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Division of Reproductive Endocrinology (S.F.P.), Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut; Division of Reproductive Sciences (A.B.T., E.Y.A.), Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah; Department of Obstetrics and Gynecology (A.H.), Sheba Medical Center, Tel Aviv University, Sackler School of Medicine, Tel-Hashomer, Ramat-Gan, Israel; and Division of Endocrinology and Metabolism (J.D.V.), University of Virginia Health Sciences Center, Charlottesville, Virginia
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Abstract |
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 Top Abstract I. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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I. Introduction
II. The Nonprimate Ovary as a Site of Estrogen Reception
III. The Nonprimate Ovary as a Site of Estrogen Action
A. The rat and mouse
B. The hamster
C. The rabbit and guinea pig
D. The pig
E. Possible interactions of estrogen with other putative ovarian regulators
IV. Lessons Learned from the Estrogen-Resistant Model- Estrogen Receptor Null Mutants (ERKOs)
V. Lessons Learned from the Targeted Disruption of the CYP-19 (Aromatase) Gene-Aromatase Null Mutant (ArKO)
VI. The Primate/Human Ovary as a Site of Estrogen Reception
A. Molecular probing
B. Immunohistochemical studies
VII. Is an Estrogen-Free (or at Least Poor) Intrafollicular Environment Compatible with Follicular Development, Ovulation, and Corpus Luteum Formation?
A. Follicular "expansion" vs. follicular "growth": an important conceptual distinction
B. Lessons learned from 17
-hydroxylase/17–20 lyase deficiency
C. Lessons learned from the 3ß-hydroxysteroid dehydrogenase (3ß-HSD) deficiency
D. Lessons learned from aromatase deficiency
E. Lessons learned from the intensely hypogonadotropic model
F. Impact of estrogen deficiency on oocytic and early embryonic development
VIII. The Primate/Human Ovary as a site of Estrogen Action
IX. Estrogen Reception and Action: The Nonclassical Alternative(s)
X. Summary
XI. Directions for Future Research
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I. Introduction |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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In this regard, the evidence relevant to the human will be compared to
that of other species, to be presented in the first part of this
review. The role(s) of estrogens will be examined not only by analyzing
the physiological evidence to the effect that these hormones control
ovarian cell function and follicular growth, but also by summarizing
the molecular evidence for the existence and distribution of the
cognate receptors. Based on cumulative data in several species, an
operational model appears to be emerging, in large part due to the
discovery of a new estrogen receptor (ER) type (i.e.,
ERß), the identification of human examples of specific mutations in
enzymes of the steroidogenic pathway, as well as the generation of
specific null mice mutants for the ER, ERß, and the aromatase
genes.
It must be pointed out, however, that even though this review will question the role of estrogens in primate/human follicular development, one must remain cognizant of the unassailable extraovarian roles of estrogens in these species. Clearly, estrogens play a vital role in supporting the growth and differentiation of the Mullerian (3) and mammary (4, 5) complexes. Of equal import is the role played by estrogens in the synchronization of ovarian follicular development with the midcycle gonadotropin surge (6) and the preparation of the uterus for implantation (7, 8, 9). Somewhat less well characterized are the "nonreproductive" effects of estrogenic substances, which may include effects on the cardiovascular (10, 11) and skeletal (12, 13) systems. Intriguing research has also been conducted to describe the role of estrogens in the central nervous system wherein it appears that estrogens may play a physiological role in the modulation of mood, cognition, and behavior (14, 15).
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II. The Nonprimate Ovary as a Site of Estrogen Reception (Table 1 )
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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-hybridizing mRNA in the mouse ovary much smaller than the
classical 6.5-kb ER mRNA. This mRNA species hybridized to probes
specific for the steroid receptor binding regions of the mouse ER
(domains E and F) and was enhanced in granulosa cells vs.
residual ovarian tissues. Wu et al. (25), in turn, reported
ER transcripts in the mouse oocyte as detected by RT-PCR.
Clemens and Richards (26) provided preliminary documentation of ER
transcripts in rat granulosa cells. Specifically, primers were designed
to amplify a 517-bp fragment from exon 3 to exon 5 of the rat ER. After
reverse transcription of granulosa cell RNA, PCR was performed in the
presence of labeled nucleotides, thereby allowing the identification
and quantification of the 517-bp product and the splice variants
involving exons 3–5. Subject to these limitations, no significant
ER splice variants were detected. In vivo and in
vitro studies revealed the granulosa cell ER transcripts to be
down-regulated by 17ß-estradiol and even further reduced by the acute
effects of the LH surge. As such, these findings provide a
documentation of the existence of hormonally responsive ER
transcripts in rat granulosa cells.
While the ER gene has been recognized since first cloned from the
human breast cancer cell line MCF-7 in 1985 (27), the ERß cDNA was
only isolated in 1996. The initial tissue sources of ERß transcripts
were the rat prostate and ovary (28) and the human testis (29). After
the cloning of ERß in rats and humans (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46), the same was
accomplished for mice (47, 48), sheep (49), cows (50), and nonhuman
primates (51). Ovarian expression studies of the ERß gene have since
been accomplished in rats (52, 53, 54), mice (55, 56), monkeys (57, 58),
humans (59, 60), and, more recently, cows (50).
In rodent reproductive tissues, the ERß gene has been shown to be
predominantly expressed in the ovary (61, 62, 63). ER transcripts, in
turn, appear to predominate in the uterus, cervix, and vagina (53, 54, 64). Localization of ERß transcripts to the rat ovary was
convincingly demonstrated by both in situ hybridization and
RT-PCR (28, 29, 56, 65, 66, 67, 68, 69, 70, 71). Although ERß transcripts predominate in
rat ovaries (68), both the ER and ERß genes have been shown to be
expressed in the ovaries albeit mostly in distinct cellular
compartments (54, 62, 65).
The expression of the ER gene in the mouse ovary dates back to fetal
life, while ERß gene expression appears to be initiated at birth. The
latter was shown to increase with age (55, 63), possibly due to the
onset of follicular maturation. In the rat ovary, ERß gene
expression was apparent by fetal day 14, a sharp increase being
noted during the first week of neonatal life (53).
In rat ovaries, ERß proved highly localized to granulosa cells of
healthy follicles, from the primary to the preovulatory stage (54, 62, 72) (Figs. 1 and 2). ERß transcripts were also expressed
in theca cells although more weakly when compared with their expression
in granulosa cells (65). Some studies have described scattered ERß
gene expression in the ovarian stroma (65). The primordial follicle,
the oocyte, and the germinal epithelium do not appear to be sites of
ERß expression (73).
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and ERß transcripts do not display a complete nonoverlapping
pattern of expression in the rat ovary (73). The ER gene is
expressed in granulosa cells albeit less abundantly (64, 66). When
compared with the ERß gene, the ER gene is expressed mainly in
theca and stroma cells (53, 54), as well as in the germinal epithelium
(53, 54, 64). Primordial follicles, oocytes, and corpora lutea (54, 61)
do not seem to express ER transcripts (73).
Some studies suggested that ERß gene expression in the rodent
ovary may be under LH/hCG control (67). Indeed, preovulatory follicles,
as well as newly formed corpora lutea of mice, express lower levels of
ERß transcripts, a phenomenon possibly due to the preovulatory LH
surge (70) (Fig. 3). The ability of
LH/hCG to down-regulate ERß gene expression has
also been evident in studies using cultured rat granulosa cells (67).
Unlike the ERß gene, ovarian ER gene expression proved
consistently uniform throughout the rat ovarian cycle. The pregnant rat
ovary is a site of ER reception (54, 69).
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mRNA, may imply that the intraovarian effects of estrogen are
primarily mediated by ERß.
In summary, the ability of estrogens to influence the above mentioned
activities in the rat ovary is due to the action of two receptors (
and ß) described to date. The rodent granulosa cells contain
predominantly, if not exclusively, the ß-subtype, whereas the ER
subtype was detected mainly in the theca layer and in the interstitium.
Although ER and ERß are not expressed in the rat ovary in a
complete nonoverlapping pattern, the predominance of specific subtypes
in different cellular compartments within the ovary suggests that each
ER subtype may be responsible for distinct downstream activities in the
cell types in which they are expressed.
Although not thoroughly characterized, porcine ovarian follicles
express high-affinity nuclear estrogen binding sites in classical
equilibrium binding assays with in vitro dissociation
constants of approximately 0.6 to 1.1 nM (74, 75). There is little or no knowledge of specific subtype ( or ß)
expression and differential regulation of ERs in porcine theca or
granulosa cells. However, estrogen exerts consistent actions on
selected facets of follicle development and/or cytodifferentiation (see
below).
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III. The Nonprimate Ovary as a Site of Estrogen Action |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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) and mouse
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). These observations led Ingram to
conclude that the trophic effect of estrogen is attributable to its
ability to retard the loss of developing follicles in the
hypophysectomized rat. More recently, Goldenberg and associates (93)
sequentially administered DES (in total doses ranging from 0–4 mg for
either 2 or 4 days) and FSH (a total dose of either 0, 50, 100, or 200
µg in 6 sc injections over 72 h), revealing that an early effect
of treatment with DES is to increase DNA synthesis by granulosa cells.
Moreover, treatment with DES produced an increase in the growth rates
of follicles larger than 200–300 µm in diameter while decreasing the
rate of atresia. Qualitatively comparable results were reported in
several other contributions (16, 94, 95, 96). That estrogens may, in fact,
be antiatretic has most recently been supported by the demonstration of
their ability to inhibit ovarian granulosa cell apoptosis (97). In this
study, a time-dependent increase in ovarian DNA fragmentation was
observed when hypophysectomized rats with subcutaneous capsules of DES
had these implants removed. This effect was shown to be prevented by
treatment with either estradiol benzoate (3 mg/day) or DES (0.5
mg/every 12 h). The above notwithstanding, evidence to the
contrary has been put forth (98), which illustrates the controversial
nature of this issue.
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Mice responded to DES [but less so to 17ß-estradiol cyclopenthylpropionate (ECP)] with an increase in the number of small or large preantral follicles (22), but without an increase in ovarian weight. Apart from the latter, in the few reports on the effects of estrogen in immature mice, both stimulatory and inhibitory effects on follicular development were noted (110, 111). However, since these observations were made in pituitary-intact mice, it is difficult to conclude that estrogens may have been acting directly at the level of the ovary. Fortunately, and more recently, Wang and Greenwald demonstrated unequivocally the ability of ECP, 10, 50, or 250 µg daily for 1–4 days, to stimulate the growth of preantral and antral follicles in the hypophysectomized mouse, an effect associated with a delay of follicular atresia (112). Moreover, estrogens were shown to synergize with FSH in the enhancement of follicular proliferation and differentiation as well as in the attenuation of follicular atresia (112). Taken together, it would appear that the mouse, similar to the rat, is estrogen-responsive. The precise reason(s) underlying the apparent discrepancy with earlier work remains uncertain.
B. The hamster
Although the direct effect of estrogen on rat ovarian follicular
development is well established, the role of estrogen in ovarian
folliculogenesis may be a species-specific phenomenon. Thus,
extrapolation of the rat data to other species may not be feasible.
Indeed, hypophysectomized adult hamsters, injected with estradiol
benzoate, 10 µg daily for 3 days, exhibited neither increased ovarian
weight nor enhanced effects of FSH on follicular development (113).
Thus, hypophysectomized (or intact) hamsters may not respond to
estrogen by increasing the number of large preantral follicles as
reported in the rat. Similar results were obtained for the intact or
hypophysectomized immature hamster (17). It is possible that for the
immature and adult hamster, unlike the rat, estrogens do not play a
major role in the recruitment of large ovarian preantral follicles. In
this same context, Hutz et al. (21) set out to observe the
response of granulosa cells from gonadotropin-primed hamsters to
treatment with DES or 17ß-estradiol under in vitro
circumstances. Interestingly the application of DES inhibited the
accumulation of estrogen regardless of the presence or absence of FSH
in the culture medium. In contrast, the combination of 17ß-estradiol
plus FSH augmented the accumulation of progesterone, which clearly
argues that estrogen is facilitating granulosa cell differentiation.
These findings were interpreted to mean that estrogens can be
nonstimulatory or inhibitory to the function of hamster granulosa cells
in vitro in parallel to effects shown in vivo.
Since the role of estrogens in hamster ovarian folliculogenesis is uncertain, comparisons between hamster and other animal models as well as conclusions on the effect of estrogen on ovarian follicular development are hindered by the paucity of available data.
C. The rabbit and guinea pig
Immature rabbits treated with ECP or DES for 3 days failed to
increase ovarian weight but did increase the number of small or large
preantral follicles (22). In contrast, estrogen-treated guinea pigs
displayed a significant increase in ovarian weight, a phenomenon
attributable to an increase in the number of large antral follicles
(22).
D. The pig
Experimental studies support a thesis of multipotential actions of
estrogen on follicular growth and cytodifferentiation in the pig (74, 114, 115, 116, 117, 118, 119). However, since much of this knowledge is based on
descriptive in vitro studies, which may involve risk,
interpretative complications due to 1) the presence of serum (120, 121), 2) failure to distinguish atretic from healthy follicles, 3)
assessment of a limited degree of estrogen’s interactions with other
regulators of follicle function, and (4) focus on growth and
cytodifferentiative features of the granulosa, but not theca cell, it
is difficult to make definitive assertions on the role of estrogen in
the swine ovary.
Whereas in vivo estrogen administration in the (hypophysectomized) rat clearly supports follicular and ovarian growth, this inference is confounded in the pig by well documented granulosa cell-proliferative responses at least in vitro to insulin-like growth factor I (IGF-I), the production of which by granulosa cells can be driven by GH alone or estrogen and FSH combined (122, 123, 124, 125). Moreover, IGF-I or estrogen each is capable (alone or in concert with FSH) of augmenting indices of in vitro granulosa cell proliferation in the pig, e.g., inducing proliferating-cell nuclear antigen (PCNA) expression and increasing tritiated thymidine incorporation or cellular DNA content (126, 127).
The full physiological in vivo role(s) of estrogen in
modulating ovarian cellular growth will be challenging to unmask in
swine, since multiple intrafollicular factors also appear to control
granulosa cell growth, and their possible interactions with estrogen
have not yet been explored, e.g., inhibitory [tumor
necrosis factor- (TNF) (126)], or stimulatory [relaxin (128),
transforming growth factor-ß (TGFß) (129), endothelin-1 (129),
epidermal growth factor (EGF) (127), cAMP analogs (127), and
T4 (130)].
Nonetheless, because the majority of correlative studies document an inverse relationship between intrafollicular estradiol concentrations and one or more cytological measures of atresia (e.g., Ref. 131), several important considerations arise, namely whether 1) estrogen is obligatory to maintain healthy late-follicle development; or, conversely, 2) failure of follicular development, predicated on whatever mechanistic grounds, is heralded by loss of estrogen-synthesizing capacity. Both views are supported, but not proven, by the concomitant waning of estrogen synthesis and other biosynthetic functions in early follicular atresia in the pig, e.g., inhibin production or FSH-stimulated cAMP accumulation (131). Moreover, aromatase activity in the pig is controlled by multiple factors other than FSH acting alone [e.g., inhibitory: cytokines, such as interleukin-6 (IL-6) (132), PRL (133), extracellular purines (134), insulin-like growth factor binding protein-3 (IGFBP-3) (135), and high concentrations of LH (136); or stimulatory: IGF-I or EGF (137)].
In contrast to the sparse evidence for follicle (or granulosa cell)
growth-promoting actions of estrogen in the pig (above), significant
trophic actions of estrogen on multiple endpoints of granulosa cell
cytodifferentiation are well recognized. For example, estradiol acts
trophically in vitro as a potent biological amplifier of
PRL, FSH, and IGF-I or IGF-II’s stimulation of progesterone production
by (immature) porcine granulosa cells in monolayer sparsely
serum-supplemented or serum-free first-passage cultures (74, 118, 119, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151). The mechanisms underlying such singular and synergistic
actions of estradiol include enhanced low density-lipoprotein
(LDL)-receptor-expression, increased cholesterol uptake and utilization
(152), augmented cytochrome P450scc enzymic
activity (148, 150, 152), increased conversion of pregnenolone to
progesterone, heightened de novo synthesis of cellular
cholesterol via 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
(146), and augmented steroidogenic acute regulatory protein
expression, without evident inhibition of progesterone’s metabolism to
5-dihydroprogesterone (74). These in vitro trophic
actions of estradiol occur over 36–48 h or longer without any
measurable proliferative effects. Rather, inhibition of porcine
granulosa cell proliferation with a noncompetitive (suicide) inhibitor
of polyamine biosynthesis amplifies the daily rate of in
vitro progesterone-expressed biosynthesis per granulosa cell
(153), suggesting an inverse relationship between granulosa cell
proliferation and cytodifferentiation (154).
Estrogen’s modulation of progesterone biosynthesis by pig granulosa cells is bipotential in a time-dependent sense (74, 75, 118, 119, 138, 139, 140). Short-term (2–18 h) exposure to estradiol consistently inhibits progesterone biosynthesis, apparently by directly antagonizing pregnenolone’s conversion to progesterone via 3ß-hydroxysteroid dehydrogenase (74, 153). This direct action is mimicked by a nonmetabolizable estrogen, moxestrol, and is not antagonized by the antiestrogen, keoxifene (144). Since inhibition can be reproduced in broken cell (microsomal) preparations, it is likely that direct steric inhibition of enzyme catalysis occurs. The mechanisms underlying delayed (34–36 h) escape from this acute inhibition are not yet known (74, 118, 119, 138, 139). However, an analogously rapid inhibition of steroidogenesis by estrogen is described in pig theca cells (155, 156, 157). Such steroidogenic autoinhibitory actions of estrogen may be relevant as the preovulatory LH surge unfolds, when intrafollicular 17ß-estradiol concentrations approach or exceed 1 µg/ml (74, 158), thereby (inferentially) limiting a premature elevation of progesterone concentrations before corpus luteum formation and/or thereby restraining the preovulatory (theca cell-derived) androgen surge.
E. Possible interactions of estrogen with other putative ovarian
regulators
Whereas estrogen is the primary focus above, its critical
interactions with other known and putative intrafollicular regulators
will be essential to unravel the complete understanding of the ovarian
follicular development in the pig and other species (130, 159, 160).
For example, a reciprocal relationship emerges between growth (cell
proliferation) and cytodifferentiation in response to certain
modulators, e.g., endothelin-1 (129, 161, 162), polyamines
(153), and TNF (126). This observation, while not universal (IGF-I
tends to promote both granulosa cell proliferation and differentiation)
illustrates the potential complexity inherent in in vivo
interpretations of estrogen’s acting in combination with other potent
regulators of folliculogenesis, such as the intraovarian IGF-I system
(145). Estrogen-growth factor interactions are of particular interest,
when folliculogenesis is viewed as a continuum of changing cellular
commitment to replication vs. cytodifferentiation.
How estrogen interacts, if at all, with specific cell-surface adhesive proteins, such as connexin, laminin, or integrin, in their presumptive maintenance of commitment to granulosa cell lineage (120, 122, 163) is likely of significance in the maturing follicle, as well as possibly in the earliest stages of preantral follicle formation (164, 165).
Estrogen’s presumptive modulation of the calcium-calmodulin, C-kinase, and A-kinase effector pathways in granulosa and theca cells will also require further study (125, 140, 141, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184). In this light, it will be critical to establish whether, when, and how estrogen participates in rescuing follicles from early or incipient atresia (131, 185). Differential hybridization of pig granulosa and theca cell mRNAs collected in untreated vs. estrogen-treated conditions, as accomplished for FSH recently (186), could be one step toward addressing this difficult issue. In vitro whole-follicle cultures also may be useful in this context (187).
Given the pivotal role inferred to date for the IGF-I and IGF-II (and their associated binding proteins) systems in follicle development, exactly how estrogen impacts the multifaceted actions of IGF-I and IGF-II in the pig will require further clarification (135, 149, 150, 151, 152, 181, 182, 188, 189, 190, 191). In addition, estradiol’s interactions with FSH receptor-mediated cytodifferentiative actions on granulosa cells should be evaluated further, given the unequivocally central role of this gonadotropin in the follicle development in this and other species (190).
Further knowledge of how estradiol modulates intrafollicular production and actions of the multiple cytokines and activin/inhibin glycoproteins (and the structurally unrelated follistatin) may also help better elucidate estrogen’s role in folliculogenesis in the pig. Although of interest for other investigative purposes, the recent development of pig granulosa cell clonal lines is not likely to be so rewarding in dissecting the foregoing broader physiological issues (192). Lastly, we suggest that the potent luteotropic effects of estrogen in the pig, albeit well supported by available data in this species, require better mechanistic understanding (193, 194, 195, 196, 197). The foregoing queries will eventually also need to be addressed with respect to the nature and mechanisms of estrogen’s modulation of theca and/or interstitial cell function in the ovary. The latter proposition assumes our thesis that folliculogenesis progresses under the dual orchestration of the intrafollicular and perifollicular milieus, which likely jointly coordinate follicle development.
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IV. Lessons Learned from the Estrogen-Resistant Model-Estrogen Receptor Null Mutants (ERKOs) |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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gene. The patient was tall and
normally masculinized. However, estrogens proved to be of import for
bone maturation and for mineralization. Although no examples of
estrogen resistance exist in the primate/human female, the recent
flurry of activity in this area might indicate that such an occurrence
is not an impossibility. Clearly, once such an individual is
identified, significant lessons could be learned with respect to the
relative importance of estrogens to ovarian follicular maturation in
the human female.
Short of such primate/human models, note must be made of the recent
works reporting the generation null mutants for the ER (199, 200, 201, 202, 203, 204, 205)
and ERß (206) genes by way of gene targeting (homologous
recombination) technologies (207). Compound null mutants were also
generated. A detailed account of this work was recently offered (207).
Therefore, only a brief discussion of the relevant points will appear
here.
ER-/- mice survived to adulthood featuring a normal gross
phenotype. However, null-mutant female mice proved infertile,
displaying hypoplastic uteri and hyperemic ovaries with no detectable
corpora lutea (205). Histological sections of ovaries from homozygous
null mutants revealed no defects in germ cell formation or migration.
Further, developmental progression through the primordial, primary, and
antral follicle stages appeared normal. However, functional maturation
to preovulatory follicles was arrested resulting in atresia or in
"anovulatory follicles," which in many cases formed large,
hemorrhagic cysts containing few, if any, granulosa cells. Stated
differently, ovarian follicles of adult ER-/- mice may progress to
a "Graafian" state albeit in the face of abnormal stratification of
granulosa cells, some areas of the follicle being surrounded by
multiple layers of cells, other regions featuring a single layer of
squamous-appearing cells (205, 208) (Fig. 5). Estrogen actions, such as the
attenuation of apoptosis or the amplification of the LH receptor
content in granulosa cells of antral follicles, appear preserved,
probably due to mediation by the ERß receptor.
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-/- mice is probably attributable, in part,
to chronic exposure to abnormally high levels of LH (207). Support for
this hypothesis can be drawn from several studies. Prolonged treatment
with antiestrogens, which possess the ability to cross the blood brain
barrier and therefore produce chronically elevated levels of LH, have
produced a similar ovarian phenotype (209, 210, 211). Similarly, targeted
transgenic overexpression of the LHß subunit gene resulted in
increased serum levels of LH and an identical ovarian phenotype (212, 213). In addition, prolonged treatment of the ER-/- mice with a
GnRH antagonist reduced serum LH levels and prevented the cystic
ovarian phenotype (214). However, the role of LH in the evolution of the above phenotype can be questioned, given two other null mutant models in which the hemorrhagic cyst phenotype is absent (in the face of chronically elevated LH levels). For example, the null mutant for the FSHß-subunit gene (215), otherwise replete with LH, proves the importance of FSH in follicular cyst formation. Similarly, the null mutant for the P450- aromatase gene (216), characterized by high circulating levels of both LH and FSH, but absent estradiol, points out a role for estradiol in the genesis of the (hemorrhagic) cyst phenotype.
To test whether ER is required for ovulation
and corpus luteum formation, gonadotropins were used to superovulate
immature ER-/- mice and wild-type siblings
(208, 214). Gonadotropin therapy resulted in ovulation in both
ER-/- and wild-type mice. However, fewer gonadotropin-treated null
mutants ovulated. In addition, ER-/- mice yielded significantly
less oocytes. Surprisingly, ovulated/ruptured ovarian follicles of null
mutants developed into corpora lutea of apparent normal morphology
boasting 3-fold the concentrations of serum progesterone as compared
with controls. However, adult ER-/- mice
could not be induced to ovulate, probably due to the elevated
circulating levels of LH and the development of hemorrhagic ovarian
cysts (200).
Null mutants for the ERß gene survive to adulthood and exhibit a
phenotype distinct from that of their ER-/- counterparts. Unlike
ER-/- mice, the ERß-/- female mouse proved fertile, albeit
with apparently less frequent ovulation (206). Consequently, fewer
corpora lutea were apparent, the litter size being smaller than in
wild-type mice. Not unlike the ER-/- mice,
gonadotropin-treated ERß-/- mice responded with fewer oocytes
released than their wild-type counterparts (206).
Histological analysis of ovaries of ERß-/- mice was mostly normal, except for an increased number of early atretic follicles and the sparse presence of corpora lutea, suggesting arrested folliculogenesis. Superovulation disclosed a reduced ovulatory capacity of ERß-/- mice as compared with their wild-type counterparts. The histology of the ovaries from superovulated ERß-/- mice revealed the presence of numerous unruptured preovulatory follicles, indicating deficiency in the response to the gonadotropin surge (hCG). The relative deficit of spontaneous ovulation in the ERß-/- mice may be due to diminished up-regulation of ovarian progesterone receptor (PR) levels by gonadotropins, or an alteration in gonadotropin synthesis or secretion (207, 217). Dupont et al. (218) described a similar phenotype for ERß null mutant, but their data reveal that only half of the superovulated female ERß null mutants ovulate, in contrast to the study of Krege et al. (206) who reported that 80% of the superovulated animals ovulated.
Recently, mice lacking both ER and ERß were generated (219).
ERß-/- mice exhibited normal reproductive tract
development but proved infertile. Ovaries of adult ERß-/-
mice exhibited follicle transdifferentiation to structures
resembling seminiferous tubules of the testis, including
Sertoli-like cells expressing Mullerian inhibiting substance, sulfated
glycopropein-2 and Sox9 (biochemical markers of Sertoli cell
differentiation). In some follicles, a recognizable but degenerating
oocyte was present, whereas others featured no evidence of germ cells.
These findings indicate that both receptors are required for the
maintenance of germ and somatic cells in the postnatal ovary.
Interestingly, serum LH levels were higher than those observed in
ER-/- mice, suggesting that both ERs are required for
estradiol-mediated regulation of LH secretion (219).
In summary, these findings suggest that prenatal reproductive tract
development in the mouse can occur in the absence of ER or ERß, an
observation consistent with traditional theory. However, reproductive
function is undoubtedly affected. Since, ovarian folliculogenesis,
ovulation, and corpus luteum formation can occur in the absence of
ER or ERß, albeit less effectively than in
wild-type mice, it might be suggested that neither one of the known ERs
is essential, but that each may play a facilitatory role in ovarian
follicular development and maturation.
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V. Lessons Learned from the Targeted Disruption of the CYP-19 (Aromatase) Gene-Aromatase Null Mutant (ArKO) |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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VI. The Primate/Human Ovary as a Site of Estrogen Reception |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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A. Molecular probing (Table 3)
Billiar et al. (232), relying on Northern blot
analysis, made use of a cDNA probe corresponding to the 1.8-kb open
reading frame of the ER expressed by the MCF-7 human breast cancer
cell line (232). A 7-kb ER mRNA species was detected when using a
poly(A)+-enriched mRNA fraction representing
whole ovarian material from two animals. Unfortunately, the use of
whole ovarian material all but precluded the localization of ER mRNA
to discrete follicular or perifollicular components. Consequently, one
cannot rule out that the positive signal reflects germinal epithelial
contamination.
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and
which crossed two introns. The identity of the resultant products was
confirmed by sizing and Southern blot analysis. Using this approach,
the authors were able to demonstrate ER transcripts in whole ovarian
material. Similarly, both oocytes and COCs were found to be
ER-positive. However, when isolated granulosa cells or cumulus
masses devoid of oocytes were examined, both were judged to be
ER-negative. As such, these data suggest that the human oocyte, but
not the human cumulus granulosa cell, is a site of estrogen reception.
These observations do not exclude the possibility that noncumulus
(i.e., membranous) granulosa cells may also be
ER-positive. The interpretation of the oocyte/COC data must also
take into account the fact that the oocytes in question failed to
fertilize in vitro and thus may not be representative. The
authors do state, however, that all oocytes were judged to be mature
(as determined by the breakdown of the germinal vesicle and the
extrusion of the first polar body within 48 h after aspiration)
and that the apparent failure to fertilize was most likely due to a
severe male factor. It also remains possible that the ER positivity
may be the result of contamination of the COC samples with nonovarian
ER-positive material as a result of transvaginal ovarian follicular
aspiration (e.g., vaginal mucosa or ovarian germinal
epithelium).
More recent reports by Hurst et al. (234, 235), using RT-PCR
technology applied to highly luteinized human "granulosa cell"
mRNA, provide data in apparent conflict with those reported by Wu
et al. (233). To identify the possibility of genomic DNA
contamination, the oligonucleotide primers were so designed as to
correspond to base pairs 570–852 in the B and C domains of the human
ER cDNA, a stretch of nucleotides known to span intron 1. Southern
blotting of the amplified products with a
32P-labeled ER mRNA probe confirmed the
existence of ER transcripts (Fig. 6). The
data suggest the existence of ER transcripts in follicular aspirates
consisting largely of luteinized granulosa cells. However, since the
granulosa cells studied were obtained via transvaginal needle
aspiration of ovarian follicles in the course of oocyte retrieval in
conjunction with in vitro fertilization procedures, the
signal detected may correspond to cells other than granulosa cells
known to be present in follicular aspirates (e.g.,
representatives of the white blood cells series). Consequently, there
exists the strong possibility of contamination by vaginal tissues or by
ovarian germinal epithelium, which may be ER-positive, thereby
reducing the level of confidence in the conclusions from this study.
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mRNA has also
been applied to rhesus monkey ovaries (236). Before collection of
tissue, each animal was stimulated with 60 IU purified human urinary
FSH (Metrodin, Serono, Norwell, MA) during menstrual cycle days
1–6 followed by 60 IU of a purified mixture of human urinary FSH and
LH (Pergonal, Serono) for days 7–9. Animals were divided into
groups, one of which received an ovulatory stimulus of hCG 1000 IU im
on day 10. Oocytes and granulosa cells were collected by follicular
aspiration of follicles at laparotomy. A Percoll gradient was used to
enrich the granulosa cell sample after oocyte removal. Samples were
dissected to yield germinal epithelium-enriched material. Corpora lutea
were obtained by luteectomy from early, mid, and late luteal
phase animals (as judged by days post-LH surge). Total RNA was isolated
from uterine myometrium (designed to serve as a positive control),
spleen (designed to serve as a negative control), whole ovary, germinal
(surface) epithelium- enriched cortical and medullary compartments
of the ovary, granulosa cells of preovulatory follicles before and
after an ovulatory stimulus, and corpora lutea from the early, mid, and
late luteal phase of the menstrual cycle. Primers were chosen to
bracket a 360-bp sequence corresponding to the human ER
steroid-binding domain and to span an area containing an intron to
identify the possibility of genomic contamination. Amplified products
of the expected size for an ER were obtained from myometrial RNA and no
product was obtained from spleen. ER mRNA was detected in whole
ovary and in germinal epithelium-enriched cortical compartments, with a
barely visible product occasionally observed in medullary compartments
of the ovary. ER mRNA was not detected in any corpora lutea,
throughout the luteal phase or in granulosa cells obtained by
follicular aspiration before or after an ovulatory stimulus. Sequence
analysis of the ER product revealed 99% homology to the cDNA for
the hormone-binding region of human ER. The apparent absence of
ER mRNA in various ovarian compartments was taken to suggest a
lesser, if any, role for estrogen in the primate ovarian life cycle. In
agreement with immunohistochemical studies (56), no ER mRNA was
detected in ovarian tissues other than the germinal epithelium. In
fact, although whole ovarian material proved ER mRNA positive,
specimens devoid of germinal epithelium proved ER mRNA negative. The
intriguing finding of the possible presence of medullary ovarian ER
transcripts remains to be explained. It is possible that this signal
originates in oocytes or migratory lymphocytes assuming the stromal
cells themselves are ER negative.
Revelli et al. (237) also undertook to evaluate the
expression of ER transcripts in the ovaries of 25 healthy eumenorrheic
women. Ovarian biopsies were taken in different phases of the menstrual
cycle during laparotomy or operative laparoscopy performed for
extraovarian benign diseases. Using in situ hybridization
targeted at ER transcripts, Revelli and associates documented ER
transcripts in 17.1% of primordial follicles. The proportions of
preantral and antral follicles positive for ER transcripts were 30.7
and 37.5%, respectively. No more than 25% of theca cells proved ER
transcript-positive. Active corpora lutea stained positive for ER
transcripts in 50% of the cases. Corpora albicantes always stained
negative. In all subjects, the stroma surrounding both follicles and
corpora lutea contained several fibroblast-like cells that stained
positive for ER transcripts. Oocytes and blood vessels stained
negative in all cases.
Brandenberger et al. (238) compared the expression profiles
of ER and of ERß transcripts in the midgestational human fetus by
semiquantitative RT-PCR. ER was detected in the human fetal ovary.
However, the relative amounts of ER transcripts in the human fetal
ovary were substantially reduced as compared with the levels observed
in the uterus. In contrast, significant amounts of ERß transcripts
were present in fetal ovaries. In relative terms, ERß transcripts
were far more prominent than their ER
counterparts. The presence of ERß transcripts in ovarian granulosa
cells of adult women was also convincingly demonstrated (239). Similar
amounts of ER and ERß mRNA were reported for normal ovaries in all
age groups from 33 to 75 yr (59). Luteinized granulosa cells also
expressed a significant level of ERß mRNA (59).
Note is also made of the contribution of Pau et al. (240),
who were able to observe (by means of RT-PCR and in situ
hybridization) weak to moderate signals of ERß and strong signals of
ER mRNA within the granulosa layer of primate ovarian follicles.
Similarly, whole ovarian mRNA yielded appropriate amplicons
corresponding to ER and ERß after RT-PCR.
Pelletier et al. (57), in an effort to clarify the expression of ERß in the reproductive organs of primates, performed in situ hybridization studies in ovaries of adult female Cynomolgus monkeys (Macaca fascicularis). ERß mRNA localized to granulosa cells of follicles at different stages of development, including small growing and secondary (antral) follicles. Primordial follicles, however, were devoid of ERß expression. The theca interna cells were also strongly labeled. It was not possible to identify any preovulatory follicle. The corpora lutea appeared to be a site of ERß gene expression, whereas interstitial cells were consistently negative. A strong autoradiographic reaction obtained for the ovarian capsule, i.e., the surface epithelium and the stroma cells.
Mention must also be made of studies on the regulation of ER and
ERß transcripts in the superovulated primate follicle (241). ER
transcripts did not change whereas ERß transcripts decreased 12–36 h
after the ovulatory stimulus. Steroid ablation reduced ER
transcripts 12 h after hCG, an effect partially reversible by
progestin replacement. ERß transcripts were unaffected by steroids.
These data demonstrate hCG-initiated, steroid-dependent (ER) and
-independent (ERß) expression of receptor transcripts in primate
granulosa cells during the periovulatory interval. Differences in
patterns of expression may relate to the possible diverse roles of
steroid hormones in periovulatory events.
Recent in vitro studies on ER and ERß gene expression
in human granulosa-lutein cells have improved the understanding of the
hormonal regulation of ERs in the ovary (242). Although transcripts and
protein are identified for both ER and ERß in human granulosa cell
cultures, ER is expressed at a lower level than ERß. Basal
expression studies indicated that ER mRNA levels remained unchanged,
whereas ERß mRNA levels increased with time in culture. Treatment
with hCG significantly attenuated the expression of both receptors.
Similarly, treatment with GnRH led to inhibition of the ER and ERß
transcripts and proteins. These results suggest that ERß more than
ER may play a dynamic role in regulating corpus luteum formation in
the human.
It has been observed that both human ER genes give rise to a
significant number of mRNA isoforms, which, in turn, exhibit
differential expression among the tissues in which they reside (41, 243). In human ovaries, the C and F mRNA isoforms of the ER gene
appear to be the major forms detected among six human mRNA isoforms,
A–F. Moore et al. (41), in evaluating the expression of the
five mRNA isoforms for the human (h) ERß gene (hERß 1–5), was able
to identify hERß-1 and -2 as most abundant in the normal ovary,
whereas hERß-4 could not be detected (41). In addition, a novel human
estrogen receptor ß isoform, ERßcx, has been identified: one that
is truncated at the C-terminal region but possesses an extra 26 amino
acids due to alternative splicing. The ERßcx transcript is expressed
in the adult human ovary (244). Interestingly, ERßcx displays no
ligand binding ability and fails to form any shifted complex in gel
shift assays. Moreover, in a transient expression assay, ERßcx shows
no ligand-dependent transactivation ability of a basal promoter and
also cannot interact with a cofactor, TIF1, in the presence or
absence of 17ß-estradiol. ERßcx preferentially forms a heterodimer
with ER rather than with ERß, inhibiting DNA binding by ER.
Thus, ERßcx may potentially inhibit ER-mediated estrogen action.
In summary, the nonhuman primate ovary is a site of expression of ER
transcripts, mainly at the level of the germinal epithelium (236). The
granulosa cell layer may also be a site of ER gene expression (240).
The adult human ovary, in turn, may express ER transcripts at the
level of the oocyte (233) and the luteinized granulosa cell (234) and,
as recently documented, the germinal epithelium (60). Revelli et
al. (237), however, failed to document ER transcripts at the
level of the human oocyte; instead, they reported ER transcript
positivity in a portion of follicular granulosa cells and in a minority
of theca cells. The status of the oocyte notwithstanding, it would
appear that the adult human ovary is a site of ER expression. ER
transcript positivity was also noted in 50% of active corpora lutea
(237). The fetal human ovary is a site of ER and ERß gene
expression wherein the ERß transcripts are more abundant (238).
Recent probing establishes the presence of ERß transcripts in the
adult human ovary at the level of the granulosa cell (59, 239) and the
germinal epithelium (60). In a recent study, both ER and ERß
transcripts colocalized to the human corpus luteum (245). In the
nonprimate ovary, ERß transcripts are consistently expressed in
granulosa cells of growing follicles as well as in the theca cell layer
and germinal epithelium (57). Given the recent discovery of the
inhibitory version of an ER, i.e., ERßcx (244), it is
reasonable to presume that the net estrogenic impact at the level of
the human ovary may be determined by the relative expression and
interaction of the various ER subtypes.
B. Immunohistochemical studies (Table 4)
The first reported attempt to identify ERs in the primate ovary
was performed by Hild-Petito et al. (246) using adult rhesus
(Macaca mulatta) and cynomolgus (Macaca
fascicularis) monkeys. Ovaries were collected from adult animals
in the early, mid, and late follicular phase as well as in the luteal
phase of the menstrual cycle (n = 3–6 ovaries per stage).
Specific monoclonal antibodies, directed against the human ER (H222
and D75), were employed (247, 248). Surprisingly, immunoreactive ER was
only localized to the ovarian germinal epithelium, an observation
applicable to all stages of the cycle (Fig. 7). Indeed, all the other structures in
the ovary were immunonegative, regardless of the phase of menstrual
cycle. In contrast, progesterone receptors (PRs) were readily
demonstrated using similar techniques in many of the (ER-negative)
ovarian tissues studied. As such, these findings lend further credence
to the experimental conclusions by arguing against the possibility that
the apparent paucity of ER reflects technical shortcomings.
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A similar immunohistochemical study was performed by Iwai et
al. (249) using human ovarian material collected at the time of
laparotomy. Subjects studied were in various stages (follicular and
luteal) of the ovarian cycle. Immunohistochemical staining involved the
use of a commercially available hER-directed monoclonal antibody
(H222). For the majority of the ovarian samples studied, the results
were in agreement with those reported by Hild-Petito et al.
(246) in that most tissues examined were ER negative. For
example, granulosa cells of primordial and preantral follicles as well
as stroma and theca cells from all cycle phases were ER negative. In
contrast, midfollicular phase antral follicles contained many granulosa
cells, which stained weakly to moderately positive for ER. Moreover, by
the late follicular phase (pre-LH surge), the dominant preovulatory
follicle contained granulosa cells, which stained intensely positive
for ER. Interestingly, two dominant follicles taken from women at the
time of the LH surge (LH = 73.8 and 63.4 mIU/ml, respectively) and
judged to be immediately preovulatory were only faintly ER positive.
All luteal phase cells were ER negative including newly formed corpora
lutea secured on menstrual days 15 and 16. When nondominant follicles
were examined, only faint ER immunoreactivity of granulosa cells was
noted during the preovulatory period. However, when examined at the
time of the LH surge, granulosa cells of nondominant follicles were
judged intensely ER positive. Similarly, granulosa cells taken from
nondominant follicles of corpora lutea-bearing ovaries were only
faintly ER positive. All atretic follicles at all stages of the ovarian
cycle were ER negative. Interestingly, however, the authors do not
comment on the status of the germinal epithelium. Of some note is the
apparent rapid disappearance of ER positivity after the LH surge. This
phenomenon may correspond to the previously documented postovulatory
decrease in the ER content of rat (16, 26, 66, 67) and rabbit (250)
granulosa cells in response to high levels of LH.
In assessing the validity of this study (249), note must be made of the
fact that the fidelity of the technique was confirmed by demonstrating
ER staining of control tissues (samples of endometrium and commercially
supplied ER-positive tissues). However, negative controls were not
used. It is unclear whether the apparent differences between the
preceding two studies, in which the same ER
antibody (H222) was used, reflect species specificity or technical
differences. Therefore, at the least, early follicular development
would seem to occur independent of classical ER-mediated mechanisms.
A subsequent study yielded similar results (251). As part of an investigation of the immunohistochemical localization of the androgen receptor (AR) in the human ovary throughout the menstrual cycle, ovarian samples were also stained for ER and PRs. Using the same methodology as in the previous study, the authors were able to demonstrate immunoreactive ER in granulosa cells of dominant follicles. Here again, a decrease was noted in the degree of positive staining of dominant follicles after the LH surge. Importantly, however, all primordial and primary follicles proved ER negative by this technique as were all luteal and stromal tissues. The authors do not comment on the status of the germinal epithelium or of nondominant follicular material.
That same year Billiar et al. (232) reported on their
studies using baboon (Papio anubis) ovaries obtained from
five nonpregnant adult animals in the mid or late follicular phase of
the menstrual cycle. The methodology employed to detect ER was similar
to that used by Hild-Petito et al. (246), i.e.,
immunohistochemical staining with ER-directed monoclonal antibodies
D75 and H222. Immunohistochemical staining of baboon ovaries revealed
ER positivity for nuclei of approximately 30–40% of granulosa cells
from ostensibly healthy antral follicles. In contrast, presumptively
healthy preantral and atretic graafian follicles exhibited relatively
low levels of positive labeling. Stromal and interstitial cells were
uniformly ER negative. However, rare thecal nuclei were ER positive.
By 1993, Hutz et al. (252) reported on an autoradiographic
and immunohistochemical study designed to detect ER in the rhesus
monkey (Macaca mulatta). Here, six animals were studied of
which three were normally cycling and in the luteal phase as judged by
the presence of a corpus luteum. For liquid emulsion autoradiography,
sections were incubated in the presence of 11.4
pM of
[2,4,6,7-3H(N)]-17ß- estradiol. The
authors report significant binding to the corpus luteum as well as less
intense binding to an antral follicle. However, the latter observation
may be compromised by the fact that similar intensity did not appear to
differ from that observed in nonspecific controls (i.e.,
section incubated with unlabeled DES). Overall, signals were considered
saturable and were inhibited by coincubation with unlabeled estrogen
agonists or antagonists. Immunohistochemical staining was carried out
with the human ER-directed monoclonal antibody H222 at a
concentration of 0.5–2.0 µg/ml. Here, however, incubation with a
second goat antirat IgG antibody and a peroxidase-amplifying system was
employed with an aim to increase signal intensity. The authors report
significant positivity for the germinal epithelium as well as for the
granulosa cells of antral follicles, interstitial cells, and
theca-lutein cells. The use of positive or negative control tissues was
not reported.
More recently, Suzuki et al. (253) set out to correlate the
expression of immunoreactive ER, PRs, and ARs with the expression of
steroidogenic enzymes in the human ovary. Fifty specimens of apparently
normal human ovaries from women of reproductive age were removed in the
course of surgery for uterine or cervical carcinoma. The phase of the
menstrual cycle was determined by a combined classification system
comprised of histological endometrial dating, serum 17ß-estradiol,
and serum progesterone concentration. Using human ER-directed
monoclonal antibody ER1D5, positive immunoreactivity was observed only
in granulosa cells from aromatase-positive antral or preovulatory
follicles (n = 2). Importantly, the ability of this antibody to
recognize the ERß variant could not be established at the time and
remains uncertain at the time of this writing. Subject to these
limitations, primordial, primary, and preantral follicles as well as
all stromal/thecal tissue, corpora lutea, and degenerating follicles
were negative.
Revelli et al. (237) have undertaken to analyze the
expression of immunoreactive ERs in the ovary of 25 healthy
eumenorrheic women. Specific monoclonal antibodies, directed against
the human ER, were employed in the course of the immunohistochemical
evaluation. Granulosa cells stained positively for ER. The proportions
of preantral and antral follicles with ER-positive granulosa cells were
at 23.1 and 37.5%, respectively. For theca cells, no more than 25%
stained positive for ER. Active corpora lutea stained positive for ER
in 50% of cases. Corpora albicantes always stained negative. In all
subjects, the stroma surrounding both follicles and corpora lutea
contained several fibroblast-like cells that stained positive for ER.
Oocytes and blood vessels stained negative in all cases.
The identification of a second intracellular ER in 1996 (29), ERß, has stimulated interest in the role of this newly recognized receptor in ovarian physiology. In rodents, after all, several studies have suggested that the ERß transcripts predominate in the ovary (61, 64). Moreover, granulosa cells proved to be the major cellular compartment that is home to ERß transcripts within the ovary (61, 73).
Despite these striking findings on the expression of ERß in the rat
ovary, only few data are available on the pattern of expression of this
ER subtype in the primate ovary. Taylor and Al-Azzawi (254) may have
provided the first relevant results on the expression of the ER and
ERß proteins in normal adult human ovaries. ERß localized to cell
nuclei of multiple ovarian cell types, including granulosa cells of
small, medium, and large follicles, theca cells, and corpora lutea.
ER, in turn, was only weakly expressed in the nuclei of granulosa
cells, but was not expressed in the theca cells or in corpora lutea.
Duffy et al.(255), performing Western Blot analysis to
evaluate the expression of the ER and ERß proteins in the Rhesus
monkey corpus luteum, observed that ERß displayed peak of
expression at the mid-late luteal phase and declining levels by the
late luteal phase. Unlike ERß, the inconsistent detection of ER
protein by Western blot suggests that the levels of ER in the
primate corpus luteum are very low.
Saunders et al. (58), studying immunoreactivity of both
nuclear ERs in the ovaries of human and monkeys (Callthrix
jacchus) (Fig. 8), used three
antibodies directed against different peptide segments of the human
ERß protein. The specificity of these antibodies was validated by
performing Western blot analysis. Importantly, the antibodies in
question were able to bind recombinant human ERß, but were unable to
do the same for human ER. The pattern of expression of both
receptors in the marmoset was mirrored by that of the human ovaries.
Immunoreactive ER was noted in the nuclei of granulosa cells of
medium and large follicles. Small follicles (one or two layers of
granulosa cell) proved negative. Given medium-sized follicles, ER
protein expression appeared weaker than noted for ERß. In contrast, a
clearly defined layer of granulosa cells adjacent to the basal lamina
proved ER positive when examined in antral
follicles. Unlike ER, immunoreactive ERß was identified in nuclei
of granulosa cells as early as in small follicles (one or two layers of
granulosa cell) as well as in medium-sized and large antral follicles.
Not unlike ER, ERß revealed a striking pattern of expression
within antral follicles wherein the protein appeared to be mainly
expressed close to the basal lamina. Expression for both receptors was
noted in theca cells surrounding preantral and antral follicles as well
as in the germline epithelium.
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was noted for
the theca, interstitial gland, and the ovarian surface epithelium.
Recent in vitro studies on ER and ERß gene expression
in human granulosa-lutein cells have improved the understanding of the
hormonal regulation of ERs in the human ovary (242). Using Western blot
analysis, both receptors were identified in cultured human granulosa
cells. Treatment of the latter with hCG and GnRH led to down-regulation
of the ER and ERß proteins. These results suggest that ERß and
ER may be involved in the regulation of corpus luteum formation in
the human (242).
In summary, then, the nonhuman primate ovary is a site of expression of
immunoreactive ER at the level of the germinal epithelium (246, 252)
and the granulosa cells of healthy antral follicles (232, 252). Both ER
subtypes appear to be expressed at the level of granulosa cell, theca
cell, and germinal epithelium in nonhuman primates. However, ER
immunoreactivity, unlike ERß, is restricted to granulosa cells of
antral follicles (58). The human ovary, in turn, may be a site of
expression of immunoreactive ER at the level of the healthy antral
granulosa cell and particularly the preovulatory granulosa cell from
"dominant" follicles (237, 249, 252, 253). Granulosa cells of
nondominant follicles may also be ER-positive at the time of the LH
surge (249, 252). As for nonhuman primates, immunoreactive ER and
ERß were identified in granulosa cells, theca cells, and the ovarian
surface epithelium (58, 254, 256). Active corpora lutea stain positive
for both ERs (254). The human oocyte was noted as negative (237).
Clearly, additional immunohistochemical studies are required using
specific antisera for both ERs to establish the relative preponderance
of ER and ERß receptor proteins in the ovary. Careful delineation
of the cross-reactivity of the antibodies employed appears essential.
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VII. Is an Estrogen-Free (or at Least Poor) Intrafollicular Environment Compatible with Follicular Development, Ovulation, and Corpus Luteum Formation? |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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B. Lessons learned from the 17-hydroxylase/17–20 lyase
deficiency
To further elucidate the role of estrogen in primate/human
folliculogenesis, use can also be made of "experiments of nature"
i.e., women afflicted with inborn errors of metabolism that
prevent the biosynthesis of estrogens. Of special interest is the
example of the 17-hydroxylase/17–20 lyase deficiency form of
congenital adrenal hyperplasia (257), a defect associated with marked
impairment of glucocorticoid, androgen, and estrogen biosynthesis.
Although the women in question suffer from hypergonadotropic
hypogonadism and sexual infantilism, early reports noted the presence
of many primary and secondary follicles in ovarian material (258). In
fact, many of the patients displayed bilateral multicystic ovaries at
the time of laparotomy (259, 260, 261). Although substantial follicular
atresia has been noted, follicular development up to and including the
antral stage has been observed. However, preovulatory follicles are not
identified and frequent atretic small graafian follicles are seen
(259).
Of particular relevance to this review is a patient afflicted with
virtually complete 17-hydroxylase/17–20 lyase deficiency who,
despite castrate levels of estrogens, underwent an apparently
successful induction of ovulation associated with progressive
follicular "expansion." Oocyte retrieval, in vitro
fertilization, and early embryonic cleavage followed suit (262). Before
treatment, the patient displayed undetectable plasma 17ß-estradiol
levels (E2 < 20pg/ml). Of concern however, is
the observation that the circulating gonadotropin levels (FSH and LH
levels of 28.5 and 9.7 IU/liter, respectively) were lower than might be
expected in a patient with this condition. Conceivably, this may
represent a partial enzyme deficiency. Vaginal sonography revealed
multiple ovarian follicle-like structures (8–26 mm). Subsequent
treatment was carried out against the backdrop of adrenal suppression
with prednisone designed to ensure blood pressure control as well as to
decrease the circulating levels of adrenal-derived progesterone, the
potentially adverse impact of which on endometrial (and perhaps
follicular) maturation was projected. Controlled ovarian stimulation
entailed GnRH-mediated pituitary down-regulation upon which "pure"
urinary hFSH was superimposed for 14 days at increasing doses of up to
300 IU/day. Ovulation was triggered with hCG (Fig. 9). Follicular development was monitored
by vaginal sonography.
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). Despite the backdrop of
profound hypoestrogenism, sonographic monitoring revealed progressive
follicular "expansion," the largest follicle exceeding 18 mm in
diameter (Fig. 11). Several follicles
were noted to develop. Overall, three apparently antral follicles were
aspirated, each yielding an oocyte surrounded by a corona/cumulus
complex. Two of the three oocytes fertilized and cleaved (albeit
suboptimally) to the seven-cell stage in 60–75 h after aspiration,
only to be arrested thereafter.
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Cultured granulosa cells derived from the index patient were able to
survive, grow, and proliferate in vitro despite the virtual
absence of estrogenic support (264). When compared with granulosa cells
from normal controls, those from the affected patient produced, as
expected, higher levels of progesterone but lower levels of
testosterone, androstenedione, and 17-hydroxyprogesterone. Basal
17ß-estradiol production by the relevant granulosa cells was also
significantly lower relative to controls (0.11 vs. 3.67
pg/105 cells) as was the total content of
estrogenic substances by EBE (0.14 vs. 4
pg/105 cells). Similar differences were noted
after in vitro supplementation of granulosa cells with
exogenous testosterone or androstenedione. Specifically, the addition
of 10-7 M testosterone or
androstenedione led to an approximately 100-fold increase in
17ß-estradiol production by control and affected granulosa cells,
thereby suggesting the presence of significant endogenous aromatase
activity in the face of an extreme hypoestrogenic environment.
In summary, then, markedly reduced to nonexistent intrafollicular and circulating concentrations of 17ß-estradiol are compatible with follicular "expansion," retrievable and fertilizable oocytes, as well as with cleavable and apparently transferable embryos. However, the actual viability of the embryos remains to be demonstrated.
C. Lessons learned from the 3ß-hydroxysteroid dehydrogenase
(3ß-HSD) deficiency
To further examine the role of estrogen in primate/human
folliculogenesis, Zelinski-Wooten et al. subjected a group
of cycling female Rhesus monkeys to ovulation induction with hFSH
(Metrodin, 30 IU im twice daily, days 1–6 beginning with menstruation)
and hFSH + hLH (Pergonal 30 IU im twice daily beginning on day
7), in the absence or presence of the 3ß-HSD inhibitor trilostane
(TRL) given on days 1–8 of the menstrual cycle (265). Follicular
development was monitored by transabdominal sonography, ovulation being
triggered with hCG (Profasi, 1,000 IU im on day 8). Oocytes were
obtained by follicular aspiration 34 h after hCG administration.
As a measure of the utility of TRL, the authors assessed the circulating levels of pregnenolone, the immediate precursor/substrate of 3ß-HSD. The circulating levels of pregnenolone were shown to be substantially, enhanced (66-fold) in TRL-treated primates in keeping with an apparently effective blockade of 3ß-HSD activity. Importantly, treatment with TRL led to a reduction in serum 17ß-estradiol levels to 7% of that of control animals throughout the follicular phase. Despite this dramatic reduction in 17ß-estradiol levels, neither the total number of large antral follicles per animal (17 ± 1 vs. 18 ± 2) nor their size distribution differed significantly from TRL-untreated controls. Furthermore, the overall maturation pattern of collected oocytes (atretic, prophase I, metaphase I, or metaphase II) was not altered by TRL treatment. Note was made, however, of a reduction in the percentage of metaphase II oocytes that were successfully fertilized (15 vs. 65%). Moreover, metaphase I oocytes that required more than 8 h to complete meiosis in vitro failed to fertilize in three of four animals receiving TRL relative to controls (31%). Taken together, these observations suggest that follicular development and the completion of meiosis may be unaffected by the low estrogen levels but that cytoplasmic oocyte maturation and/or function could be unfavorably affected. Indeed, the acquisition of oocyte competence for fertilization may require adequate amounts of intrafollicular steroids.
In summary, then, markedly reduced circulating concentrations of 17ß-estradiol are compatible with seemingly normal follicular "expansion" as well as retrievable oocytes of seemingly normal quantity and maturity. However, fertilization rates appear to be adversely affected. Consideration must also be given to the possibility that TRL-treated animals may display nonspecific side effects relative to the therapeutic agent independent of its 3ß-HSD-inhibitory activity. Clearly, no evidence exists to either support or negate such a possibility. However, complete interpretations of the findings of this study must include consideration of this issue. Consideration must also be given to the realization that low circulating levels of estrogen need not be equated with low intrafollicular concentrations of estrogen. This important qualification was pointed out by Mannaerts and associates (266) in their studies of the immature hypophysectomized rat. Under those circumstances, treatment with recombinant FSH led to significant follicular "expansion" in the face of moderately reduced circulating levels of 17ß-estradiol. However, under these very same circumstances, systemic therapy proved capable of promoting an increase in the intraovarian 17ß-estradiol content, a phenomenon possibly causally related to follicular "growth" and to increased ovarian weight.
D. Lessons learned from aromatase deficiency
In yet another experimental model, the aromatase inhibitor
1,4,6-androstatrien-3,17-dione (ATD) was used as a means to inhibit
estrogen production during gonadotropin-mediated ovarian stimulation
(267). Specifically, cycling female rhesus monkeys (n = 6) were
treated beginning at menses with a regimen of 30 IU hFSH im twice daily
given on days 1–6 followed by 30 IU hFSH and 30 IU hLH for 3 days. On
day 10, hCG (10,000 IU im) was given to induce preovulatory follicular
maturation. In addition, four animals received twice daily oral
(1.0–1.25 g) ATD (Steraloids, Wilton, NH), an aromatase
inhibitor. Follicles were aspirated at laparoscopy 27 h after hCG,
the resultant oocytes being subjected to fertilization in
vitro. The follicular and hormonal response to stimulation as well
as the numbers and function of the oocytes obtained were evaluated.
As expected, animals treated with ATD displayed a drastic reduction in serum 17ß-estradiol levels to 37% of that of controls within 8 h of ATD treatment and to 16% of control by the day of hCG injection. In turn, the circulating levels of androstenedione rose. Despite the drastic reduction in the circulating levels of 17ß-estradiol and the increase in the circulating levels of androgens, the overall number of large antral follicles (16 ± 3 for controls and 20 ± 3 for ATD-treated) and their size distribution (as assessed by ultrasonography) proved comparable for control and ATD-treated animals. Similarly, no difference was noted in the number of oocytes collected or in the proportion of oocytes reinitiating meiosis (MI at the time of collection). In contrast, ATD-treated animals displayed a marked increase (31 vs. 11%) in the proportion of prophase I oocytes. Moreover, ATD-treated oocytes displayed retarded in vivo completion of maturation to MII (4% vs. 26%). Interestingly, the latter retardation was not observed in vitro. Furthermore, two of the four ATD-treated animals yielded oocytes that were morphologically abnormal. Finally, oocytes from ATD-treated animals displayed significantly reduced rates of fertilization (9% vs. 25%) as compared with controls. However, the cleavage rate after successful fertilization was similar for ATD-treated vs. ATD-untreated controls. In vitro cultures of granulosa cells collected at the time of oocyte aspiration revealed equivalent 24-h progesterone production in treated and control animals.
Overall, these observations suggest that the acute reduction in the circulating levels of 17ß-estradiol during the terminal stage of gonadotropin-induced stimulation had little effect upon follicular recruitment and "expansion." However, an apparent detrimental effect upon gametogenic function may, in fact, exist. In this respect, these observations differ from those made in the aromatase-inhibited rat for which a reduction was noted in the number of follicles that completed maturation and were able to ovulate in vivo (267). In part, however, this latter difference may reflect the timing and duration of aromatase inhibition.
More recently, Selvaraj et al. (268, 269) and Shetty et al. (270) reported on a related experimental paradigm designed to examine the effects of blocking estrogen biosynthesis during the follicular phase on follicular maturation in the adult female bonnet monkey. The experimental design called for the administration of the aromatase inhibitor CGS 16949A by Alzet mini-pump from day 3 of the menstrual cycle. This approach resulted in 53% and 70% reduction in the basal and surge levels of 17ß-estradiol, respectively. However, no obvious effect was noted on follicular maturation, ovulation, and luteal function as assessed by serum hormone profiles as well as by laparotomy. Moreover, the concurrent administration of FSH and an aromatase inhibitor resulted in the suppression of the FSH-induced increase in the circulating levels of 17ß-estradiol (by 100%). Still, no effect was noted on either the number of follicles developed or their size relative to control. Granulosa and theca cells, removed on day 9 of the treatment cycle, proved responsive to gonadotropins under in vitro circumstances, disclosing no evidence to the effect that cellular development and maturation of follicular cells were significantly affected.
It may be important to note, however, that the above mentioned experimental paradigms, not unlike some of the preceding paradigms, represent a hypoestrogenic but by no means an estrogen-free circumstance. Above and beyond this qualification, interpretation of the findings is further confounded by the difficulties in distinguishing between the impact of decreased circulating levels of 17ß-estradiol and those attributable to increased circulating levels of androgens. Last, but not least, consideration must be also given to the possibility that ATD-treated animals may display nonspecific side effects relative to the therapeutic agent independent of its estrogen-inhibitory activity. Although there is little evidence to either support or negate the latter possibility, complete interpretation of the findings of this study must include consideration of this issue. Moreover, whether abrogation of oocyte nuclear maturation after aromatase inhibition in vivo is due to androgen excess and/or a reduction of 17ß-estradiol remains to be determined.
More recently, an extreme example of complete aromatase deficiency in the adult human female was described (271, 272). Specifically, two mutations were detected in the CYP 19 gene in an 18-yr-old 46 XX female afflicted with ambiguous external genitalia, primary amenorrhea, sexual infantilism, and multicystic ovaries. Evaluated at birth for ambiguous external genitalia, the patient was judged to be afflicted with a nonadrenal form of female pseudohermaphroditism. The fetal masculinization could, in retrospect, be ascribed to defective placental conversion of C19 steroids to estrogens leading to increased levels of C19 steroids, which can be converted to testosterone peripherally. At 17 months of age, normal internal female genital structures were identified at laparotomy. The ovaries at that time were grossly and microscopically normal. At puberty, the clitoris had progressively enlarged to 4 x 2 cm, and pubic and axillary hair were Tanner stage III. Basal plasma testosterone levels were elevated at 95 ng/dl, androstenedione levels were 185 ng/dl, and plasma estrone and 17ß-estradiol levels were undetectable. ACTH and dexamethasone tests indicated a nonadrenal source of testosterone and androstenedione. Plasma FSH and LH hormone levels were markedly elevated. Quantification of urinary steroids by gas chromatography-mass spectrometry indicated normal levels of C19 and C21 steroids but very low levels of estrone and 17ß-estradiol. Sonography and magnetic resonance imaging showed multiple bilateral 4 to 6 cm ovarian cysts. Thus, the pubertal failure and the development of multicystic ovaries at the normal age of puberty may be attributed to aromatase deficiency and the consequent elevation of FSH and LH levels. Estrogen treatment resulted in a decrease in plasma gonadotropins, breast development, a prepubertal growth spurt, menarche, and regression of the ovarian cysts.
Most recently, Morishima et al. (273) reported on the
aromatase deficiency in a 28-yr-old 46 XX proband followed since
infancy. This woman, afflicted with ambiguous genitalia at birth, went
on to develop progressive signs of virilization at puberty with no
evidence of estrogen action. In addition, note was made of
hypergonadotropic hypogonadism, multicystic ovaries on pelvic
sonography, and tall stature. Physical examination disclosed Tanner
stage 2 pubic hair, facial comedones and acne, but no breast
development. The bone age proved marginally retarded. The basal
concentrations of plasma testosterone, androstenedione, and
17-hydroxyprogesterone, were elevated whereas plasma 17ß-estradiol
was low. Cyst fluid from the multicystic ovaries displayed a strikingly
abnormal ratio of androstenedione and testosterone to 17ß-estradiol
and estrone. Hormone replacement therapy led to breast development,
menses, resolution of ovarian cysts, and suppression of the elevated
FSH and LH values. Analysis of genomic DNA in transformed lymphoblasts
indicated a single mutation in exon IX of the CYP19 gene associated
with marked reduction of aromatase activity (0.2% of the aromatase
activity of the wild-type enzyme). Around age 13, the patient underwent
abdominal exploration revealing several multiloculated cystic masses in
both ovaries, the largest of which measured 8 x 6 cm. Follicular
cyst fluid displayed very low concentrations of 17ß-estradiol and
estrone (< 1/1500 th of the normal value). Biopsy specimens of the
cystic masses revealed both stages of involuting follicles, excessive
atresia of follicles, increased thickness with more collagen in the
tunica, and dense fibrotic subcortical stroma. The pathology was judged
consistent with multicystic ovaries.
It is clear, once again, that follicular "expansion" is possible in the virtually complete absence of estrogens, presumably under the influence of elevated gonadotropins. Although these findings support the thesis that follicular antrum formation is, in fact, possible in the face of complete estrogen deficiency, the health of such follicles and their oocytes is subject to question. Yet another more recent case, reported by Mullis et al. (274), proved confirmatory. The relevant literature was recently summarized by Bulun (275).
E. Lessons learned from the intensely hypogonadotropic model
A relevant clinical study was performed by Couzinet et
al. (276) involving women with surgically induced
panhypopituitarism (n = 6) or congenital isolated gonadotropin
deficiency (n = 4). All subjects had previously been hospitalized
so as to establish the absence or presence of pulsatile LH secretion.
All subjects displayed no detectable LH pulsation. Plasma LH and FSH
levels proved to be <2.5 mIU/ml, with none of the patients responding
with gonadotropin increments to iv GnRH administration. It is this
population, which was subjected to a cross-over study, performed with
partially purified urinary hFSH + hLH (hMG-Inductor, Searle, Paris,
France) and highly purified urinary hFSH (Fertiline, Searle, 0.8%
endogenous LH activity). A mouse Leydig cell in vitro
bioassay was used to assess the LH activity of the purified hFSH
preparation revealing bioactivity of 0.064 IU/ampoule (i.e.,
LH content 0.09%).
Patients were treated with 225 IU hFSH/day im for 10 days, hCG being
given 24 h after the last hFSH dose. After a 3-month waiting
period the patients were switched to the hMG arm of the study.
Importantly, given treatment with hFSH, plasma LH levels remained
undetectable, as did urinary LH excretion. The mean basal serum
17ß-estradiol level, 11 pg/ml, increased slowly to a day 10
(preovulatory) peak of 207 pg/ml (Fig. 12). The latter was judged
significantly lower when compared with levels reached in both normally
cycling women and in the hypogonadotropic women treated with hMG. Serum
estrone levels remained low, rising from 14 to 82 pg/ml, while
circulating androstenedione and testosterone levels were not
significantly increased (androstenedione rose from 20 to 40 ng/dl;
testosterone rose from 20 to 30 ng/dl). Ovulation was presumed to have
occurred in six of nine women treated with gonadotropins who received
hCG, an inference made from the post-hCG plasma levels of
17ß-estradiol and progesterone.
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Note must also be made of a case study by Shoot et al. (277)
of a patient with congenital isolated gonadotropin deficiency. The
purpose of this phase I study was to assess the pharmacokinetics and
safety of a recombinant hFSH preparation. Conception was not attempted.
The recombinant form of human FSH (recFSH) is, by definition, devoid of
LH activity, and therefore an excellent tool for the investigation of
the effects of truly pure FSH. An in vitro mouse Leydig cell
testosterone bioassay revealed recFSH to possess intrinsic LH
bioactivity of less than 0.025 mIU LH/IU FSH as compared with 2.4 mIU
LH/IU FSH activity for partially purified urinary FSH (278). Baseline
serum studies confirmed the patient’s diagnosis by revealing FSH
levels of 1.2 IU/liter and LH levels of 0.37 IU/liter (Fig. 13). The corresponding circulating
17ß-estradiol levels were 17 pg/ml. Interestingly, baseline
ultrasound examination revealed numerous ovarian follicles <4 mm in
diameter even before ovarian stimulation was begun. Treatment consisted
of the administration of recombinant hFSH (75 IU/day im during the
first week followed by 150 IU/day im during the second week). On
treatment day 13, gonadotropins were discontinued according to protocol
when the leading follicle reached 14 mm in diameter. Six days later,
ultrasound examination revealed multiple ovarian follicles (six of
which were 12–18 mm in diameter), the three leading ones being
subjected to aspiration. hCG (10,000 IU) was then administered
intraperitoneally, and hormonal and follicular sonographic monitoring
was continued for 3 additional weeks. For comparison purposes,
follicular fluid was obtained by aspiration of dominant and nondominant
follicles from 23 normally cycling women.
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). Serum 17ß-estradiol
levels (Fig. 14) peaked at only 64
pg/ml on day 15 (day of maximal follicular size), endometrial thickness
increasing by only 2 mm during treatment (Fig. 13). Not unexpectedly,
no oocytes were obtained at the time of follicular aspiration.
Follicular fluid LH and FSH levels were undetectable. Similarly,
follicular fluid 17ß- estradiol levels were limited to an average
of 1.48 ng/ml (Fig. 14). Such intrafollicular 17ß-estradiol
concentrations contrast sharply with those encountered in unstimulated
controls, i.e., 3.965 ng/ml for large follicles.
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More recently, Shoham et al. (279) reported on two volunteer
women afflicted with hypogonadotropic hypogonadism who underwent
successful follicular "expansion" with recombinant hFSH as part of
an open phase I clinical trial study designed to assess the safety and
pharmacokinetics of a recFSH preparation. Both patients were diagnosed
with isolated hypogonadotropic hypogonadism with FSH and LH levels of
less than 1.5 IU/liter. The treatment protocol and response are
demonstrated in Fig. 9. Both patients refrained from exogenous estrogen
therapy for a period of 30 days before the study. Stimulation with
recombinant hFSH (Organon 32489 with specific activity 15 IU/mg
protein) was continued until a follicle of 14 mm was detected by
transvaginal ultrasound. Since neither patient desired pregnancy, hCG
was not given. Gonadotropins were administered in a stepwise fashion.
Patients received 75 IU hFSH for cycle days 1–7 followed by 150 IU for
days 8–14, followed by 225 IU for days 15–17. One patient received an
additional 4 days of treatment at the 225 IU/day dosage. Final
steady-state FSH levels were 11.8 and 10.1 IU/liter. Mean serum LH
levels for the two patients did not differ from those at baseline (0.11
IU/liter vs. 0.15 IU/liter). Similarly, serum
androstenedione and testosterone levels also did not significantly
increase from baseline. Neither patient displayed detectable serum
levels of progesterone at any point in the stimulation protocol. No
change in endometrial thickness was observed in either patient during
the study period. Each patient demonstrated a small, but finite,
increase in serum 17ß-estradiol levels to 21 and 38 pg/ml,
respectively.
At the conclusion of the stimulation period, the first patient displayed three ovarian follicles as detected by ultrasound ranging from 11–17 mm. The second patient displayed six follicles ranging from 8–17 mm. It must be noted that a single follicle (13 and 15 mm) was detected in each of the patients before stimulation. As such, these observations and ones reported earlier (280) further support the notion that follicular "expansion" can occur in a strikingly hypoestrogenic environment.
Schoot et al. (281) also reported on the ovarian response of intensely hypogonadotropic women to recombinant hFSH. Some of the seven women under study had previously been subjected to hypophysectomy. Others suffered from isolated gonadotropin deficiency or from hypogonadotropic hypogonadism of the Kalmann type. Circulating serum FSH and LH levels in these subjects were 0.25 and 0.06 IU/liter, respectively. The study protocol called for the injection of recombinant hFSH for 3 weeks, the dose being increased from 75, to 150, to 225 IU/day in weekly increments. Whereas FSH concentrations were increased as expected, serum LH concentrations remained unchanged. Interestingly, two of the subjects in question displayed absent follicular development, but a labile rise in the circulating levels of immunoreactive inhibin. These patients were deemed to suffer from ovarian failure or the resistant ovary syndrome, prompting their exclusion from the study protocol. The remaining five subjects displayed circulating immunoreactive inhibin concentrations compatible with levels encountered during the normal late follicular phase. In contrast, circulating 17ß-estradiol levels only increased to a mean of 21 pg/ml the range being 5–57 pg/ml. Importantly, sonographic follicular "expansion" to preovulatory size was clearly apparent despite the limited 17ß-estradiol response. As such, these findings are compatible with the concept that follicular recruitment and "expansion" are possible in the face of minimal circulating estrogen concentrations. Comparable findings were reported by Balasch et al. (282).
Akin to the above studies are several others wherein intact subjects were first pretreated with a GnRH agonist to down-regulate pituitary gonadotropin secretion only to be stimulated thereafter with either (crudely purified) hFSH/hLH, partially purified urinary FSH preparations, or rec FSH (281). Although this paradigm is a theoretically valid hypoestrogenic model and may provide for interesting data, one must keep in mind that current GnRH agonists may not effect complete pituitary gonadotropin suppression (283). Moreover, even modest concentrations of serum LH are compatible with normal ovarian follicular development (283, 284). Clearly, partially purified urinary FSH preparations are not without some residual LH activity.
Another drawback related to the conclusions that may be extracted from the above mentioned hypoestrogenic model is the fact that the human ovary is the site of expression of genes for GnRH and its receptor (285, 286, 287). It is even possible that the human ovary possesses an intrinsic GnRH axis, although a full understanding of the role of this axis within the human ovary is yet to be realized (286). Thus, the administration of pharmacological doses of GnRH agonists (or antagonists) might directly perturb ovarian physiology that cannot be underestimated. Consequently, FSH-primed/GnRH agonist-treated subjects may not constitute a truly representative model of an estrogen-free environment.
GnRH antagonists, in turn, may produce more complete suppression of pituitary gonadotropin levels and may theoretically eliminate the residual LH activity that confounds studies with GnRH agonists (288). While ovulation induction with rechFSH in intact GnRH antagonist-pretreated animals is theoretically an excellent hypoestrogenic model, even this paradigm represents, at best, an estrogen-attenuated circumstance. Here, cynomolgus monkeys were treated with the GnRH antagonist antide (Nal-Lys-GnRH, 3 mg/kg/day) for 20 days beginning in the midluteal phase. After 10 days of treatment with the antagonist, animals were randomized to treatment with either (289) rechFSH (10 IU/day; n = 3), rechFSH (20 IU/day; n = 3), or hFSH +hLH (20 IU/day; n = 3). At the lower dosage, the rechFSH failed to stimulate either follicular growth (as judged by ultrasonography) or 17ß-estradiol production. At the higher dosage of rechFSH, however, multiple follicular development was documented (by ultrasonography), in the face of relatively low circulating levels of 17ß-estradiol (257 ± 53 pg/ml on the day of follicular aspiration). Animals treated with both FSH and LH displayed equivalent numbers and sizes of follicles in the face of significantly higher circulating 17ß-estradiol levels. Follicular fluid 17ß-estradiol levels were consistent with the serum levels. These observations were taken to mean that in this relatively LH-deficient primate model, FSH alone is capable of stimulating ovarian follicular growth despite markedly reduced 17ß-estradiol production.
In a related publication, Zelinski-Wooten et al. (290) evaluated the role of recombinant human FSH with and without recombinant LH after 90 days of GnRH antagonist (Antide) treatment in macaques. Multiple follicular growth required a longer interval after recombinant FSH than recombinant FSH plus recombinant LH, but the total number of follicles per animal did not differ between groups. The circulating levels of 17ß-estradiol were 4-fold lower in recombinant FSH-treated animals as compared with those subjected to combination therapy. In contrast, more oocytes completed meiosis to metaphase II and fertilized after recombinant FSH therapy as compared with combination therapy. Follicular growth and maturation in LH-deficient macaques occurred with FSH alone, thereby confirming the role of FSH in follicular expansion. Interestingly, however, the apparent higher fertilization rates associated with the FSH alone stimulation argues for a possible deleterious effect of LH in this regard, a notion currently under active investigation.
F. Impact of estrogen deficiency on oocytic and early embryonic
development
Several of the studies cited above appear to indicate that an
estrogen-poor or estrogen-free intraovarian environment in the primate
may adversely affect oocyte quality and early embryonic development
(265). These findings could be in keeping with the apparent presence of
ER transcripts in human oocytes (233). In this context, an
experimentally- induced 3ß-HSD deficiency in the primate model
was associated with a marked reduction in the fertilization rate (265).
Likewise, systemically induced aromatase blockade was associated with a
detrimental effect on oocyte function (267). Similar conclusions were
also reached by Couzinet et al. (276) whose findings
suggested that the health and functional capacity of the follicle might
be related to the intrafollicular level of estrogens.
To assess the quality of FSH-primed/estrogen-free follicles, Wang and Greenwald (291) undertook to treat adult hypophysectomized mice with recombinant hFSH (4 µg/day) twice a day for 3 days, beginning 12 days after hypophysectomy. Concurrent with the last FSH injection, hCG was injected to induce ovulation. Animals were mated and killed 1–4 days later. Interestingly, treatment with FSH alone produced large preovulatory follicles. The ovulation rate proved virtually normal for hCG-triggered mice (90% as distinct from 100%). However, successful mating was noted in only 45% of the hCG-triggered mice, fertilization being limited to 56% of the oocytes so generated. The significantly decreased percentage of successful mating is striking. With a 1:1 ratio of mating females and males, the fertilization rate should be about 90% since the ovulation rate is 100% in hCG-triggered groups. Moreover, only 5% of ovulated eggs developed to four-cell stages in vivo by day 3 after hCG triggering. Comparable results were secured in vitro. Indeed, embryos formed consequent to hCG triggering displayed significant meiotic arrest manifested by the observation that only 22% (rather than 80%) of two-cell embryos were converted to blastocysts by 96 h (rather than 72 h) of culture. Eleven percent of one-cell embryos divided to the four-cell stage. Although hCG triggering resulted in a virtually immediate increase in the secretion of progesterone, a post-hCG increase in 17ß-estradiol levels was not noted until day 2. Thus, in hypophysectomized adult mice, FSH alone induces the growth of follicles to preovulatory stages under estrogen-free circumstances. Subsequent ovulation induced by hCG results in limited fertilization and limited development of preimplantation embryos in vivo and in vitro. These abnormalities could be due to the insufficiency of follicular steroids, the immaturity of the oocytes, and the presumably abnormal oviductal environment.
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VIII. The Primate/Human Ovary as a Site of Estrogen Action |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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A particularly elegant model is represented by the immature cynomolgus macaque paradigm. In a series of studies reported by Koering et al. (292), use was made of this hypogonadotropic model to assess the impact of systemic estrogen therapy on ovarian function. Given the presumed quiescence of the hypothalamic-pituitary axis, the effects of estrogen on the ovary could be deemed direct in nature. In an elegant cross-over experimental design in which each animal served as its own control, animals were treated with 20 mg/kg/day of DES for 14 days. One ovary each was removed before and after treatment and numbers of follicles were counted and compared. Examination of the ovaries revealed that after treatment with DES 1) numbers of primordial follicles were unchanged, 2) preantral follicles decreased in number, 3) antral follicle numbers decreased slightly, and 4) numbers of early atretic follicles remained unchanged. This was in sharp contrast to the many well documented studies carried out in hypophysectomized immature rats wherein treatment with DES caused a clear stimulation of follicular development. The finding of no increase in atresia rates suggests that DES treatment arrested the development of antral follicles, thereby diminishing developmentally associated atresia. Similar observations were reported in a follow-up publication (293).
A significant focused line of studies, summarized by Hutz et al. (294) made use of the adult cycling rhesus monkey (Macaca mulatta). In this model, treatment with estrogens led to atresia of the dominant follicle. Specifically, Silastic 17ß-estradiol-containing capsules were inserted subcutaneously for 24–48 h. Treatments that resulted in peripheral 17ß-estradiol plasma levels in excess of 300 pg/ml for greater than 24 h were associated with irreversible atresia of the dominant follicle. Unfortunately, this treatment regimen may also be associated with suppression of the circulating levels of FSH followed by a rise in gonadotropins with removal of the capsules. Thus, it is possible that a central effect (i.e., withdrawal of FSH support) is the cause of the observed follicular atresia.
In an effort to address the limitations of the in vivo paradigm, Hutz and associates (295) assessed whether 17ß- estradiol in amounts similar to those found in monkey follicular fluid directly alters in vitro progesterone accumulation by granulosa cells aspirated from the follicles of cycling rhesus monkeys. Follicular contents were aspirated from three to five animals on each of days 8–13 of the cycle. Granulosa cells were incubated with zero or 2–200 ng/ml of 17ß-estradiol and the cultures maintained for 72 h. These experiments revealed that 17ß-estradiol, at concentrations found in follicular fluid, can inhibit progesterone output by monkey granulosa cells, thereby supporting a direct effect and by inference independence from a central phenomenon.
Further complexity was introduced by the observations of Harlow
et al. (296) whose in vitro studies made use of
the common marmoset monkey. Specifically, granulosa cell cultures from
reproductively suppressed monkeys were cultured for 48 h in the
absence or presence of hFSH, with or without various sex steroids.
Expectedly, treatment with hFSH produced dose-dependent stimulation of
granulosa cell steroidogenesis. As was previously documented in the
rodent, the concurrent addition of testosterone produced substantial
amplification of the hFSH effect. Similar amplification was noted for
5-dihydrotestosterone, a nonaromatizable androgen, but not for
17ß-estradiol, thereby suggesting specific androgenic synergism with
FSH. These studies were taken to mean that androgen may play a local
role in the regulation of FSH-stimulated granulosa cell function during
follicular development in primates. Moreover, these findings suggested
that unlike the rodent (42, 43, 44, 45, 46, 47, 48, 49), estrogen is incapable of modulating
FSH hormonal action as it relates to the cytodifferentiation in the
primate granulosa cell. However, as demonstrated by Shaw and Hodges
(297), the above conclusions must be tempered by the recognition that
the findings are highly contingent upon the experimental design in
question. Indeed, given an identical experimental model, prior
17ß-estradiol priming was associated with an increase in FSH and
cAMP-supported steroidogenesis. Moreover, concurrent pretreatment with
tamoxifen produced complete abrogation of this estrogenic
effect. Taken together, these observations suggest that subject to the
experimental design of the study, estrogen may either prove stimulatory
to FSH-supported granulosa cell steroidogenesis or else prove inert.
To further distinguish between the latter possibilities, Shaw and
associates (298) undertook to reinvestigate the role of estrogen and
IGF-I in the modulation of FSH action in cultured marmoset granulosa
cells. Animals underwent oophorectomy in the follicular phase, and all
follicles 
0.5 mm were dissected and granulosa cells harvested.
Granulosa cells were cultured in the presence of increasing
concentrations of IGF-I (1–30 ng/ml), with and without 17ß-estradiol
(10-7 M for 48 h) up to a
maximum of 6 days. While IGF-I was able to increase progesterone
production after 4 days of culture, the addition of
17ß-estradiol was without effect. This is in contrast to aromatase
activity for which the addition of an 17ß-estradiol/IGF-I combination
was associated with a 13- to 18-fold increase after 2 days of culture.
Results were corrected for cell number to control for any potential
effects of 17ß-estradiol on granulosa cell proliferation. Taken
together, these observations suggest yet another set of select
circumstances wherein estrogen may be stimulatory to marmoset granulosa
cell steroidogenesis. The apparent divergent actions of estrogen in the
context of the mammalian ovary were elegantly summarized by Hutz (299),
whose observations provide the additional benefit of a comparison with
an avian paradigm.
Taken together, the preceding observations, partially reviewed elsewhere (300), clearly document the primate antral follicle as a site of estrogen action. However, no uniform pattern appears to emerge in that the outcome was often highly contingent upon the experimental paradigm under study. The relatively limited nature of these conclusions is further compounded by the recognition of the paucity of data as they relate to the human paradigm. Clearly then, additional studies would be required to clarify the precise role estrogens play in the modulation of the function (as opposed to growth) of the somatic ovarian cell in both the primate and human ovary.
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IX. Estrogen Reception and Action: The Nonclassical Alternative(s) |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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In addition to the conventional hormone-dependent regulation of the
activity of members of the steroid/thyroid receptor family, some
studies demonstrated substantial interaction between signal
transduction pathways and steroid receptors. In some cases, the
modulation of kinase/phosphatase activity in cells caused the
activation of steroid receptors in the absence of hormone. Nuclear
receptors have been reported to be activated by different signaling
pathways, including those stimulated by the neurotransmitter dopamine
(301, 302), growth factors such as EGF (303, 304, 305), transforming growth
factor- (TGF-) (303), and IGF-I (303, 306). The so-called
ligand-independent mechanism of steroid receptor activation has been
described for different steroid receptors, such as for progesterone
(307), androgen (308), and ERs (309). Although a complete understanding
of the molecular mechanisms underneath the ligand-independent
activation of steroid receptors is yet to be uncovered, in the case of
the ER there is evidence that altered receptor phosphorylation may play
a role in this process (310, 311).
Recent evidence from diverse lines of investigation has suggested that novel forms of steroid hormone receptors may exist and act in heretofore unrecognized mechanisms. In support of this possibility, Kudolo et al. demonstrated an additional class of low-affinity cytosolic ERs in rat granulosa cells (312, 313, 314). Importantly, this receptor could not be demonstrated in the nucleus. It has also been suggested that there exists a family of membrane-associated rapidly acting steroid receptors, which function independently of protein synthesis. Indeed, membrane-associated receptors have been suggested for aldosterone (315, 316), glucocorticoids (317, 318, 319, 320, 321), androstenedione (322), testosterone (323, 324), and progesterone (325, 326, 327, 328, 329, 330, 331, 332, 333, 334) in human and nonhuman cells, including the Xenopus oocyte. These receptors have been demonstrated to mediate rapid nongenomic effects (in vitro), which may be transduced via guanine nucleotide-binding protein(s) or ion channels. Similar receptors have been localized to the membrane of amphibian and mammalian neurons and may be the route of action of "neurosteroids" (i.e., steroids that are produced and act locally in the CNS), which appear to have a rapid onset of action (335, 336, 337, 338, 339, 340).
Special attention must also be given to the work of Pietras and Szego (341, 342, 343), who observed that estrogen may have a rapid membrane-mediated action in the rat endometrial cell. Specifically, estrogen administration proved capable of inducing luminal morphological changes in microvilli within 30 sec (344). Further studies by this group have partially isolated ostensible "estrogen receptors" from the hepatocyte plasma membrane (343). Cardiovascular examples were also described (345).
In this context, it has been recognized that the effects of estrogen on
the cardiovascular system may be produced through either the classical
(genomic) activation of transcription factors or newly identified rapid
(nongenomic) direct actions on the vasculature (346, 347, 348). Animal and
human studies have disclosed that physiological levels of estrogen can
rapidly cause vasodilation (346). Reports in human subjects support
that this effect is largely mediated by activation of endothelin nitric
oxide synthase (eNOS) (349, 350). Indeed, it appears that ER
mediates the short-term effects of estrogen on eNOS activity (351, 352). In cells lacking ER and eNOS, the acute response of eNOS to
estradiol may be achieved by cotransfection of cDNA for those two
proteins (351).
Note should be made of the contribution of Morley et al. (353), who investigated the effects of steroids on the intracellular calcium ionic concentration in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. The results indicate that estrogens almost instantaneously trigger the release of calcium from intracellular stores, an effect that may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal calcium mobilization is consistent with the activation of a cell surface receptor, which is different from the conventional nuclear ER.
As is true for the granulosa cell, one must not exclude the possibility
of membrane-mediated nongenomic estrogenic effects at the level of the
oocyte. In this respect, the demonstration of rapid
17ß-estradiol-induced alteration in intracytoplasmic calcium economy
(354) strongly supports such a possibility. Specifically, it has been
suggested that local intrafollicular estrogen may directly and
nonclassically (i.e., nongenomically) affect the
developmental potential of human oocytes. Tesarik and Mendoza (354)
observed 17ß- estradiol-mediated modulation of intracellular
calcium oscillations at the time of germinal vesicle breakdown (GVB),
in a process analogous to that noted in amphibians. Human oocytes were
aspirated in conjunction with micromanipulation-assisted fertilization
procedures. Those oocytes that had not undergone GVB, and which did not
display signs of degeneration, were loaded with the calcium indicator
dye fluo-3. Relative changes in the intracellular concentration of
calcium were visualized via confocal scanning microscopy of emitted
fluorescence. No spontaneous calcium fluctuations were observed in
control oocytes incubated in the absence of estrogen. In contrast,
oocytes incubated in the presence of 17ß-estradiol (1 µmol/liter)
displayed a transient increase in calcium followed by a series of
secondary calcium oscillations lasting 1–6 h (Fig. 15). These calcium oscillations were
observed in both intact and zona-free oocytes, yet occurred with a
quicker onset in zona-free cases. The triggering of calcium
oscillations was specific to estrogens and was not observed after the
administration of androstenedione or progesterone. The site of
17ß-estradiol action was apparently the oocyte plasma membrane in
that membrane-impermeant estradiol-17ß-estradiol conjugated to BSA
was as effective a stimulus as free 17ß-estradiol. A suggestion was
also made as to an effect of 17ß-estradiol on the developmental
potential of the oocyte. A subgroup of oocytes were incubated in
vitro in an effort to promote maturation to the MII stage.
Although 17ß-estradiol supplementation had no effect on the percent
of oocytes that matured in vitro, those oocytes that were
matured in the presence of 17ß-estradiol were more likely to develop
two pronuclei and to cleave at least once (following in
vitro insemination with spermatozoa) than oocytes incubated in the
absence of 17ß-estradiol. This observation suggests a potential
effect of estrogen upon early oocytic or embryonic development.
|
or ERß cDNA. The
nuclear estrogen-binding component was attributed to nuclear
localization of both ER and ERß. Competitive binding studies of
the nuclear and membrane fractions revealed very similar dissociation
constants. Estrogen binding to ER and ERß in CHO cells resulted in
the rapid activation of Gaq and Gas proteins as well as induction of
inositol phosphate production and adenylate cyclase activity. The
induction of these signaling molecules subsequently led to the
activation of the mitogen-activated protein kinase ERK. c-Jun
N-terminal kinase activity was stimulated by estrogen in cells
expressing the ERß isoform. This study suggests that plasma membrane
and nuclear forms of the ER arise from a single transcript and that the
membrane-associated receptors are linked to downstream signaling
entities. |
X. Summary |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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1. Is the primate/human granulosa cell a site of estrogen
reception? Given what is currently known, the answer to this question
is an affirmative one. On the one hand, molecular probing failed to
detect classical ER in granulosa cells of human origin (233) or in
granulosa cells of primate origin (236). Another study (234), however,
suggested the presence of ER transcripts in luteinized human
granulosa cells. Some of the above studies contrast, however, with
immunohistochemical studies wherein both the primate (246) and human
(237, 249, 251, 253) granulosa cells have been noted to be ER positive,
particularly at midcycle and in dominant follicular structures. It is
possible, since the primate/human granulosa cell is also a site of
ERß expression, that the immunoreactivity noted in some of the
preceding studies constitutes cross-reactivity with ER-directed
antisera. Yet another primate study, however, failed to note
immunoreactive ER in granulosa cells (252). The precise reasons
underlying the apparent discrepancies between the various studies
remain uncertain at this time. The fetal human ovary is a site of ER
and ERß gene expression, the ERß transcripts being more abundant
(238). Recent probing establishes the presence of ERß transcripts in
the adult human ovary and the adult granulosa cell (59, 239). Note must
also be made of the newly discovered ERßcx the potential role of
which in inhibiting ER-mediated estrogen action must be taken into
account (244). Finally, consideration must also be given to the
possibility that estrogen may be acting at the level of the granulosa
cell by means other than classical nuclear ERs. The convincing
demonstration of an apparent nongenomic action of estrogen at the level
of the chicken granulosa cells (rapid release of intracellular calcium)
supports such a possibility (353). Thus, on balance, estrogen action at
the level of the primary/human ovary is undoubtedly representative of
the net impact generated by the relative contribution of the various ER
subtypes at the genomic or nongenomic levels.
2. Is the primate/human oocyte a site of estrogen reception? Given what
is currently known, the answer to this question is uncertain. Indeed, a
single study wherein molecular probing (PCR) was employed (233)
detected ER transcripts in the human oocyte. Revelli et
al. (237), however, failed to document ER transcripts at the
level of the human oocyte. No immunoreactive data are available to
confirm or negate these observations. No information exists at this
time as to the possibility that the human oocyte is a site of ERß
gene expression. Given the apparent import of estrogen to normal
cytoplasmic gametogenic maturation (112, 199, 267, 269, 354), it is
possible that classical oocytic ERs may subserve a meaningful
functional role. However, as is true for the granulosa cell, one must
not exclude the possibility of membrane-mediated nongenomic estrogenic
effects. In this respect, the demonstration of rapid
17ß-estradiol-induced alteration in intraoocytic calcium economy
strongly supports such a possibility (354).
3. Is an estrogen-free/poor intrafollicular environment compatible with follicular development, ovulation, and corpus luteum formation in the primate/human? To the extent that follicular "development" entails antrum formation and therefore sonographically detectable "expansion," the answer is an affirmative one. Indeed, in a manner not unlike that observed in the rodent, an estrogen-free/poor environment is compatible with FSH-induced increments in ovarian size, weight, and antral volume. Thus, the antrum-forming property of FSH, long suspected and herein confirmed once again, is perhaps best illustrated by an environment that may possibly be estrogen free (or at least estrogen poor), i.e., the case of the aromatase-deficient woman (271, 272, 273, 274, 275). However, it is not immediately apparent that follicular "expansion" needs to be equated with true optimal follicular development, the ingredients of which must include enhanced granulosa cell division and differentiation as well as gametogenic maturation. In contrast, however, the very process of follicular rupture and the process of corpus luteum formation appear to proceed unperturbed, despite the fact that the antecedent follicular phase was characterized by an estrogen-free/poor intrafollicular environment (265, 267).
4. Is an estrogen-free/poor intrafollicular environment compatible with gametogenic maturation in primates/humans? The answer to this question is probably a negative one. Indeed, a number of primate studies appear to indicate that an estrogen-free/poor intrafollicular environment is associated with marked decrements in the rates of meiotic maturation and fertilization (82, 265). Data derived from rodent models further suggest a compromise in early embryonic development (291).
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XI. Directions for Future Research |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
|---|
More dynamic in vitro circumstances may be employed to
address the same issues. For example, the development of long-term
human granulosa cell lines may prove helpful in this context. However,
the maintenance of the differentiated phenotype by the latter, ideally
representative of the different phases of follicular development, is
not attainable at this time. Moreover, long-term cultures of follicles
in vitro, an emerging technology, may also be put to good
use under circumstances that ensure either the complete suppression of
aromatase or the antisense-mediated blockade of the corresponding
transcripts. Such an approach may well provide substantial insight into
the relative role of estrogens in folliculogenesis and in the
maturation of the oocyte. Reliance on follicular material from ER or
ERß knockout mouse models may prove indispensable.
In this context, continued in vivo studies of the hypogonadotropic state may prove useful in conjunction with the employment of recombinant FSH preparations. Specifically, GnRH antagonist-treated subjects may be assessed for their estrogen-independent follicular potential. Still, it must be emphasized that such experimental settings may fall short of perfection, thereby failing to yield unequivocal results. Further evaluation will also have to be undertaken in settings wherein the intraovarian concentrations of 17ß-estradiol are approaching or indeed zero (e.g., aromatase deficiency). It must be pointed out, however, that circulating estrogen deficiency cannot be equated with intraovarian estrogen deficiency (266). Hence, it is conceivable that some intraovarian estrogen formation may be possible even in the complete absence of LH (266), a poorly understood phenomenon possibly attributable to extant intraovarian or circulating androgenic substrates.
Finally, one must keep an open mind as to the possibility that ovarian estrogen action is mediated, in part, through a nongenomic mechanism. With rapid advances in membrane and ion channel physiology, there is every reason to believe that new insight will be derived in this evolving area.
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Acknowledgments |
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|
Footnotes |
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1 Supported in part by NIH Research Grants RO1 HD-30288 (now
inactive), R01 HD-39432, and R01 HD-37845 (E.Y.A.), American Physician
Fellowship Award (A.H.), and CAPES/Brazil BEX 1007/99-8 (A.B.T.). 
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References |
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TopAbstractI. IntroductionII. The Nonprimate Ovary...III. The Nonprimate Ovary...IV. Lessons Learned from...V. Lessons Learned from...VI. The Primate/Human Ovary...VII. Is an Estrogen-Free...VIII. The Primate/Human Ovary...IX. Estrogen Reception and...X. SummaryXI. Directions for Future...References |
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