of ERß mRNA (98). Still, previous studies employing
radiolabeled ligand do not corroborate this difference in the rat
anterior pituitary between the sexes at the level of estradiol binding
(370, 371). Furthermore, there are contrasting reports concerning the
cellular localization of the ERß transcripts in the rat anterior
pituitary. Mitchner et al. (99) reported varied levels of
ERß mRNA throughout the different cell types of the anterior
pituitary. In contrast, Wilson et al. (98) describe the
detection of both ERß and ER mRNAs in the gonadotrophs, whereas
the lactotrophs appear to possess ER only. Nonetheless, as
previously mentioned, we find low to undetectable levels of ERß mRNA
in the pituitary of the adult mouse, including that of the ERKO
(93). However, in light of the above studies described in the
prepubertal rat, which indicate a possible developmental role for the
ERß, similar assays in the mouse are warranted.
Although the rate of gonadotropin secretion from the anterior pituitary
is directly modulated by the hypothalamus, the level of circulating sex
steroids is the most important physiological determinant of serum
gonadotropin levels in animals and humans (reviewed in Ref. 251). The
positive and negative regulatory loops of the
hypothalamic-pituitary-gonadal axis have been reviewed in detail and
will be summarized briefly here (251, 362, 372). In short, the
hypothalamus stimulates the synthesis and subsequent secretion of the
gonadotropins into the circulatory system, which then act to induce
gametogenesis and steroidogenesis in the gonads. The functional
gonadotropins, FSH and LH, are composed of dimers of the common
-glycoprotein subunit (GSU), with a distinct ß-subunit that
confers specificity to the hormone, i.e., active FSH
consists of a FSH-ß (FSHß)/GSU dimer, whereas LH consists of a
LH-ß (LHß)/GSU dimer (373). Hypothalamic stimulation of the
anterior pituitary is via the release of GnRH, a deca-peptide that
functions to positively regulate the synthesis and secretion of the
gonadotropins from the anterior pituitary (365). A reciprocal
hypothalamic factor that may act to inhibit pituitary secretion of
gonadotropins has not yet been found. Hypothalamic secretion of GnRH is
not tonic but rather in the form of pulses, driven by a poorly
understood oscillating pulse generator (374, 375). The pulsatile
stimulation of the anterior pituitary by GnRH thereby results in
pulsatile gonadotropin release, which has been shown to be necessary
for gonadal function and reproductive success (374, 375).
Gonadotropin stimulation of the gonads subsequently results in
gametogenesis and the synthesis of gonadal steroid and peptide
hormones, which then feed back to the hypothalamus and pituitary to
regulate FSH and LH secretion (251). However, differences in the system
of feedback loops are apparent between the sexes. While there exists a
tonic system to maintain a constant level of gonadotropins in both
sexes, the female has been endowed with the ability to produce a surge
of gonadotropin secretion to produce transient levels of hormone that
are several fold higher than the tonic level (365). This massive
gonadotropin surge provides for the reproductive cycle in the female
and is critical to ovulation.
1. Female-negative gonadotropin regulation. There is ample
experimental evidence in several species that estradiol can suppress
the secretion of gonadotropins from the anterior pituitary (reviewed in
Refs. 251, 372, 373, 376, 377). Ovariectomy of the female rodent is
known to result in significant elevations in serum FSH and LH that can
be returned to intact levels with physiological treatments of estradiol
(376, 378, 379). The effects of ovariectomy are mirrored in the
gonadotrophs of the anterior pituitary, which exhibit equally elevated
mRNA levels for the gonadotropin subunit genes (373, 376). Studies in
the female rat indicate that by 21 days post ovariectomy, LHß mRNA
levels rise to as high as 20-fold, whereas the increases in FSHß and
GSU mRNA levels plateau at 4- to 5-fold (373). Daily treatments with
estradiol will return the gonadotropin subunit mRNAs to the
pregonadectomy levels within 7 days in the rat (373, 376).
The ability of estradiol to maintain a tonic level of gonadotropins via regulation of both the expression of the gonadtropin subunit genes and the ultimate secretion of the peptide hormones is well accepted. However, the precise site at which the steroid exerts this effect has been difficult to ascertain. This is partly due to the fact that the ER has been detected in both the hypothalamic regions controlling pituitary function as well as the anterior pituitary itself, as previously discussed. Although there is evidence to support a direct effect of estradiol on both components of the neuroendocrine axis, it is believed that the hypothalamus may be the primary site of action in the negative feedback actions (reviewed in Refs. 251, 372, 373, 376). In the laboratory animal, it is believed that castration principally results in an increased frequency of GnRH pulses and therefore increased tonic levels of gonadotropins, both of which can be restored to normal with exogenous estradiol replacement (373, 380, 381, 382, 383).
Numerous studies have produced data to indicate that estrogen
regulation of hypothalamic GnRH secretion may be the predominant
pathway by which transcription of the gonadotropin subunit genes and
gonadotropin secretion is maintained (373, 376). The most prominent
include the characterization of transgenic mice that possess, within
the anterior pituitary, a measurable reporter gene under the regulation
of a gonadotropin subunit gene promoter, including the promotor of the
human GSU gene (384), the rat GSU gene (385), and the bovine
LHß gene (386). As expected, ovariectomy of the transgenic females
resulted in increased transcription and activity of the transgene
reporter gene. Keri et al. (386) illustrated that estradiol
was able to reduce the postovariectomy rise in transcription of the
reporter gene despite the lack of detectable DNA binding of the ER
to the gonadotropin promoter sequences of the construct. Furthermore,
the postovariectomy rise in promoter activity could be prevented by
administration of a GnRH antagonist to the transgenic animal, thereby
inhibiting GnRH action at the gonadotrope. Therefore, a loss of
estradiol action via ovariectomy appeared to result in increased GnRH
release from the hypothalamus, which in turn stimulated increased
transcription of the reporter constructs. However, the possibility of
estradiol actions at the level of the gonadotrope that may directly
effect gene transcription or alter GnRH responsiveness can not be ruled
out.
Regardless of the precise site at which estrogens negatively regulate
gonadotropin expression and release, genetic disruption of the ER
gene was expected to have effects in the female hypothalamic-pituitary
axis that mimic ovariectomy. Northern blot analysis of RNA from
pituitaries of intact wild-type and ERKO females indicates this to
be true. Scully et al. (282) demonstrated that in the
ERKO female, the levels of the GSU transcript are elevated
4-fold, whereas FSHß and LHß mRNAs are as high as 7-fold compared
with wild-type. Ovariectomy in wild-type female littermates produced
elevated gonadotropin subunit mRNAs that approximated the levels
observed in the intact ERKO, indicating that the effects of an acute
loss of estrogen action are similar to those produced by a hereditary
loss of ER (282). Therefore, despite the fact that the
hypothalamic-pituitary axis of the ERKO female is chronically
exposed to elevated levels of estradiol, the significantly increased
level of all three gonadotropin subunit transcripts resemble those of
an ovariectomized female. These data provide strong support for a
critical role of ER, rather than ERß, in the negative regulation
of transcription of the gonadotropin subunit genes. However, at this
time, studies of the ERKO have not provided data to further
elucidate the precise mechanism or site at which a loss in ER action
has resulted in this effect.
Although the transcript levels of both the LHß, FSHß, and GSU
are significantly elevated in the ERKO pituitary, this effect does
not extend to the serum levels of the gonadotropins. Whereas serum LH
is elevated 4- to 7-fold in the adult ERKO female, levels of FSH
appear to be within the normal range (Table 2) (252). A similar effect
is reported in the PRKO female mice, although the serum LH levels in
this mutant female are not nearly as elevated as those in the ERKO
female (387). In addition, ovariectomy in the PRKO female results in a
further elevation of serum LH (387), most likely due to the loss of
serum estrogens. This effect is not observed in the ERKO female
(252), indicating that estradiol is the predominant steroid hormone
maintaining tonic levels of LH in the female.
As shown in Table 2, the serum gonadotropin levels in the ERKO
female indicate that only LH is significantly elevated, whereas FSH is
within the wild-type range. This is in contrast to the levels of FSHß
mRNA in the anterior pituitary of the ERKO, which are elevated
7-fold and equal to those exhibited by an ovariectomized wild-type
female. Furthermore, assays of pituitary homogenates from intact
ERKO females for FSH protein indicate levels within the wild-type
range, suggesting that the divergence between gene expression and serum
levels for FSH does not appear to be due to a decreased secretory rate
of the hormone but rather at the level of translation. This is in
contrast to the ArKO female mouse, in which serum levels of both
gonadotropins are reportedly elevated 3-fold compared with the
wild-type (257).
There are a number of possible explanations for this observation in the
ERKO female. Whereas estradiol treatment of ovariectomized rats has
been reported to completely block the expected increases in LHß mRNA
and LH secretion, it appears to be only partially effective in reducing
transcription of the FSHß gene and secretion of FSH (376). It is now
known that FSHß gene expression and FSH secretion is selectively
regulated in a positive or negative nature by the peptides activin and
inhibin, respectively (reviewed in Ref. 388). This is illustrated in
the knockout mouse model of the activin receptor type II gene, which
exhibits suppressed FSH levels in the adult of both sexes, supporting a
role for activin in the positive regulation of FSH secretion (248).
Although it is believed that the reciprocal effects of the
activin/inhibin peptides are mediated at the level of the anterior
pituitary, the precise mechanisms of action remain unclear. Inhibin has
been shown to alter the levels of GnRH receptor (389) and decrease
FSHß mRNA levels (390, 391, 392), as well as inhibit translation of FSHß
mRNA (391, 393). Activin appears to utilize similar mechanisms to exert
opposite effects on FSHß mRNA transcription and translation and
ultimate secretion of the hormone (394, 395). Therefore, the normal
levels of FSH in the pituitary and serum of female ERKO mice may
indicate that disruption of the ER gene has no effect on the pattern
of activin and inhibin secretion. This is supported by the apparent
negative regulation of FSHß at the translational level in the ERKO
female, suggesting the presence of an active inhibin-signaling pathway.
Further support is provided by studies indicating that ovariectomy of
the ERKO female, and therefore a loss of ovarian inhibin secretion,
results in elevated levels of serum FSH similar to those seen in
ovariectomized wild types (252). Estradiol replacement was partially
effective in reducing the serum FSH in the ovariectomized wild-type but
completely ineffective in the ovariectomized ERKO (252). These
studies provide for at least two conclusions: 1) the partial
effectiveness of estradiol in reducing serum FSH levels in the
wild-type was likely via functional ER in the hypothalamus that may
complement the actions of inhibin in the intact female; and 2) ovarian
factors other than estradiol, most likely inhibin, are maintaining
normal serum FSH levels in the ERKO female, possibly by mechanisms
that override the loss of ER action. It is also possible that both
activin and inhibin synthesis and secretion may be altered in the
ERKO female, but do not result in a net difference in serum FSH
levels. Future investigations to determine the serum levels of the
activin/inhibin subunit peptides and their functional dimers in the
ERKO female are warranted.
Another possible explanation for the selective increase in serum LH in
the ERKO female may be that a lack of ER action has resulted in
aberrations in the GnRH pulse frequency and amplitude that are more
conducive to LH secretion from the anterior pituitary. Normal levels of
GSU transcripts can be maintained with constant GnRH stimulation of
the anterior pituitary (376). However, normal expression of both
gonadotropin ß-subunit genes and gonadotropin secretion requires
pulsatile stimulation from the hypothalamus, and each responds
differently depending on the amplitude and frequency of GnRH
stimulation (373, 376). Varied expression of the GnRH receptor on the
cell surface of the gonadotrophs also varies with the level of
hypothalamic stimulation (396). This mechanism, by which the level GnRH
receptors can be differentially regulated and thereby modify the
gonadotropin responsiveness to GnRH, has been proposed to be a key
element in the differential regulation of the two different
gonadotropins by the same releasing hormone (397). Studies in the rat
have revealed that more rapid GnRH pulses (15–60 min) favor the
secretion of LH, whereas slower pulses (120 min) allow for secretion of
FSH (251, 376). Positive regulation of LH synthesis and secretion is
also more sensitive to the amplitude of GnRH stimulation (251, 376).
Therefore, a loss of ER action during development and maturation of
the hypothalamic-pituitary axis may have resulted in a pattern of GnRH
secretion that favors the translation and secretion of LH rather than
FSH. The influence that ERß may have, including a possible
compensatory role in the ERKO hypothalamus, remains to be evaluated.
2. Female-positive gonadotropin regulation. In addition to maintaining tonic levels of serum gonadotropins, estradiol also plays a central role in the preovulatory gonadotropin surge mode in the female (reviewed in Refs. 263, 264, 377). Differentiation of the neuroendocrine system results in the development of mechanisms necessary to produce a dramatic preovulatory rise in serum gonadotropins in response to the positive feedback of ovarian steroids. In the rodent, developmental differentiation of this pathway in the neuroendocrine system is unique to the female (365). The surge in serum LH and FSH is the hallmark of the female cycle and is critical to ovulation as well as the synchronized induction of appropriate sexual behavior and, therefore, is vital to the female ovarian cycle and fertility.
Numerous studies have demonstrated that estradiol is required for the preovulatory gonadotropin surge, and that the timing and dose of estradiol exposure may be the most critical parameters (reviewed in Ref. 377). As with the negative regulatory effects of estradiol, the precise site of action for the sex steroids during the preovulatory gonadotropin surge also remains unclear. However, it is believed to be the result of the combined effects of external and internal cues transduced from the brain and the positive feedback of gonadal hormones that provide for a synchronized pattern of GnRH secretion upon an anterior pituitary that has been rendered transiently hypersensitive to the releasing hormone (reviewed in Ref. 377). In the monkey, destruction of the neurons involved in GnRH production can be overcome with pulsatile administration of exogenous GnRH, whereupon a gonadotropin surge can be produced with exogenous estradiol, indicating the pituitary as the predominant factor in the surge (398). However, this is not possible in the rodent, apparently due to a greater level of interdependency among the components of the neuroendocrine axis (365).
Therefore, although the rises in ovarian estrogen secretion are
critical to the generation of a gonadotropin surge, the pathways
involved are poorly understood. A number of mechanisms involving direct
actions of estrogen in the brain have been proposed and recently
reviewed (264). However, it is unlikely that the actions of estrogen
are via direct interaction with GnRH neurons since these processes
appear to be devoid of ER (264). Therefore, the actions of estradiol
appear to result in indirect stimulation of the hypothalamic neurons
that synthesize and release GnRH. Possible mechanisms include steroid
interaction with receptor-positive monoaminergic and opoid neurons that
may mediate the ultimate effects to GnRH neurons, possibly via
modifications in the levels of catacholamines, glutamate,
-aminobutyric acid, neuropeptide Y, ß-endorphins, and galanin
(reviewed in Refs. 263, 264). At the level of the anterior
pituitary, the preovulatory increases in estradiol may act in concert
with GnRH to enhance gonadotrope sensitivity to the forthcoming rise in
releasing hormone by increasing the levels of GnRH receptor (263, 377).
Furthermore, the 5'-flanking region of the rat LHß gene has been
shown to possess an imperfect estrogen-responsive element that is able
to bind ER and confer estrogen responsiveness to a chimeric
promoter-reporter gene construct in vitro (399). Therefore,
the estradiol-ER complex may also act to directly increase LHß
mRNA levels before the LH surge (251).
Attempts to elicit an LH surge in the ovariectomized model with acute estradiol treatments have been reported to be only partially effective, indicating that ovarian factors other than estradiol are also required for a full physiological response (reviewed in Ref. 263). It is now known that the actions of progesterone and the PR are also a necessary component in the induction of the gonadotropin surge (reviewed in Ref. 263). The role of progesterone and PR in facilitating the preovulatory surge may be to induce a rapid release of GnRH from the hypothalamus, as well as possibly mediate a decrease in ER levels in the anterior pituitary, thereby possibly counteracting the inhibitory effects of estradiol (263). Although the precise mechanism of action may be unclear, the complete lack of a preovulatory surge in intact PRKO female mice provides strong support for the requirement of this steroid receptor (387).
A cooperative role between the estrogen- and progestrone-signaling
pathways in the induction of the preovulatory surge may include the
ability of estradiol to stimulate increased PR levels in both the
hypothalamus and anterior pituitary, thereby increasing the sensitivity
of these tissues to progesterone (400, 401). Shughrue et al.
have shown that estradiol-induced increases in PR expression in the
preoptic nucleus are possible in the ERKO female (Fig. 8) and suggest that this may be a
compensatory action of ERß (400). These same studies demonstrated
that the preoptic nucleus of the hypothalamus in intact ERKO females
possessed a significantly greater level of PR mRNA when compared with
wild-types, perhaps due to chronic stimulation of ERß by the elevated
serum estradiol (400). Evidence to support this hypothesis is the
apparent decrease in the level of PR transcripts observed after
ovariectomy in the ERKO, which can be returned to intact levels
6 h after a single treatment with estradiol (Fig. 8) (400).
|
ERKO males possess a population of dopaminergic neurons
more characteristic of a wild-type female, confirming a critical role
of ER in this differentiation process. Furthermore, the numbers of
dopaminergic neurons in the female ERKO are only slightly reduced
when compared with wild-type, indicating a morphologically normal AVPV
region (405). Therefore, with the ERKO female exhibiting an apparent
preservation of estrogen-induced increases in hypothalamic PR and a
wild-type-like female phenotype in the AVPV region, it is conceivable
that the hypothalamic mechanisms required for induction of the
preovulatory surge may be intact. 3. Males: gonadotropin regulation. Because of the more prominent role of testosterone in the male, certain issues specific to the male hypothalamic-pituitary axis are worthy of discussion. Of course, lower aromatase activity in the testis results in circulating levels of estradiol in the male that do not approach those observed in the intact cycling female. Therefore, it would be expected that distinct mechanisms of steroid feedback and regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis have evolved in males, presumably one likely to be more dependent on testosterone. This difference is thought to occur at the level of the hypothalamus since the anterior pituitary generally exhibits no sexual differentiation (365) and possesses receptors for all sex steroids (339).
A critical role of testosterone and AR-mediated actions in the negative
regulation of gonadotropin secretion in the male is illustrated by the
elevated serum LH levels in Tfm mice (301) and in humans
with androgen insensitivity syndromes (38). As in the female,
transcription of the gonadotropin subunit genes is significantly
elevated after castration in the male, although peak levels are reached
much earlier (
7 days) (373). Furthermore, FSHß mRNA levels appear
to return to precastration levels by 28 days, whereas the levels of
LHß and GSU transcripts remain elevated (373). Estradiol is
equally effective as testosterone in reducing serum LH levels that
result after castration in the male (reviewed in Ref. 373). These data,
along with the documented presence of P450arom activity
(reviewed in Refs. 406, 407) and wide distribution of ER in the
hypothalamic-pituitary axis support a role for locally synthesized
estradiol and ER action in male gonadotropin synthesis and/or secretion
(88, 339). Furthermore, adult male ArKO mice exhibit elevated levels of
serum LH despite possessing significantly high circulating testosterone
(257). Therefore, the roles of estradiol and testosterone often appear
overlapping as well as distinct, making obvious the complexity of the
steroid-feedback mechanisms that exist in the male.
Any specific role the ER may play in the regulation of gonadotropin
synthesis and secretion in the male would be expected to become
apparent in the ERKO. Adult ERKO males exhibit levels of
hypothalamic GnRH, pituitary FSHß mRNA, and serum FSH that are within
the normal range when compared with wild-type littermates (Table 2)
(317). The normal levels of FSHß mRNA in the pituitary of ERKO
males are in stark contrast to the significantly elevated levels found
in the female ERKO mice (282). This contrast between the sexes may
reflect differences in the inhibin/activin levels or may represent a
definitive sexual differentiation in the transcriptional regulation of
the FSHß gene in the mouse, indicating that androgens are the primary
acting steroids in the male. However, although not as extreme as those
found in the ERKO female, pituitary LHß mRNA and serum LH levels
are increased 2-fold in the adult ERKO male (Table 2) (317).
In a series of experiments, Lindzey et al. (317)
demonstrated that castration results in the expected elevated levels of
serum LH in the wild-type, and a further increase in the already
elevated LH levels in ERKO males. The rise in serum LH that occurs
upon castration even in the ERKO male suggests that either
estradiol-ERß or androgen-mediated mechanisms are maintaining the
lower LH levels in the intact animal. Once again however, the mouse
pituitary (including the ERKO) appears to possess very little if any
ERß mRNA (93), although ERß is expressed normally in the
hypothalamic regions in the ERKO (93, 352). Estradiol treatment of
castrated animals over a period of 3 weeks reduced the serum LH levels
to normal in the wild-type males, whereas no effect was observed in the
ERKO, indicating a requirement for ER in this process (317).
Although treatments of similar castrated males with testosterone was
completely effective in producing an inhibitory effect on LH release in
the wild-types, it was only partially effective in the ERKO (317).
The authors therefore concluded that the ability of testosterone to
fully restore normal levels of LH in the sera of castrate wild-type
males but only partially in the ERKO males suggests that
local aromatization of testosterone to estradiol and subsequent
activation of ER-mediated pathways act to enhance the negative
feedback effects of androgens in the male hypothalamic-pituitary axis
(317). However, the inability of testosterone to completely suppress
the serum LH in the ERKO male may be related to the dosage used in
these studies. Strong evidence of AR-dependent regulation of LH
secretion in the male ERKO is found in preliminary experiments in
which treatment with an antiandrogen (flutamide) increased serum LH by
3- to 10-fold in wild-type and ERKO males, respectively. This
suggests that ERKO males have come to rely entirely on AR-mediated
actions to regulate LH secretion, whereas the ER continues to play a
role in the wild-type.
In these same studies, Lindzey et al. (317) illustrated that
prolonged treatment with DHT, the more potent and nonaromatizable
androgen, resulted in no reduction in the castrate levels of serum LH
in wild-type but was partially effective in the ERKO male. However,
the DHT was effective in restoring hypothalamic GnRH content levels to
normal in castrate males of both genotypes (317). Therefore, the
enhanced effect of DHT in negatively affecting the
hypothalamic-pituitary regulation of serum LH, including the inhibition
of hypothalamic GnRH release, remains a puzzling phenomenom unique to
the ERKO male. It is possible that a lack of ER action during
development resulted in a "re-organization" of the
hypothalamic-pituitary axis in the ERKO male, and thereby somehow
allowed for an increased sensitivity to androgens (317). Further
studies in the ERKO as well as the ßERKO males may help elucidate
these unexpected results.
4. PRL regulation. PRL possesses more biological actions than all of the other anterior pituitary hormones combined. A recent review by Bole-Feysot et al. (279) thoroughly covered the current knowledge of the diverse actions of PRL, including its functions as a hormone, growth factor, neurotransmitter, and immunoregulator. A reflection of the multiple functions of PRL is the equally broad distribution of PRL-binding sites throughout the many physiological systems in vertebrates (279). The well known effects of PRL in reproduction include a critical role in the differentiation and function of the lactating mammary gland, as a luteotrophic hormone in the function of the corpus luteum and thereby as a promotor of blastocyst implantation, and an overall enhancement of the physiological functions in the tissues of the male reproductive tract (279). Nonreproductive roles of PRL include an involvement in osmoregulation; promotion of growth, development, and differentiation in several tissues; enhancement of metabolic activities in the brain, liver, pancreas, and adrenals; and various actions in immunoregulation (279).
It has long been known that estradiol is a critical hormone in the
regulation of PRL synthesis and secretion from the lactotrophs in the
anterior pituitary. Estradiol has also been shown to stimulate
lactotroph cell growth (reviewed in Ref. 281) and has been implicated
as a possible factor in the promotion of PRL-secreting tumors in humans
(reviewed in Ref. 339). Furthermore, the lactotrophs of several species
have been shown to possess significant levels of ER, strongly
suggesting that the actions of estradiol are receptor-mediated
(reviewed in Ref. 339). As discussed above, recent descriptions have
indicated the presence of ERß in the lactotrophs of the rat anterior
pituitary, although contrasting reports exist, possibly due to strain
variations. Whereas Wilson et al. (98) describe the presence
of only ER in lactotrophs, Mitchner et al. (99) report
variable levels of ERß mRNA throughout the various cell types of the
rat anterior pituitary. Shupnik et al. (100) have also
reported the detection of ERß transcripts in human PRL-secreting
tumors. Once again, we find only low to undetectable levels of ERß
mRNA in the pituitary of the adult mouse, including the ERKO (93).
The upstream regulatory sequences of the rat PRL gene have been found
to possess an estrogen-responsive element that binds ER and
functions synergistically with the pituitary-specific factor, Pit-1, to
promote expression (281, 408, 409). The required function of the ER
in the positive regulation of the PRL gene is nicely illustrated in the
ERKO mouse. The ERKO females exhibit a 20-fold decrease in PRL
mRNA levels in the anterior pituitary, whereas the ERKO males
exhibit a 10-fold decrease when each is compared with sex-matched
wild-type controls (282). Although not as drastic, this reduction in
the expression of the PRL gene is mirrored in the serum levels of the
hormone in the ERKO female, which possess an approximate 5-fold
reduction in serum PRL (Table 2). Therefore, given the plethora of
roles in which PRL is involved, it is likely that several of the
phenotypes observed in the ERKO mice may be due to a direct loss of
or simply enhanced by the concurrent decrease in PRL signaling.
Interestingly, the extremely low levels of PRL mRNA in the anterior
pituitary of the ERKO female are even significantly less than that
observed 14 days after ovariectomy in the wild-type (282). Therefore,
the loss of ER during development and differentiation of the
lactotrophs in the anterior pituitary has resulted in a phenotype that
is more severe than that induced by postpubertal ovariectomy, possibly
due to a decrease in lactotroph cell number. It has been proposed that
the lactotroph and somatotroph cell types of the adult anterior
pituitary may be derived from a common cell that expresses both the
genes for GH and PRL during development (reviewed in Ref. 410). The
factors that may be involved in the terminal differentiation of this
stem cell into a distinct cell type secreting only one of the
respective hormones remain elusive. Because the appearance of the ER
and the ontogeny of PRL expression appear to coincide in the developing
pituitary, estrogen action has been proposed as a possible factor
(410, 411, 412). However, a defect in the cell lineage of the lactotrophs
that may be expected due to a loss of ER action was not apparent in
the ERKO, as immunostaining for both PRL and GH localized expression
of the genes to distinct cell types (282).
Furthermore, estrogen has also been shown to stimulate proliferation of
the lactotrophs and PRL-secreting cell lines (reviewed in Ref. 339).
Therefore, since the marked difference in PRL mRNA levels observed
between the ERKO female and the ovariectomized wild-type is not
apparently due to a defect in the differentiation of the lactotrophs,
it may possibly be due to a decreased number of lactotrophs in the
anterior pituitary of the ERKO. Scully et al. (282)
provided evidence against this hypothesis, by once again employing
immunohistochemical staining to illustrate only a modest decrease in
lactotroph cells in the anterior pituitary of the ERKO mice.
Therefore, ER action does not appear to be required for either
differentiation or proliferation of the lactotrophs in the mouse
anterior pituitary. However, a recent report by Chun et al.
(413) has illustrated a distinct contrast in the level of occupied ER
required to elicit proliferation and that required for PRL synthesis in
PR1 cells, a PRL-secreting cell line. Whereas approximately 50% of the
cellular pool of ER was required to be complexed with estradiol for
half-maximal stimulation of the PRL gene, only 0.1% was required to
induce cellular proliferation (413). These results suggest that the
mechanisms required for estrogen-induced lactotroph proliferation are
hypersensitive in this cell line compared with the mechanisms involved
in regulation of the PRL gene (413). Therefore, it is possible that the
small amount of the active ER splicing variant known to be present
in the ERKO (see Section II.C.) has allowed for
sufficient estrogen signaling and lactotroph proliferation during
develpment, resulting in the apparent lack of a somewhat expected
phenotype of decreased lactotroph cell number in the pituitary of
ERKO mice.
B. Behavior
There are obvious effects of the gonadal steroids on sexual
behavior in vertebrates; however, a more defined knowledge of these
actions has become evident from a series of classical experimental
schemes. These laboratory studies often relied on perinatal castration
and/or developmental exposure to exogenous steroids followed by studies
of the activational abilities of the different steroids during
adulthood. The majority of such investigations have been carried out in
the rat, but similar results have been described in other species (345, 414). Breifly, studies on sexual behavior in the rat have shown that 1)
castration on the day of birth results in a feminized adult male that
exhibits a female pattern of behavioral responses when treated with
estradiol and progesterone, and 2) neonatal testosterone or estradiol
treatment of a female results in a masculinized adult that exhibits a
male-like pattern of behaviors and is refractory to estradiol and
progesterone (131, 337). The culmination of the data collected from
such experimental schemes has led to the conclusion that testosterone
secreted from the perinatal testes during a critical developmental
window results in permanent changes in the hypothalamic nuclei of the
brain that mediate male sexual behavior. However, the data indicating
that developmental exposure to estradiol results in an adult phenotype
that is similar to that elicited by testosterone suggest that many of
the masculinizing effects of perinatal testosterone may be via local
aromatization of the hormone to estradiol and subsequent activation of
the ER signaling pathway (reviewed in Refs. 406, 407). In addition,
estradiol is also necessary for normal development of the female brain,
although in lower amounts (131). Therefore, sex steroid-mediated sexual
differentiation of the various regions of the brain that are critical
to behavior relies not only on the nature of the steroid ligand, but
also on the dose and timing of exposure (348).
Before the availability of the ERKO mouse, McCarthy et
al. (415) employed an elaborate technique of infusing anti-ER
oligodeoxynucleotides into the neonatal rat hypothalamus to elucidate a
direct role for ER in the sexual differentiation of the female
brain. This experimental scheme was based on the hypothesis that the
presence of specific ER antisense oligodeoxynucleotides in the
hypothalamus would interfere with proper expression of the ER gene
during a critical period of differentiation (415). The experimental
groups included neonatal rats treated with testosterone plus or minus
the infusion of the ER antisense oligodeoxynucleotides. As adults,
those females infused with the ER antisense oligodeoxynucleotides
exhibited more female sexual behavior compared with those treated with
androgen alone. The investigators thereby concluded that the reduced
ER expression protected the infused female rats from the
masculinizing effects of testosterone exposure (415), providing strong
evidence that local aromatization and subsequent estradiol activation
of the ER pathway plays a primary role in the masculinization of the
rat brain. However, the experimental scheme of McCarthy et
al. does not allow for a direct comparison with the ERKO
female, due to the caveats discussed (see Section II.C.1).
It is important to recognize that the ERKO are deficient in ER
throughout development, whereas McCarthy’s scheme produced a lack of
ER action that was only transient and most likely not as complete.
The use of technologies to target individual genes has created numerous
models available for studies in the behavioral sciences. A recent
review by Nelson and Young (128) summarized and compared the behavioral
or lack of behavioral phenotypes in a select 50 murine knockout models.
Due to the lack of any grossly apparent behavioral phenotypes in the
ßERKO mice, only those studies concerning the ERKO will be
discussed here.
1. ERKO female. The dependence of female sexual behavior on
the synchronized fluctuations in estradiol and progesterone that occur
during the ovarian cycle have been described in detail (reviewed in
Ref. 416). In the rodent, circulating estrogens continue to rise as
ovulation approaches, eventually leading to the gonadotropin surge that
not only triggers the release of the oocyte from the ovary but also a
marked increase in serum progesterone. This dramatic rise in
circulating progesterone is required for an optimal display of the
lordosis posture, a measurable response required for successful
copulation (416). The gonadotropin surge from the
hypothalamic-pituitary axis is due to the postive-feedback actions of
estradiol. The development of this pathway in the rodent is unique to
the female as a result of sexual differentiation of the neurons in the
anteroventral periventricular nucleus of the preoptic area that serve
to regulate hypothalamic function (264, 365, 405). As discussed above,
feminization of the brain involves the actions of estradiol during
fetal and neonatal development that may rely heavily on the dose and
time of exposure. Therefore, knockout models for ER and ERß, as
well as the PR, have and will continue to serve as invaluable resources
for dissecting the role of each receptor-signaling system in female
sexual behavior.
In general, evaluation of the aberrant sexual behaviors of the ERKO
female must consider not only the absence of ER signaling, but also
the elevated levels of serum testosterone that exist in the adult
female (Table 2). Despite the presence of the hormones presumably
required for sexual behavior, intact adult ERKO females exhibit
behavior that resembles that of a male in terms of parental,
aggressive, and sexual activities (417, 418). When placed in the
presence of a stud male, ERKO females display a complete lack of
sexual receptivity, measured as prelordotic behavior and a lordosis
posture (418). In fact, intact ERKO females were often treated as
intruders and attacked by the stud male (417). However, similar studies
using ovariectomized females indicate that this behavior of the stud
male was most likely elicited by the significantly elevated levels of
circulating testosterone in the ERKO female (418, 426). These same
studies, employing ovariectomized females coupled with steroid
replacement of varied combinations, illustrated a complete resistance
to estradiol (418, 419) and a minimal effect of progesterone in
inducing a lordosis response in the ERKO female (418).
Although the above studies have indicated a prominent role for ER in
sexual behavior in the mouse, the precise pathways disrupted by a lack
of ER remain unclear. Not surprisingly, the PRKO female mice also
exhibit a lack of normal sexual behavior and are unable to produce a
lordosis posture even when treated with doses of either estradiol
and/or progesterone (44). However, this same study reported an
inability of estradiol alone to induce lordosis but rather a
requirement for both estradiol and progesterone in wild-type females
(44). This is in contrast to the capacity of estradiol to solely induce
lordosis in the wild-type controls employed by Rissman et
al. (419) and may be a reflection of the differences in background
strain and/or experimental design employed in the two studies.
The ERKO and PRKO models nicely illustrate a requirement for both
estradiol and progesterone action for a full lordotic response. As
early as 1939, estrogen exposure before progesterone treatment was
found to be required for a full display of sexual behavior in the rat
(420). As in the uterus, the expression and induction of PR in certain
regions of the brain is under estrogen regulation. Studies have
indicated that estradiol elicits detectable increases in PR in the
hypothalamus, strongly suggesting that this estrogen-action is required
for ultimate progesterone-induced sexual behaviors (346, 421, 422).
Therefore, disruption of the ER gene may be expected to cause
abnormally low levels of PR expression in those areas of the brain that
mediate female sexual behavior and therefore may explain the lack of
such behavior in the ERKO mouse. However, two separate reports have
described the ability of estradiol to induce increases in PR
transcripts in the forebrain of the ERKO female mouse, including
regions of the preoptic area (400) (Fig. 8) arcuate nucleus, caudal
ventromedial hypothalamus, and posterodorsal medial hypothalamus (423).
However, the extent of estrogen-induced increases in hypothalamic PR in
the ERKO female is slightly attenuated compared with that observed
in similarly treated wild-type mice (400, 423). It is possible that
this observed estrogen action in the ERKO female is either mediated
by a splicing variant of the disrupted ER gene or by ERß.
Regardless, the level of estrogen-induced PR in the ERKO female does
not appear to allow for a response to progesterone that is sufficient
to elicit sexual behavior. These data support the conclusion that
normal expression of female sex behavior requires the sequential
activation of the ER- and PR-signaling pathways.
Significant deficits in parental behavior and a greater tendency toward
infanticide is also observed in ERKO females compared with wild-type
littermates (418). These phenotypes do not dramatically differ between
intact and ovariectomized ERKO females; however, levels of
infanticide were reduced in tests carried out after a prolonged
post-gonadectomy period (65 days) (418). The ERKO female exhibits
aggressive behaviors that are significantly elevated compared with the
wild-type female littermates, and in stark contrast to the dramatically
reduced aggressive behavior displayed by the ERKO male (to be
discussed below) (418). Remarkably, acute treatment of ovariectomized
females with estradiol resulted in the expected reduction in aggressive
behavior in both the wild-type and ERKO females (418). Previous
studies have shown that whereas both testosterone and estradiol can
elicit aggressive behaviors in the castrated male mouse, only
testosterone is effective in the ovariectomized female mouse (424, 425). These studies, in combination with the findings in the ERKO,
indicate that differentiation as well as activation of aggressive
behaviors in the female mouse are testosterone dependent. However, the
preserved ability of estradiol to reduce aggression in the
ovariectomized ERKO female is puzzling and may indicate an
ERß-mediated pathway.
The elevated levels of infanticide and aggressive behavior exhibited by
the ERKO females may be contributed to by the elevated levels of
testosterone secreted by the acyclic ovary (Table 2). As discussed
earlier, experimental evidence suggests that disruption of the ER
gene has resulted in a hypothalamic-pituitary axis with an enhanced
capacity to respond to androgens in the ERKO male. In support of
this possibility, Ogawa et al. (418, 426) reported their
preliminary finding of increased androgen receptor levels in the brain
of the ERKO female as early as 12 days of age.
2. ERKO male. Given the apparent role of ER-mediated
estrogen actions in the masculinization of the brain, it was expected
that the ERKO males would exhibit a female-like behavioral
phenotype. Surprisingly, however, Ogawa et al. (318)
observed that a lack of hypothalamic ER during development has
little effect on the sexual behavior of the intact ERKO male in
terms of mounting and sexual attraction toward wild-type females. In
contrast, Wersinger et al. (427) report that tests of male
sexual behavior carried out in a neutral arena, as opposed to the
male’s home cage as done in the study of Ogawa et al.,
demonstrates that the number of mounting attempts exhibited by the
ERKO males is reduced. Interestingly, the studies of Ogawa et
al. illustrated that ERKO males, however, exhibit an almost
complete lack of intromission and ejaculation, even though the number
and frequency of mounts were similar to those of wild-type males (318).
Furthermore, treatment of castrated males with estradiol or the
nonaromatizable androgen, DHT, resulted in no differences in sexual
behavior compared with the findings in the intact ERKO males (428).
These results differ from those described by Ono et al.
(429) and Olsen (131) in the androgen-insensitive Tfm mouse,
which exhibits no male-like sexual behavior including a lack of
mounting as well as intromission and ejaculation. However, the ERKO
and Tfm males are similar in terms of exhibiting complete
insensitivity to the effects of both estradiol and testosterone as
behavioral activators during adulthood.
The ERKO male behavioral phenotype described above is obviously a
contributing factor to the infertility that results after disruption of
the ER gene. The culmination of the studies indicate that a discrete
component of sexual behavior in the male mouse, i.e.,
consummatory activity, is dependent on the actions of ER, whereas
testosterone or possibly ERß-mediated estradiol actions may regulate
motivational aspects (428). Although reports of categorical studies on
sexual behavior are not available, both the ßERKO (47) and ArKO (257)
male mice appear to be fertile and able to sire multiple litters,
suggesting a minor role for ERß in sexual behavior. The possibility
of compensatory mechanisms mediated by ER in the ßERKO cannot be
ruled out. It is interesting, however, that the ArKO males, presumably
lacking physiological levels of estradiol throughout life, show no
obvious deficits in sexual behavior that result in infertility (257).
It might be expected, given the apparent need of ER-mediated
estrogen actions illustrated by the ERKO, that the ArKO male would
display a similar phenotype. Perhaps, exposure to maternal steroid
hormones during gestation in the ArKO mouse has allowed for the proper
"organization" of the neuronal circuitry regulating sexual
behavior. More detailed studies may elucidate subtle behavioral
phenotypes that exist in both the ßERKO and ArKO models and will help
further define the precise role that estradiol and testosterone play in
the regulation of sexual behavior.
A dichotomy similar to that observed for the elements of male sexual
behavior in the ERKO is observed when behavioral assays for
aggression and parental instincts are considered. Intact ERKO males
demonstrate a relatively normal pattern of parental behavior as
measured by levels of infanticide when placed in the presence of
newborn pups (428). However, despite the fact that ERKO males
possess serum testosterone levels that exceed the norm by as much as
2-fold and show no reduction in the levels of AR (430) or ERß
(93, 352) in the brain, they consistently exhibit a significant deficit
in all male aggression indices tested (428, 430). Therefore, as in the
case of certain components of sexual behavior, ER-mediated actions
appear critical to the development and/or activation of aggressive
behaviors, whereas parental instincts appear to be independent of ER
action (428).
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VII. Phenotypes in Peripheral Tissues |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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ERKO, and eventually the ßERKO.
A. Skeletal system
The link between the onset of osteoporosis and the decreasing
estrogen levels associated with menopause has been realized since the
report of Albright et al. in 1941 (432). Postmenopausal
estrogen replacement therapy is currently the most commonly prescribed
drug treatment in the United States (433, 434). In most patients, the
increased risks associated with long-term estrogen replacement therapy,
such as breast and endometrial cancer, are strongly overshadowed by the
well established reduction in the risk of osteoporosis and bone
fracture (433). Bone is a dynamic tissue that is constantly being
resorbed to serve as a mineral source for the body and remodeled to
replace this reservoir as well as maintain skeletal strength.
Osteoporosis is a defined pathology characterized by a loss in bone
mass and strength and is believed to be due to a disruption in the
equilibrium between bone resorption and formation (435). Current
evidence supports the hypothesis that excess bone resorption occurs in
the postmenopausal years, acting to strip the bone of mass and further
remove the foundation upon which new bone may be formed (435). Several
therapies are known to reduce the postmenopausal increases in bone
resorption, including the intake of calcium and vitamin D, calcitonin,
bisphosphates, and estrogens (435). The obvious beneficial effects of
estrogen replacement therapy have evoked an intense research effort for
bone-specific estrogen agonists that lack the potentially harmful side
effects in the breast and reproductive tissues. Ironically, the
currently available "selective ER modulators" or SERMs, as these
drugs have come to be termed, have been selected from the pool of
nonsteroidal estrogen antagonists (reviewed in Refs. 8, 434, 436).
For several years it was believed that the effects of estrogens on bone
physiology were indirect, inferred from the inability to detect ER
within bone and bone cell cultures. However, in 1988, Komm et
al. (437) and Eriksen et al. (438) simultaneously
reported the detection of high-affinity, competable estradiol binding,
ER mRNA, and the induction of estrogen-responsive genes in cultured
rat and human osteoblast-like cells, the bone-forming cell. We have
reported similar findings, including the inhibition of estradiol
transactivational activity with antiestrogens, in two separate
osteoblast-like cell lines from the rat (439). Oursler et
al. (440) have since demonstrated ER and estrogenic activity in
cultured avian osteoclasts, the bone-resorbing cell type, including
estrogen induction of the genes for c-fos and c-jun.Using in situ RT-PCR analysis, Hoyland et
al. (441) demonstrated the presence of ER mRNA in both
osteoblasts and osteoclasts in bone grafts from human females.
Immunocytochemical methods have also been used to demonstrate the
presence of ER in multiple bone cell lines (442). Recently, Bodine
et al. (443) reported significant increases in the levels of
ER transcripts during dexamethasone-induced differentiation of rat
osteoblasts in vitro. Therefore, there is adequate
experimental evidence to support the presence of a direct
ER-mediated estrogen-signaling pathway in bone.
The discovery of the ERß introduced renewed vigor in the search for
SERMs, allowing for the greater possibility of finding a
receptor-selective agonist. The distinct expression pattern of the two
ERs among various tissues has further enhanced the possibility
of finding tissue-specific SERMs. Several recent studies have reported
the detection of ERß in bone cells. Onoe et al. (444)
employed RT-PCR to demonstrate the presence of both ER and ERß
mRNA in immortalized as well as primary osteoblast cell cultures from
the rat. Similar to reports of ER, both Onoe et al. (444)
and Arts et al. (95) report significant increases in ERß
mRNA levels during in vitro dexamethasone-induced
differentiation of osteoblasts derived from the rat and human,
respectively. Therefore, the generation of mice lacking ER or ERß
will once again prove invaluable in delineating the roles of the two
receptors in bone physiology.
The majority of animal studies concerning the role of estrogens in bone
morphology and metabolism have been carried out in the rat (reviewed in
Ref. 445). Ovariectomy in the rodent results in increased bone turnover
similar to that seen in postmenopausal women; however, the mechanisms
of action may differ between the species (445). The effects of
ovariectomy in the rat include decreases in bone mineral density,
cancellous bone area, and bone strength, whereas increases are observed
in radial and longitudinal growth, osteoblast and osteoclast activity,
and overall rates of bone turnover (445). Estrogen replacement,
including those compounds with mixed agonist/antagonist
activity, has been shown to reverse several of the effects induced by
ovariectomy (445). However, the extent and direction of the changes
induced by ovariectomy, as well as the protection provided by estrogen
replacement, vary depending on the bone parameter, sex, and type of
bone being evaluated, e.g., femur, tibia, calvaria, or
vertebrae (445). Interestingly, a study of the androgen-resistant
Tfm rat describes a bone phenotype similar to a wild-type
female, i.e., shorter and thinner femurs, indicating that
androgen action may also be critical to longitudinal and radial bone
growth in the male rat (446). However, endogenous gonadal estrogens
were able to maintain a normal cancellous bone mass in the
Tfm rat (446). It is noteworthy that the first description
of a human case of estrogen insensitivity due to a spontaneous mutation
of the ER gene exhibits severe osteoporosis as well as significant
increases in longitudinal growth of bones (see Section
VIII.C) (116).
Unfortunately, few studies of the effects of steroids on bone
physiology have been carried out in the mouse. Analysis of femoral bone
length in ERKO mice indicates a significant decrease in length and
diameter in females and a slight decrease in males, when compared with
age- and sex-matched wild-type controls (447). However, measurements of
bone density and mineral content indicate the opposite effect,
i.e., ERKO males exhibited significant decreases
throughout the femur (448), whereas the ERKO females demonstrate
just slight and localized decreases (447). In agreement with the
ovariectomized rat model, ERKO female mice exhibit increased bone
resorption-remodeling rates (448). However, the decreased femur length
observed in the ERKO is in contrast to that reported in the
ovariectomized rat and the ER-deficient human male. Interestingly, a
series of studies by Migliaccio et al. (449, 450)
illustrated that prenatal and neonatal exposure to the synthetic
estrogen, DES, also results in significantly shorter femur lengths as
well as increased cortical bone thickness and increased trabecular bone
at the epiphysis of the femur during adulthood in female mice.
Therefore, it appears that in the mouse, aberrant estrogen exposure
during development or a hereditary loss of ER action leads to
decreased longitudinal bone growth, contrasting experimental schemes
resulting in a similar phenotype.
These data indicate that pathways other than ER may mediate the
negative regulatory effects of estradiol on bone growth,
suggesting a possible role for ERß. Longitudinal bone growth is a
poorly understood process that depends on chondrocyte activity,
including proliferation, hypertrophy, and the secretion of
extracellular matrix at the growth plate (445). Estrogen is thought to
slow this process by reducing the recruitment, proliferation, and
synthetic activity of chondrocytes, thereby resulting in a maturation
of the epiphyseal plate and inhibition of further longitudinal growth
(445). The detection of both ER (451) and ERß (452) in human
epiphyseal chondrocytes has recently been described. Therefore, it is
possible that ERß, in the context of significantly elevated levels of
estradiol, results in an inhibition of long bone growth in the ERKO
mouse. It is also possible that the significantly elevated levels of
serum androgens in the ERKO female may be playing an influential
role. This may also be true in the ERKO male, which exhibit slightly
shorter femur lengths in the context of only a 2-fold increase in serum
androgens. The possibility that a loss of ER during bone development
results in abnormal genomic imprinting and increased ERß and/or AR
levels as a compensatory mechanism must also be considered.
B. Cardiovascular system
Since the early part of this century, a remarkable gender-related
contrast in the risk of cardiovascular disease has been known. Although
women generally possess a greater incidence of the multiple risk
factors associated with cardiovascular disease when compared with men,
e.g., obesity, diabetes, elevated blood pressure, and plasma
cholesterol, epidemiological studies continue to indicate their
relative risk of developing this disease is significantly lower (453, 454). It is now believed that the protective factor against
cardiovascular disease in females is their inherently increased
exposure to estrogens (reviewed in Refs. 433, 454). The protective
effects of estrogens have been documented in a number of
epidemiological studies documenting the reduced rate of cardiovascular
disease in postmenopausal women receiving estrogen replacement therapy
(433, 454). This correlation between the administration of estradiol
and a reduced risk of vascular disease has been reproduced in
laboratory animal studies involving several different species (454).
The possible mechanisms by which estrogens reduce vascular disease remain unclear but are likely to include positive modifications of a number of physiological parameters that are believed to impinge upon the development of cardiovascular pathlogy (reviewed in Ref. 454). Most notable of these is the ability of estrogen replacement therapy to significantly lower total cholesterol, with a profound effect on levels of the more damaging low-density lipoproteins (LDLs) (454). Hypercholesterolemia is strongly associated with the development of cardiovascular disease. The ability of estrogen therapy to reduce total cholesterol levels is believed to account for a large portion of the cardiovascular protective effects of the hormone (433, 454). Lundeen et al. (455) reported that the significant decreases in serum cholesterol are specific to estrogen action in the ovariectomized rat, whereas other gonadal steroids tested had little effect. Furthermore, cotreatment with the antagonist, ICI-182,780, was shown to inhibit the cholesterol-lowering effects of estrogens, strongly indicating that this is a receptor-mediated effect (455). One possible site at which the actions of estradiol may converge with the pathways of cholesterol metabolism is via the up-regulation of the apo E protein and the apolipoprotein (apo) B/E LDL receptor within and on the cell surface of hepatocytes, respectively (454). During hydrolysis of triglycerides in the circulation, the apo E protein is integrated into the apo B-LDL complexes and thereby functions as a ligand for the hepatocyte apo B/E LDL receptor (454). When the apo E-containing LDL complex is bound by the apo B/E LDL receptor on the cell surface of hepatocytes, the complex is internalized, thereby providing for a means of clearing LDL from the blood (454). The importance of the apo E protein in maintaining serum cholesterol levels and providing protection against vascular disease is evident from studies in transgenic mice that either overexpress the protein or possess a targeted disruption of the apo E gene. Transgenic mice in which an apo E gene is overexpressed are significantly protected from atherosclerosis (456), whereas the apo E knockout is highly susceptible to the vascular disease (457).
Therefore, much of the cholesterol-lowering ability of estradiol is
thought to be due to increased clearance of LDL from the circulation
via the mechanism described above (454). Regulation of the apo E gene
by estradiol has been demonstrated in the rat (458, 459). Furthermore,
Srivastava et al. (460) recently reported that hepatic
levels of apo E mRNA are similar in wild-type and ERKO male
littermates, although a slight decrease in serum levels of the protein
was observed in the ERKO. However, 6 days of estradiol exposure (via
implantation of a 3 µg E2/g body weight/day pellet)
elicited an almost 2-fold increase in serum apo E levels in the
wild-type compared with a 1.2-fold increase in the ERKO (460).
Therefore, these data provide evidence that ER, acting at the level
of translation, is required to mediate the estradiol up-regulation of
apo E expression in mice (460).
In addition to the favorable effects of estrogens on the lipid profile,
it is now believed the steroid may also play a beneficial function
directly at the level of the vasculature. The proposed mechanisms of
estrogen action on the vasculature include modulations of vascular
adhesion molecules, chemoattractants, vasodilators (e.g.,
nitric oxide), vasoconstrictors (e.g., endothelin-1), as
well as possibly acting as an antioxidant (reviewed in Refs. 454, 461). Several of the effects of estrogens, as well as other steroid
hormones, on blood vessel physiology have been proposed to be
nongenomic and independent of the classical nuclear activational
pathway of steroid receptors (reviewed in Ref. 355). However, ER has
been detected in both the endothelial and smooth muscle cells of the
vasculature in several species, suggesting a role for receptor-mediated
actions as well (reviewed in Refs. 431, 454, 462). Furthermore, a
recent study reported that the level of ER in atherosclerotic plaques
from human coronary arteries was reduced compared with levels found in
nonlesioned sections (463). Studies have indicated the presence of
ERß in the vasculature as well. Analysis by ribonuclease protection
assay for ER and ERß mRNA in the mouse indicate only detectable
levels of ER transcripts in the aorta (93). However, Iafrati
et al. (464) report the detection of ERß transcripts by
RT-PCR in mouse aorta and blood vessels, including those of the
ERKO. The apparent contrast in ERß detection between these two
reports in the ERKO vasculature is most likely due to differences in
the sensitivity of the techniques used, since ERß mRNA was detected
only by the more sensitive RT-PCR method. This is likely a reflection
of the low levels of this receptor compared with ER. In the rat
aorta, Petersen et al. (70) described the detection of
full-length and variant ERß mRNA by RT-PCR. Recent reports also
describe the detection of ERß transcripts by RT-PCR in aortic smooth
muscle cells and coronary artery (465), aorta, and cardiac muscle (96)
from monkey. An intriguing report of the tissue distribution for ER
and ERß mRNA in human vasculature was that of Tschugguel et
al., which described the presence of ER mRNA only in cultures
from larger blood vessels, i.e., aorta and pulmonary artery,
whereas ERß transcripts were predominant in cultures from the
endothelium of smaller vessels, such as those from the uterus (466).
Therefore, with the apparent variations that exist in the distribution
of the two forms of ER in the vasculature, depending on the species and
possibly even the vessel of study, the ERKO mice provide a unique tool
to discern the direct role that ER and/or ERß may play on vascular
biology.
One of the initial studies of the vasculature in the ERKO mice was that
by Rubanyi et al. (467) in which the level of the
vasodilator, nitric oxide, was characterized in the aortic rings of the
ERKO male. This study demonstrated that the male C57BL/6J mice, the
background strain of the ERKO, possess a significantly greater
amount of ER in the aorta than female littermates, when quantified by
radiolabeled E2 binding (467). Furthermore, the basal
levels of endothelial-derived nitric oxide were also found to be
significantly higher in the male tissue compared with female,
suggesting that the level of ER, rather than the amount of ligand, may
be the most critical parameter in modulating endothelial nitric oxide
production (467). In agreement with this hypothesis was the observation
of significantly reduced basal nitric oxide levels in aortic tissue
from male ERKO mice (467). However, the levels of stimulated
vasodilatation achieved after treatment with acetylcholine to induce
endogenous nitric oxide release or nitroglycerin, a nitric oxide donor,
did not differ between the sexes or ER genotypes (467). Therefore,
it appeared that only the ability to synthesize basal nitric oxide
levels was altered by ER gene disruption, whereas the capability of
the vasculature to respond to the vasodilator effects of nitric oxide
were not altered (467). Interestingly, the susceptibility to
hypercholesterolemia-induced atherosclerosis in this strain of mice is
in direct correlation with the level of ER detected, i.e.,
male C57BL/6J mice appear less likely to develop the vascular disease
than female counterparts (468). Therefore, it may be speculated that
the ERKO mice possess an inherent susceptibility to vascular disease
as well. These data also indicate that a contributing factor to the
protective effects of estrogen action on the vasculature may be caused
by a convergence of the estrogen and nitric oxide systems and may, in
fact, be due to ligand-independent ER actions.
In contrast to the above description of a measurable vascular defect in
the ERKO mice, Mendelsohn et al. (464) demonstrated no
apparent difference in the response between wild-type and ERKO
female mice in a carotid vascular injury model. This model
involves the monitoring of endothelial and smooth muscle cell growth
and proliferation that is spontaneously elicited in carotid arteries
after artificial endothelial denudation (469). This response has been
shown to be inhibited by estradiol, suggesting a mechanism by which
estrogens may protect the vessel from atherosclerosis (462). Removal of
estrogen action via ovariectomy in wild-type and ERKO mice resulted
in similar levels of endothelial and smooth muscle cell proliferation 2
weeks after carotid injury, as measured both morphometrically as well
as with BrdU incorporation (464). However, daily estradiol treatments
commencing 1 week before injury and continued for the 2 weeks after
were able to inhibit the cellular proliferation to similar levels in
both the wild-type and ERKO animals (464). Therefore, the
"protective" effect of estradiol illustrated by this model system
appears to be independent of ER action and may possibly be mediated
by ERß. Similar studies in the ßERKO mice are currently underway
and will prove interesting in this regard.
Estrogens may also exert direct effects at the level of the heart and
have been proposed to play an important role in cardiac hypertrophy and
remodeling after myocardial infarction (462, 470). In a recent report,
Grohé et al. (471) described the presence of both
ER and ERß as well as the P450arom enzyme in cardiac
myocytes cultured from neonatal rats. This same study demonstrated that
androgen-induced up-regulation of ER was evident in cardiac myocytes
derived from female rats, whereas ERß levels were unaffected and
remained stable in the cells from both sexes (471). Furthermore,
estrogens may modulate cardiac contractility via regulation of
Ca2+ channel activity. Previous studies have indicated that
estrogen may reduce Ca2+ channel activity in several
tissues; however, demonstrative studies in the heart usually involved
superphysiological doses and therefore remain an issue of controversy
(355, 472). However, Johnson et al. (472) demonstrated an
increased number of L-type Ca2+ channels in ERKO male
heart, presumably due to a hereditary loss of ER. Whole hearts from
ERKO males exhibited an approximate 46% increase in the number of
L-type Ca2+ channels and a corresponding delay in cardiac
repolarization when compared with those from wild-type (472).
Therefore, estradiol ER-mediated actions appear to modulate cardiac
contractility via regulation of the number of calcium channels,
possibly providing another function to complement the protective
effects of estrogens in the cardiovascular system.
C. Adipogenesis
A role for gonadal steroid action in adipogenesis and
adipocyte function has been realized for some time (473). This is
evidenced by the well known gender differences that exist in the
distribution of white adipose tissue in humans. Whereas men tend to
accumulate fat stores in the thoracic and abdominal regions,
accumulations in women are found in the upper portions of the arms and
legs. Furthermore, obese woman that also exhibit androgen excess
demonstrate a male pattern of fat distribution, termed android
adiposity (474). Supporting evidence for a functional relationship
between the gonadal and adipogenesis systems is also provided by the
direct correlation that exists between nutrition, body mass, and fat
content with the onset of menarche in young females (475). In addition,
white adipose tissue serves not only as an energy reservoir, but is
also a site of steroid storage and metabolism that can supplement
gonadal and adrenal synthesis and thereby influence the overall
endocrine milieu (292, 473).
The roles of several members of the nuclear hormone receptor
superfamily in adipogenesis have been intensely researched, especially
the glucocorticoid receptor, peroxisome proliferator-activated
receptor, retinoic acid receptor, and the thyroid receptor (reviewed in
Ref. 473). There is little research data concerning any direct role
that ER may play in adipocyte function and distribution, but it is well
known that ovariectomy results in increased fat stores and body weight
in females of some strains of rodents (197, 476, 477). This effect can
be prevented with estrogen replacement as well as reproduced with
prolonged antiestrogen treatments in intact females (197, 476, 477).
Interestingly, a similar increased body weight is observed in ERKO
females of the C57/BL background. Gross observations upon necropsy of
adult ERKO females indicate an obvious increase in the amount of
white adipose fat in the pads of the mammary gland and those lining the
lateral-ventral portions of the body cavity. As early as 4 months of
age, ERKO females exhibit an increased body weight compared with
female wild-type littermates, at 28.7 g (±0.91) vs.
23.3 g (±0.43), respectively. This difference in body weight
between wild-type and ERKO females increases at 8 months of age, at
which time the average weight of ERKO females exceeds that of
age-matched wild-types by almost 35% (t test;
P < 0.01). However, by 12 months of age, increases in
the body weight of wild-type females appear to decrease the gap between
the two genotypes.
The fact that the increased fat stores in the ERKO female are
similar to those observed in the classical ovariectomized model
indicate that a loss of ER-mediated estrogen action may alter
metabolism and adipocyte physiology. Fisher et al. (257)
reported a similar phenotype in the ArKO female mice, in which the
weight of the gonadal and mammary fat pads were increased by 50–80%.
In both ERKO and ArKO females, serum testosterone levels are
substantially elevated but do not appear to have a lessening effect on
fat pad weight (257). Furthermore, there are no reports of increased
body weight in the androgen-resistant Tfm mice (478),
indicating that androgen action may play a lesser role than estrogen in
fat storage. It is unknown at this time whether a phenotype similar to
the ERKO and ArKO mice may also exist in the ßERKO mice. However,
sexually mature ßERKO mice of both sexes exhibit no significant
differences in body weight and appear to possess a relatively normal
distribution of white adipose tissue in the peritoneum at the ages
examined.
Although the above evidence strongly supports a role for
ER-mediated estrogen action in adipocyte physiology, the mechanisms
involved remain unclear. Adipose tissue has been shown to possess ER
(479, 480, 486, 487, 488) and the enzymes necessary for estradiol synthesis
(292, 480). Recent studies have also indicated that the gonadal sex
steroids can alter the activity of lipoprotein lipase, a critical
enzyme in adipocyte growth and fat storage (474). Interestingly, Lubahn
et al. (481) reported that ERKO female mice fed a diet
enriched with the phytoestrogen, genistein, exhibited a reduced body
weight compared with ERKO females fed the control diet. This may
indicate a non-ER-mediated yet estrogenic action of genistein, a
naturally occurring isoflavone shown to possess hormone-like actions in
mammalian cells that are devoid of ER (482). Therefore, the
ER-independent actions of genistein, such as the inhibition of
protein tyrosine kinases and influence on growth factor action,
complicate the interpretation of the above study in terms of defining a
role for ER in fat stores.
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VIII. Comparison with Human Disease and Models of Deficient Estrogen Action |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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expression. Although the causal factors remain unclear,
evidence indicates that incessant ovulation, resulting in constant
rupture and estrogen-mediated repair of the surface epithelium,
increases the probability of spontaneous genetic abnormalities that may
lead to tumorigenesis (483, 484). The exact role that estrogen and the
ER may play in the induction and promotion of ovarian carcinoma remains
unclear (reviewed in Refs. 483, 484, 485). A number of immortalized cell
lines have been generated and characterized from human ovarian tumors
and exhibit varied levels and responses to estrogen agonists and
antagonists (reviewed in Ref. 484). Brandenberger et al.
(94) recently reported the detection of ER and ERß mRNA in the
human ovary, ovarian tumors, and ovarian tumor cell lines. Their
findings include the description of ERß mRNA predominantly in the
granulosa cells of normal ovaries and a marked reduction in the levels
of ERß transcripts in ovarian carcinomas (94). In contrast, a cell
line derived from a human ovarian surface epithelium and several human
ovarian carcinomas were reported to express high levels of ER mRNA
(94). As mentioned previously, ovarian tumors are most often derived
from the outer surface epithelium, whereas granulosa cell-derived
carcinoma in humans is rare. Interestingly, we observe an approximately
40% incidence of granulosa/thecal cell tumors of the ovary in ERKO
females between the ages of 15–20 months. No such ovarian tumors have
been observed in the wild-type or heterozygous littermates of the
ERKO. A similar incidence and type of spontaneous tumor is reported
in transgenic mice possessing significantly elevated levels of LH
caused by overexpression of the LHß subunit gene (254, 255).
Therefore, hypergonadotropin stimulation appears to play a significant
role in the etiology of this type of ovarian tumor in the mouse. The
similarities in the incidence and type of tumor observed between the
ERKO and bLHß-CTP mouse indicate that ER probably plays a minor
role. Preliminary analysis in tumor samples for the ERKO females
indicates normal to elevated levels of ERß expression. Therefore,
elevated gonadotropins and estradiol may result in chronic induction of
the granulosa cells to proliferate and is the likely stimulus for the
ovarian tumors observed in both transgenic models. Future
investigations to determine the incidence of tumors in the ßERKO
ovary will prove useful in further elucidating the etiology of this
neoplasia.
B. Chronic anovulation
Targeted gene disruption of the ER genes has resulted in partial
and complete anovulation in the ßERKO and ERKO females,
respectively. In the clinical setting, chronic anovulation is often
categorized by the presence or lack of estrogen synthesis (204).
Chronic anovulation in the absence of estrogen is diagnosed in women
who experience little to no menstrual bleeding after progesterone
withdrawal and is most often associated with hypogonadotropism
resulting from impaired function of the hypothalamic GnRH neurons or
disorders of the pituitary (204). Chronic anovulation in the presence
of estrogen synthesis can be caused by several different functional
abnormalities, such as Cushing’s syndrome, hyper/hypothyroidism,
adrenal hyperplasia, and ovarian tumors (204). However, the majority of
cases of anovulation in the presence of estrogen are associated with
polycystic ovarian syndrome (PCOS), a heterogeneous series of clinical
and endocrine abnormalities thought to be due to inappropriate steroid
feedback regulation of the hypothalamic pituitary axis (reviewed in
204, 486, 487). Although the ovaries of both the ERKO and ßERKO
females synthesize estradiol, the inherent insensitivity of certain
target tissues to the hormone may render effects similar to those of
insufficient estrogen synthesis and therefore may offer some analogy to
the human syndrome. The primary defect resulting in inefficient
ovulation in the ßERKO appears to be due to defects directly within
the ovarian tissue, more precisely, an inability to appropriately
respond to estradiol in the granulosa cells of maturing follicles.
However, until further studies are carried out, an impaired ability to
produce an adequate gonadotropin surge must also be considered as a
possible contributory factor to the decreased ovulatory rates observed
in the ßERKO. Still, the reduced number of ovulations observed in the
ßERKO even after stimulation with exogenous gonadotropin suggests the
presence of a significant defect within the ovary.
Although an analogy of the ßERKO ovarian phenotype with a human
pathology may be obscure at this point, the ovarian phenotype of the
ERKO is strikingly similar to that of PCOS patients. The most common
pathological symptoms of PCOS include anovulation, hirsutism, and the
presence of bilateral enlarged, white, smooth, and sclerotic ovaries
with a thickened outer capsule (204, 486, 487). Internal
characterization of the ovaries of a PCOS patient most often indicates
the presence of follicles at various stages of atresia, a strikingly
hypertrophied thecal/stromal compartment, and the observation of rare
or absent corpora lutea (204). The follicles presented usually possess
a reduced number of granulosa cells and little aromatase activity
(204). Biochemical findings are most often characterized by elevated
serum androgen and LH levels, whereas progesterone and FSH levels
remain low to normal (204, 486, 487). However, there appear to be wide
variations in the extent and occurrence of each of these symptoms,
illustrated by the diagnosis of PCOS in women with apparently normal LH
levels and no menstrual abnormalities, indicating there is likely more
than one etiological factor involved in this syndrome (204, 487).
The onset of PCOS most often coincides with menarche in young females, suggesting the existence of endocrine abnormalities even before puberty (204, 486). The first clinical indications are dysfunctional and/or unpredictable uterine bleeding or oligo/amenorrhea (204, 486). This is often accompanied by a high incidence of hirsutism (male-pattern hair growth) in 70–90% of PCOS females, determined to be due to increased serum androgens (204, 486). However, the causal agents that lead to increased circulating steroids and the initiation of PCOS remain unclear, although a strong familial component is believed to be involved (204, 488). Franks et al. (489) have proposed that the primary cause of PCOS is ovarian in nature and that endocrine abnormalities are secondary. Others have provided evidence to indicate that inappropriate steroid feedback at the hypothalamic-pituitary axis, resulting in increased frequency and amplitude of LH pulses, results in hyperstimulation of an otherwise normal ovary (204, 486). The initiation of the abnormal steroid signaling to the hypothalamic-pituitary axis may stem from extragonadal conversion of elevated circulating androgens to estrone and estradiol (204, 486, 487). The source of the initial increases in androgen that may trigger PCOS remain unclear and may be ovarian or adrenal in nature (487). Regardless, extraglandular aromatase activity in the adipose tissue is believed to result in the initial acyclic increases in estrogens that lead to the ovarian syndrome (204, 486). Obesity is reported in 40% of PCOS patients and is thought to be a contributory factor in the adipose tissue-derived estrogen production (204, 486). The abnormally high levels of estradiol then feed back to the hypothalamic-pituitary axis in a positive fashion, resulting in chronically elevated LH levels (204, 486). Increased secretion of LH leads to hypertrophy of the ovarian thecum, a hallmark of the PCOS ovary, and further increases in androgen production (204, 486). Therefore, once initiated, it is thought that ovarian-derived androgens, followed by extraglandular conversion to estrogens, act to self-perpetuate the syndrome. Treatment of PCOS patients with a GnRH antagonist has proven to reduce circulating androgen and subsequent estrogen levels, indicating the ovary as the primary source of the androgen (204, 486). Furthermore, women with PCOS can be induced to ovulate with administration of FSH, suggesting that the primary defect is extragonadal in nature (204).
The similarities of the ERKO ovarian phenotype and those associated
with PCOS are worth noting. However, the initial stimulus of abnormal
feedback to the hypothalamic-pituitary axis that leads to the phenotype
may be different in the human and ERKO mouse. As described above in
the human female, it is believed that extragonadal synthesis of
estrogens results in acyclic positive regulation of LH secretion by the
hypothalamic-pituitary axis. In contrast, the ERKO ovary is the
principal source of both androgen and estradiol, and acyclic increases
in serum LH are primarily due to a lack of negative feedback at the
hypothalamic-pituitary axis. However, preliminary studies in the
ERKO female have indicated that the signaling pathways for positive
regulation of the LH secretion may be intact and therefore may also
contribute to the increased gonadotropin levels observed. Therefore,
although the means by which the elevated levels of LH are achieved in
human PCOS and the ERKO female mouse may differ, some features of
the ovarian phenotypes are similar. Certain morphological abnormalities
in the ovaries are comparable, i.e., polycystic follicles
with reduced numbers of granulosa cells, hypertrophied thecal and
stromal tissue, and the absence of corpora lutea, although the ERKO
ovary does not exhibit the thickened outer capsule seen in PCOS
patients. It is possible that encapsulation of the polycystic ovary in
the human is an ER-mediated event and therefore not possible in the
ERKO. However, the LH-overexpressing bLHß-CTP mice, which possess
a functional ER gene as well as several characteristics associated
with PCOS, also do not demonstrate this phenotype (254, 255). Several
of the endocrine parameters associated with human PCOS are also
observed in the ERKO, especially the presence of elevated serum
androgens and LH. Although the observation of hirsutism caused by
elevated serum androgens is not possible in mice, analogous biological
manifestations of elevated androgen levels are exhibited in the ERKO
female, including masculinized preputial (clitoral) glands and a
thickened dermis. Furthermore, obesity is reported in a large
proportion of PCOS patients and is thought to be a contributory factor
in the extragonadal conversion of androgens to estrogens. As described
above (Section VII.C), adult ERKO females significantly
outweigh their age-matched wild-type littermates because of excessive
white adipose tissue. Interestingly, insulin resistance and
hyperinsulinemia are often reported in PCOS patients and thought to
contribute to further androgen synthesis in the ovarian thecum (204, 487). Taylor and Lubahn (490) reported an abnormal glucose tolerance
test in ERKO females that could be corrected with ovariectomy,
suggesting PCOS-like insulin resistance. Therefore, the similarities of
the endocrine abnormalities and ovarian phenotypes in the ERKO and
bLHß-CTP mice with those of human PCOS patients provide support that
the cause may be extragonadal in nature, and possibly due to elevated
LH in the presence of normal FSH. Although the initial cause of the
abnormally high LH levels in ERKO females may differ from that
postulated in the PCOS patient, the resulting effects on the ovary are
similar and may allow the ERKO to be a useful experimental model in
future investigations of this human disease.
C. ER and aromatase deficiency
The first and only reported case of estrogen insensitivity in a
human is that of Smith et al. (116) in which a male (46, XY)
was determined to be homozygous for a single-point mutation in exon 2
of the ER gene that resulted in a premature stop codon. Similar
reports of human estrogen deficiency were also lacking in the clinical
literature until recently. However, five cases of aromatase deficiency
and therefore a lack of estrogen synthesis have been reported in both
human males (119, 120) and females (117, 118, 120, 491). Therefore, the
existence of humans that exhibit insufficient estrogen action due to
either a lack of functional receptor or hormone, along with the
successful generation of the ER null mice, suggests that estrogen is
not essential to embryonic and fetal development in mammals. However,
like the ER null mice, a distinct syndrome of phenotypes is apparent in
both sexes of humans lacking in estradiol or ER.
All three reports of females homozygous for inactivating mutations in
the P450arom gene describe pseudohermaphroditism,
i.e., 46XX genotype, the presence of internal female
reproductive structures but ambiguous external genitalia (117, 120, 491). This phenotype is due to the lack of aromatase activity in the
fetus and placental unit during gestation, leading to the accumulation
of androgens, which in turn elicit masculinizing effects on the fetus
(117, 120, 491). A similar phenotype of ambiguous external genitalia
was described in an infant in which placental aromatase deficiency was
determined, although enzyme activity in the fetus was not evaluated at
the time of the report (118). In the two cases of a complete lack of
aromatase, the mother also exhibited virilization during pregnancy
(120, 491), whereas a traceable level of placental aromatase activity
appeared to inhibit this symptom in the case of Conte et al.
(117). In all three cases, development of the internal structures of
the female reproductive tract did not appear to be altered by a lack of
estrogen action (117, 120, 491), although a compensatory role fulfilled
by maternal estrogens cannot be ruled out. Serum levels of androgen,
androgen precursors, FSH, and LH were elevated in the
P450arom-deficient patients as early as infancy (117, 491)
and continued to be elevated as the females approached puberty (117, 120). At 12–14 yr of age, secondary development of the breasts, a
pubertal growth spurt, and menarche were all absent, but virilization
of the external genitalia was reported (117, 120). In all three cases,
hyperstimulation of the ovary and the development of multifollicular
cysts was illustrated, even as early as 2 yr of age (117, 120, 491).
The ovarian pathology exhibited was similar to that observed in the
ERKO females and was compatible with a diagnosis of PCOS (120).
Therefore, intraovarian estrogen action does not appear to play a role
in the development of the multifollicular ovarian cysts in humans.
Estrogen and progesterone replacement therapy alleviated the above
phenotypes, resulting in normal gonadotropin and androgen levels,
regression of the ovarian cysts, and in the two pubertal patients
reported, the onset of breast development, a growth spurt, and menarche
(117, 120, 491).
The clinical syndromes exhibited by the two reported human male cases
of aromatase deficiency (119, 120) and the single known human case of
an inactivating mutation of the ER gene (116) are strikingly
similar. All three patients exhibited a normal onset of puberty and no
gender-identity disorders, as well as normal external genitalia and
prostate size (116, 119, 120). The patient lacking functional ER
exhibited a testicular volume and sperm density within the norm for men
of his age (116). A normal testicular volume was also reported in the
P450arom-deficient patient of Morishima et al.
(120). However, Carani et al. (119) reported small testis
and severe oligozoospermia and infertility in the other male lacking
aromatase, although the presence of similar findings in a brother with
a normal P450arom gene suggested other possible familial
factors for this finding. Serum levels of androgens, FSH, and LH were
all consistently elevated in the P450arom-deficient males
(119, 120), as well as estrone and estradiol in the ER-deficient
male (116). Estrogen replacement therapy resulted in the return of
androgen and gonadotropin levels to the normal range in the
P450arom-deficient males. However, similar estrogen therapy
induced no such changes in the ER-deficient male (116). In fact,
estrogen replacement therapy in the ER-deficient male achieved
circulating levels of the steroid that exceeded the norm by nearly
5-fold, yet no alleviation of the above pathologies or side effects
were observed, such as impotence and gynecomastia (116). Alterations in
the cardiovascular system have been reported recently in the
ER-deficient male, including dysfunctions in vasodilation (492) and
premature coronary artery disease (493).
The most overt phenotypes in all patients lacking sufficient estrogen
action involved the skeleton. Females homozygous for mutations of the
P450arom gene displayed a delay in bone age, a significant
decrease in bone mineral density of the lumbar spine, and absence of
the pubertal growth spurt (117, 491). Both the ER-deficient male and
the two males lacking aromatase were evaluated as adults and exhibited
strikingly similar skeletal phenotypes. All three were characterized by
tall stature (>95th percentile) with continued slow linear growth, a
low upper/lower body segment ratio (<0.88, vs. 0.96,
average for men), unfused epiphyses, bone age of 14–15 yr, and
decreased bone density (116, 119, 120). Epiphyseal closure and
increases in bone mineral density were observed after estrogen
replacement therapy in the P450arom-deficient patients
(119, 120). However, as with the phenotypes described above in the
ER-deficient male, exogenous estrogen treatments resulted in no
changes in bone physiology (116). Therefore, although it was once
believed that estrogens were the major sex steroid influencing bone
physiology in females and androgen fulfilled similar roles in the male,
the phenotypes described above strongly indicate that estrogen action
is critical to the pubertal growth spurt, bone mineralization, and
epiphyseal maturation in both males and females.
|
IX. Summary |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
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The ERKO mice continue to satisfy the confirmatory role of a
knockout quite well. As summarized in Table 4, the phenotypes observed in the ERKO
due to estrogen insensitivity have definitively illustrated several
roles that were previously believed to be dependent on functional
ER, including 1) the proliferative and differentiative actions
critical to the function of the adult female reproductive tract and
mammary gland; 2) as an obligatory component in growth factor signaling
in the uterus and mammary gland; 3) as the principal steroid involved
in negative regulation of gonadotropin gene transcription and LH levels
in the hypothalamic-pituitary axis; 4) as a positive regulator of PR
expression in several tissues; 5) in the positive regulation of PRL
synthesis and secretion from the pituitary; 6) as a promotional factor
in oncogene-induced mammary neoplasia; and 7) as a crucial component in
the differentiation and activation of several behaviors in both the
female and male.
|
ERKO must begin with the
observation that generation of an animal lacking a functional ER
gene was successful and produced animals of both sexes that exhibit a
life span comparable to wild-type. The successful generation of ßERKO
mice suggests that this receptor is also not essential to survival and
was most likely not a compensatory factor in the survival of the
ERKO. In support of this is our recent successful generation of
double knockout, or ßERKO mice of both sexes. The precise defects
in certain components of male reproduction, including the production of
abnormal sperm and the loss of intromission and ejaculatory responses
that were observed in the ERKO, were quite surprising. In turn,
certain estrogen pathways in the ERKO female appear intact or
unaffected, such as the ability of the uterus to successfully exhibit a
progesterone-induced decidualization response, and the possible
maintenance of an LH surge system in the hypothalamus. Furthermore, it
is apparent that several of the ERKO phenotypes may be aggravated by
the downstream attenuation of progesterone and PRL action or even
enhanced by increased androgen sensitivity, including those observed in
the mammary gland, gonads, bone, and behavior. In addition, disruption
of the ER gene may have unmasked estrogen-signaling systems that
were not easily detectable in the wild-type, such as 1) the apparent
estrogen actions of the 4-OH-E2 in the ERKO uterus that
are independent of ER and ERß; 2) the ability of estrogen to
induce increased levels of hypothalamic PR in the ERKO female; 3)
estradiol-induced potentiation of select neurons of the hippocampus;
and 4) the protective effects of estrogen in the carotid vascular
injury model. Finally, the concurrent descriptions of human mutations
resulting in a lack of estrogen action due to a loss in ligand or
functional receptor have illustrated the utility of the ER null mice as
a model system to further understand the pathologies that result.
Therefore, the ERKO mice have illustrated the several ways in which
data collected from a knockout model can quickly contribute to the
current knowledge concerning the function of a particular gene.
However, it is worth noting the distinct differences in thought that
occurred during the generation of the ERKO and ßERKO models. At
the time the work was initiated to generate the ERKO mice, much was
known about the many roles of estrogen and the ER; therefore, several
educated predictions of the possible phenotypes were possible and have
since been confirmed, rejected, or reevaluated. However, the conception
of the ßERKO mice occurred only 2 yr after the discovery of the
ERß. Because little was known about the function and role that the
ERß might play, it was difficult to make sound predictions.
Therefore, the ERKO mice have played a significant role in
confirming many of the roles thought to be fulfilled by estrogen,
whereas the ßERKO mice have and will continue to provide primary
insight into the functions of the ERß. The generation of both models,
as well as a forthcoming description of the ßERKO, will prove
invaluable in elucidating the precise roles fulfilled by each ER, as
well as any possible cooperative roles the two receptors might play
within the same tissue or even within the same cell.
|
Acknowledgments |
|---|
ERKO; and Drs. John Krege, Jeff Hodges, and Jan-Åke Gustafsson
and laboratory for the ßERKO. The authors are exceptionally indebted
to Sylvia Curtis, Todd Washburn, Dr. Jonathan Lindzey, Dr. Wayne
Bocchinfuso, Mariana Yates, Linwood Koonce, James Clarke, Page Myers,
Dr. Motohiko Taki, and Dr. Sean Kimbro for their efforts and
dedication. We would also like to thank our several collaborators, Drs.
Ralph Cooper, Richard DiAugustine, E. Mitch Eddy, Thomas Golding, Paul
Kincaide, Diane Klotz, Michael Mendolsohn, Robert Moss, Jeffrey Moyer,
Sonoko Ogawa, Donald Pfaff, Gabor Rubanyi, David Schomberg, Allen
Silverstone, and Harold Varmus. We would also like to acknowledge Drs.
Paul Shughrue and Istvan Merchenthaler for the contribution of Fig. 8. |
Footnotes |
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References |
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TopAbstractI. Introduction—A...II. Estrogen ReceptorsIII. Reproductive Tract...IV. Mammary GlandV. Reproductive Tract Phenotypes...VI. Neuroendocrine SystemVII. Phenotypes in Peripheral...VIII. Comparison with Human...IX. SummaryReferences |
|---|
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9. Error bars indicate
SEM. Statistical significance is indicated as follows
(t test, wild-type vs. 
