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Endocrine Reviews 22 (3): 389-424
Copyright © 2001 by The Endocrine Society

Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis?1

Steven F. Palter, Adriano B. Tavares, Ariel Hourvitz, Johannes D. Veldhuis and Eli Y. Adashi

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


    Abstract
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
The notion that estrogens play a meaningful role in ovarian folliculogenesis stems from a large body of in vitro and in vivo experiments carried out in certain rodent models, (e.g., rats) wherein the stimulatory role of estrogen on granulosa cell growth and differentiation is undisputed. However, evidence derived from these polyovulatory species may not be readily generalizable to the monoovulatory subhuman primates, let alone the human. Only recently, significant observations on the ovarian role(s) of estrogen have been reported for the primate/human. It is thus the objective of this communication to review the evidence for and against a role for estrogens in primate/human ovarian follicular development with an emphasis toward the application of the concepts so developed to contemporary reproductive physiology and to the practice of reproductive medicine. The role(s) of estrogens will be examined not only by analyzing the physiological evidence to the effect that these hormones control ovarian function and follicular growth, but also by summarizing the molecular evidence for the existence and distribution of the cognate receptors.

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{alpha}-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


    I. Introduction
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
THE NOTION that estrogens play a meaningful role in ovarian folliculogenesis stems from a large body of in vitro and in vivo experiments carried out in certain rodent models (e.g., rats) wherein the stimulatory role of estrogen on granulosa cell growth and differentiation is undisputed (1, 2). However, evidence derived from these polyovulatory species may not be readily generalizable to the monoovulatory subhuman primates, let alone the human. Only recently, significant observations on the ovarian role(s) of estrogen have been reported for the primate/human. It is thus the objective of this communication to review the evidence for and against a role for estrogens in primate/human ovarian follicular development with an emphasis toward the application of the concepts so developed to contemporary reproductive physiology and to the practice of reproductive medicine.

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{alpha}, 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).


    II. The Nonprimate Ovary as a Site of Estrogen Reception (Table 1Go)
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
For the most part, it has been assumed that the ovarian actions of estrogens are mediated via classical nuclear ERs. Specific ovarian estrogen binding (documented by way of radioligand assays) was first shown to exist in the ovaries of intact or hypophysectomized immature rats (16, 17, 18, 19, 20). Hormone binding studies by several investigators also confirmed the presence of ERs in the ovaries of intact immature hamsters, rabbits, guinea pigs, and mice (17, 21, 22). Autoradiographic studies by Stumpf (23) demonstrated the preferential localization of silver grains over nuclei of granulosa cells after in vivo administration of [3H]17ß-estradiol, thereby suggesting that the site of action of estrogen in the rat ovarian follicle is the granulosa cell. Subsequent cellular localization studies confirmed the rat granulosa cell as a site of estrogen reception (16). More recently, Arakawa et al. (20) documented (using radioligand receptor assays) the presence of ERs in antral follicles of rats.


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Table 1. The nonprimate ovary as a site of estrogen reception

 
Although conventional radioligand receptor assays localized ERs to the granulosa cells of several species (see above), molecular probing of the mouse granulosa cell gave rise to some uncertainty as to whether or not the murine granulosa cell is a site of "classical" estrogen reception. Specifically, Hillier et al. (24) detected a 1.5-kb ER{alpha}-hybridizing mRNA in the mouse ovary much smaller than the classical 6.5-kb ER{alpha} mRNA. This mRNA species hybridized to probes specific for the steroid receptor binding regions of the mouse ER{alpha} (domains E and F) and was enhanced in granulosa cells vs. residual ovarian tissues. Wu et al. (25), in turn, reported ER{alpha} transcripts in the mouse oocyte as detected by RT-PCR.

Clemens and Richards (26) provided preliminary documentation of ER{alpha} 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{alpha} splice variants were detected. In vivo and in vitro studies revealed the granulosa cell ER{alpha} 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{alpha} transcripts in rat granulosa cells.

While the ER{alpha} 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{alpha} 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{alpha} 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{alpha} 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. 1Go and 2Go). 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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Figure 1. Localization of ERß mRNA in adult rat ovary during dioestrus. Bright- (a.i) and dark-field (a.ii) images show intense signal for ERß mRNA in the granulosa of ovarian follicles (F) at different stages of development, but negative signals in the corpus luteum (CL). b, Pronounced expression of ERß mRNA in granulosa cells (G) and moderate expression in theca cells (T) of healthy follicles. c, Expression of mRNA is not detectable in the granulosa cells of degenerating follicles (DF). Bar = 200 µm (a.i), 50 µm (b and c). [Reproduced with permission from Mowa and Iwanaga: J Endocrinol 165:59–66, 2000 (54 ). © the Society for Endocrinology.]

 


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Figure 2. Localization of ERß in 23-day-old (A and B) and 60-day-old (C and D) rat ovary. Frozen sections of rat ovary were incubated with PAI P310 ERß antibody (A–C) or preadsorbed ERß antibody with the peptide immunogen (D). Note the immunostaining in follicles (F), specifically in nuclei of granulosa cells (G; panels B and C) and the lack of staining in theca cells (T), interstitial cells (I), and oocytes (O). No staining was observed when peptide-adsorbed antibody was used (panel D). Counterstained with hematoxylin; magnification, x110 (A), x800 (B), and x440 (C and D). [Reproduced with permission from Sar and Welsch: Endocrinology 140:963–971, 1999 (73 ). © The Endocrine Society.]

 
ER{alpha} and ERß transcripts do not display a complete nonoverlapping pattern of expression in the rat ovary (73). The ER{alpha} gene is expressed in granulosa cells albeit less abundantly (64, 66). When compared with the ERß gene, the ER{alpha} 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{alpha} 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. 3Go). 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{alpha} gene expression proved consistently uniform throughout the rat ovarian cycle. The pregnant rat ovary is a site of ER{alpha} reception (54, 69).



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Figure 3. Effect of hCG on protein ERß expression using immunocytochemistry. Ovary sections from rats were incubated with an ERß antiserum. ERß protein was detected in granulosa cells of small and large antral follicles in vehicle-treated (A) and PMSG-treated (B) animals. Star indicates the location of antrum in antral follicles. PMSG-treated rats were injected with an ovulatory dose of hCG and ovaries were isolated after 3 h (C), 9 h (D), 12 h (E), or 24 h (F). The expression of ERß protein in granulosa cells 9 h after hCG was reduced in large antral follicles (left, D), greatly reduced in Graafian follicles (center, D) but did not change in small antral (right, D) follicles. Similar expression was observed 12 h after hCG administration (E). One day (24 h) after hCG treatment, expression was not detected in corpora lutea (small arrowheads, F) but highly expressed in preantral (right, F) and small antral follicles. Magnification: A–F, x250. [Reproduced with permission from S. L. Fitzpatrick et al.: Endocrinology 140:2581–2591, 1999 (70 ). © The Endocrine Society.]

 
The precise role(s) of both ERs in rodent ovarian function has yet to be fully understood. The strong signals corresponding to ERß mRNA in the mice/rats ovary, in contrast to relatively weak expression of ER{alpha} 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 ({alpha} and ß) described to date. The rodent granulosa cells contain predominantly, if not exclusively, the ß-subtype, whereas the ER{alpha} subtype was detected mainly in the theca layer and in the interstitium. Although ER{alpha} 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 ({alpha} 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).


    III. The Nonprimate Ovary as a Site of Estrogen Action
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
A. The rat (Table 2Go) and mouse
In 1940, Richard I. Pencharz (76) reported on the ability of systemically administered diethylstilbestrol (DES) to produce considerable ovarian enlargement even after hypophysectomy. Moreover, the implantation of DES at the time of hypophysectomy not only maintained ovarian weight but also rendered the ovaries more responsive to exogenous gonadotropins. Indeed, a striking ovarian enlargement occurred in DES-primed animals, which were subsequently injected with CG (i.e., Antuitrin "S"). The ovaries of DES-treated animals consisted of healthy, predominantly solid (i.e., preantral), medium-sized follicles packed tightly at the expense of markedly reduced interstitial tissue. DES-primed/CG-treated animals, in turn, displayed large antral follicles, many corpora lutea, and, in two instances, hemorrhagic follicles.


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Table 2. The nonprimate ovary as a site of estrogen action

 
Similar results were reported that very year by Williams (77), followed in turn by several other contributions (78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90) that largely confirmed and, to some degree, extended the original observations of Pencharz (e.g., establishing dose-response relationship and/or examining the role of estrogens other than DES). Ingram, who also administered estrogen to hypophysectomized rats, either by using 1 mg/day of DES (91) or 5 µg/day of estradiol dipropionate (92), in both studies for 5 days, noted the same number of follicles less than 300 µm in diameter, but a considerable increase in the number of medium sized follicles (>300 µm) (Fig. 4Go). 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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Figure 4. Photomicrograph of an ovary from the immature hypophysectomized DES-treated rat. Cross-section of the entire ovary showing a near-uniform population of late secondary (type 5a and 5b) and early tertiary (type 6) follicles. The type 6 exhibits cavitation (arrows) or beginning antrum formation. Magnification, x29. [Modified from R. Sadrkhanloo et al.: Endocrinology 120:146–155, 1987 (98 ). © The Endocrine Society.]

 
Above and beyond their impact on follicular growth and atresia, estrogens have also been shown to affect cytodifferentiation, either by themselves as it is in the modulation of follicular intercellular gap junctions (99, 100), ER content (16), adenylate cyclase activity (101, 102), and cell cycle activator cyclin D2 (103), or as a result of synergy with FSH, as in FSH binding (104), LH binding (101, 104, 105), aromatase activity (106, 107), progestin biosynthesis (108), and A-kinase content (109).

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-{alpha} (TNF{alpha}) (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{alpha}-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{alpha} (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.


    IV. Lessons Learned from the Estrogen-Resistant Model-Estrogen Receptor Null Mutants (ERKOs)
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
The first and only known case of clinical estrogen insensitivity in man was reported by Smith et al. (198). This male patient displayed no detectable response to estrogen administration due to an inactivating mutation of the ER{alpha} 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{alpha} (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{alpha}-/- 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{alpha}-/- 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. 5Go). 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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Figure 5. Histological examination of adult wild-type (WT) and ER{alpha}KO ovaries. A, Histological examination of normal adult WT ovaries revealed many corpora lutea (CL) (stars). Magnification bar = 500 µm. B, Ovaries from ER{alpha}KO female mice contained many hemorrhagic ovarian cysts (asterisks). However, occasional Graafian ovarian follicles (arrow) were present. Magnification bar = 500 µm. C, Higher magnification of Graafian ovarian follicle from adult ER{alpha}KO ovary depicted in panel B reveals that there was differentiation of granulosa cells with cumulus cells surrounding the ovary and follicular antral fluid in the central portion of the ovarian follicle. However, there was abnormal stratification of granulosa cells, with a portion of the follicle lined by a single layer of cells (bracketed area). Magnification bar = 200 µm. [Reproduced with permission from C. S. Rosenfeld et al.: Biol Reprod 62: 599–605, 2000 (208 ). © Society for the Study of Reproduction.]

 
The phenotype of the ER{alpha}-/- 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{alpha}-/- 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{alpha} is required for ovulation and corpus luteum formation, gonadotropins were used to superovulate immature ER{alpha}-/- mice and wild-type siblings (208, 214). Gonadotropin therapy resulted in ovulation in both ER{alpha}-/- and wild-type mice. However, fewer gonadotropin-treated null mutants ovulated. In addition, ER{alpha}-/- 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{alpha}-/- 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{alpha}-/- counterparts. Unlike ER{alpha}-/- 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{alpha}-/- 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{alpha} and ERß were generated (219). ER{alpha}ß-/- mice exhibited normal reproductive tract development but proved infertile. Ovaries of adult ER{alpha}ß-/- 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{alpha}-/- 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{alpha} 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{alpha} 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.


    V. Lessons Learned from the Targeted Disruption of the CYP-19 (Aromatase) Gene-Aromatase Null Mutant (ArKO)
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450, the product of Cyp 19 gene. Null mutant mice for aromatase gene (ArKO) were generated (216), thereby affording the opportunity to examine the role of estrogen in the follicular development in the mouse ovary. Upon reaching sexual maturity, female ArKO mice, born phenotypically normal, develop a male body habitus with excessive internal fat deposition, underdeveloped uteri, and ovaries lacking corpora lutea. The clitoral glands, mammary glands, and gonadal fat pads enlarge. Histological evaluation of the ovaries reveal the presence of many large follicles filled with granulosa cells and evidence of antrum formation, but no corpora lutea. As expected, testosterone and LH levels are markedly elevated. The high testosterone levels presumably reflect stimulation of the theca interna cells by LH (216, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230). Although estrogen was not a prerequisite for the reinitiation of follicle growth (from the point of primordial follicle up to the antral follicle stage), note was made of a block of follicular development and absent corpora lutea. The ovarian phenotype degenerated with age upon the appearance of hemorrhagic cystic follicles and the loss of secondary and antral follicles coincident with the infiltration of macrophages and with stromal hyperplasia (228, 231). In summary, the ArKO female mouse is infertile, as a consequence of disrupted folliculogenesis and failure to ovulate.


    VI. The Primate/Human Ovary as a Site of Estrogen Reception
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
Essential to any discussion of a local paracrine/autocrine role for estrogens in primate/human ovarian physiology must be a review of the evidence relevant to the possibility that the primate/human ovary may be a site of estrogen reception.

A. Molecular probing (Table 3Go)
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{alpha} expressed by the MCF-7 human breast cancer cell line (232). A 7-kb ER{alpha} 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{alpha} mRNA to discrete follicular or perifollicular components. Consequently, one cannot rule out that the positive signal reflects germinal epithelial contamination.


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Table 3. The primate/human ovary as a site of estrogen reception—molecular probing

 
Wu et al. (233), in turn, employing the RT-PCR technique, examined unfertilized human oocytes, cumulus-oocyte complexes (COC), whole ovarian tissue, and isolated granulosa cells. Total ovarian homogenates were secured from two patients undergoing surgery for benign gynecological conditions. Oocytes, COC, and granulosa cells were obtained from patients undergoing transvaginal follicular aspiration in the course of in vitro fertilization-related procedures. The relevant DNA was digested with RNase-free DNase to minimize genomic DNA contamination. Reverse-transcribed RNA was amplified over 30 cycles with specific oligonucleotide primers defining a 263-bp cDNA fragment corresponding to the entire DNA binding domain of the human ER{alpha} 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{alpha} transcripts in whole ovarian material. Similarly, both oocytes and COCs were found to be ER{alpha}-positive. However, when isolated granulosa cells or cumulus masses devoid of oocytes were examined, both were judged to be ER{alpha}-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{alpha}-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{alpha} positivity may be the result of contamination of the COC samples with nonovarian ER{alpha}-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{alpha} cDNA, a stretch of nucleotides known to span intron 1. Southern blotting of the amplified products with a 32P-labeled ER{alpha} mRNA probe confirmed the existence of ER transcripts (Fig. 6Go). The data suggest the existence of ER{alpha} 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{alpha}-positive, thereby reducing the level of confidence in the conclusions from this study.



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Figure 6. Agarose gel demonstrating the amplification of the 282-bp ER{alpha} fragment in human granulosa cells (GC), but not in the negative control human glioblastoma cell line, JHN J889H. The ß-actin (b-A) fragment is amplified from both cell types. [Reproduced with permission from B. S. Hurst et al. : J Clin Endocrinol Metab 80:229–232, 1995 (234 ). © The Endocrine Society.]

 
The technique of RT-PCR of ER{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha} product revealed 99% homology to the cDNA for the hormone-binding region of human ER{alpha}. The apparent absence of ER{alpha} 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{alpha} mRNA was detected in ovarian tissues other than the germinal epithelium. In fact, although whole ovarian material proved ER{alpha} mRNA positive, specimens devoid of germinal epithelium proved ER{alpha} mRNA negative. The intriguing finding of the possible presence of medullary ovarian ER{alpha} transcripts remains to be explained. It is possible that this signal originates in oocytes or migratory lymphocytes assuming the stromal cells themselves are ER{alpha} 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{alpha} transcripts, Revelli and associates documented ER{alpha} transcripts in 17.1% of primordial follicles. The proportions of preantral and antral follicles positive for ER{alpha} transcripts were 30.7 and 37.5%, respectively. No more than 25% of theca cells proved ER{alpha} transcript-positive. Active corpora lutea stained positive for ER{alpha} 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{alpha} transcripts. Oocytes and blood vessels stained negative in all cases.

Brandenberger et al. (238) compared the expression profiles of ER{alpha} and of ERß transcripts in the midgestational human fetus by semiquantitative RT-PCR. ER{alpha} was detected in the human fetal ovary. However, the relative amounts of ER{alpha} 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{alpha} counterparts. The presence of ERß transcripts in ovarian granulosa cells of adult women was also convincingly demonstrated (239). Similar amounts of ER{alpha} 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{alpha} mRNA within the granulosa layer of primate ovarian follicles. Similarly, whole ovarian mRNA yielded appropriate amplicons corresponding to ER{alpha} 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{alpha} and ERß transcripts in the superovulated primate follicle (241). ER{alpha} transcripts did not change whereas ERß transcripts decreased 12–36 h after the ovulatory stimulus. Steroid ablation reduced ER{alpha} 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{alpha}) 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{alpha} 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{alpha} and ERß in human granulosa cell cultures, ER{alpha} is expressed at a lower level than ERß. Basal expression studies indicated that ER{alpha} 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{alpha} and ERß transcripts and proteins. These results suggest that ERß more than ER{alpha} 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{alpha} 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{alpha}, in the presence or absence of 17ß-estradiol. ERßcx preferentially forms a heterodimer with ER{alpha} rather than with ERß, inhibiting DNA binding by ER{alpha}. Thus, ERßcx may potentially inhibit ER{alpha}-mediated estrogen action.

In summary, the nonhuman primate ovary is a site of expression of ER{alpha} transcripts, mainly at the level of the germinal epithelium (236). The granulosa cell layer may also be a site of ER{alpha} gene expression (240). The adult human ovary, in turn, may express ER{alpha} 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{alpha} transcripts at the level of the human oocyte; instead, they reported ER{alpha} 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{alpha} expression. ER{alpha} transcript positivity was also noted in 50% of active corpora lutea (237). The fetal human ovary is a site of ER{alpha} 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{alpha} 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 4Go)
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{alpha} (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. 7Go). 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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Table 4. The primate/human ovary as a site of estrogen reception—immunohistochemistry

 


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Figure 7. Frozen sections of ovarian cortex from one monkey during the early follicular phase of the menstrual cycle. Germinal epithelium (e) exhibited intense nuclear staining when the antibodies against ER(A) or PR(B) were used. When the first antibody was replaced with antibody to antigen B of timothy grass pollen (C), the nuclear reaction product was absent. Sections were counterstained with hematoxylin. Magnification, x320. Bar is 31 µm. [Reproduced with permission from S. Hild-Petito et al.: Endocrinology 123:2896–2905, 1988 (246 ). © The Endocrine Society.]

 
Although the precise reason(s) underlying the apparent absence of primate ovarian follicular ER remains unknown, it is possible, as the authors recognized, that the limited sensitivity of the technique might be a factor. Specifically, the receptor may exist at levels below the threshold of detection of the immunohistochemical technique employed. Alternatively, the primate ovarian ER may exist in a form that did not react with the specific monoclonal human ER-directed antibody used. However, the latter possibility appears unlikely since the technique employed capably localized ER to the monkey ovarian germinal epithelium and the rabbit corpus luteum, as well as to the monkey cervix and oviduct. The preceding arguments notwithstanding, the relative importance of a second ER species (ERß) in the ovary must now be considered (see below).

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{alpha}-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{alpha} 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{alpha}-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{alpha}-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{alpha}-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{alpha}, 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{alpha} 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{alpha}, 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{alpha} 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{alpha} protein by Western blot suggests that the levels of ER{alpha} 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. 8Go), 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{alpha}. The pattern of expression of both receptors in the marmoset was mirrored by that of the human ovaries. Immunoreactive ER{alpha} 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{alpha} protein expression appeared weaker than noted for ERß. In contrast, a clearly defined layer of granulosa cells adjacent to the basal lamina proved ER{alpha} positive when examined in antral follicles. Unlike ER{alpha}, 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{alpha}, 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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Figure 8. Comparative expression of ERß, ER{alpha}, and androgen receptor (AR) proteins in marmoset and human follicles. Sections were incubated with antibodies directed against hERß (anti-P4; a, d, g, j, m, and n), hER{alpha} (b, e, h, k, o, and p), or AR (c, f, i, l, q, and r). Panels a–c are closely adjacent sections of the same ovary taken from a marmoset at the late follicular phase of the cycle; note the preantral (arrows) and large antral follicles (*). In human ovaries, ERß and AR proteins were detected in follicles with one or two layers of granulosa cells (d and f), but these cells lacked immunodetectable ER{alpha} (e). In addition, ERß and AR are present in granulosa and theca cells (arrows) in a preantral follicle (g and i) and an approximately 10-mm antral follicle (*; j and l). Expression of ER{alpha} could also be detected in the theca surrounding both follicles and in the granulosa cell nuclei of the large antral follicle (k). In the preantral follicle (h) containing multiple layers of granulosa cells, a few cells had weak immunopositive staining for ER{alpha}. In surface epithelial cells, ERß (m), ER{alpha} (o), and AR (q) were detected. Positive control tissues were as follows: marmoset prostate, ERß (n) and AR (r); and marmoset uterus, ER (p).

 
Pelletier and El-Alfy (256), assessing the cellular localization of ERs in human reproductive organs, identified ERß immunoreactivity in the nuclei of granulosa cells of growing follicles (all stages, from primary to mature follicles), in the interstitial gland and in the ovarian surface epithelium. Nuclear staining for ER{alpha} was noted for the theca, interstitial gland, and the ovarian surface epithelium.

Recent in vitro studies on ER{alpha} 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{alpha} and ERß proteins. These results suggest that ERß and ER{alpha} 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{alpha} 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{alpha} 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{alpha} and ERß receptor proteins in the ovary. Careful delineation of the cross-reactivity of the antibodies employed appears essential.


    VII. Is an Estrogen-Free (or at Least Poor) Intrafollicular Environment Compatible with Follicular Development, Ovulation, and Corpus Luteum Formation?
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
A. Follicular "expansion" vs. follicular "growth": an important conceptual distinction
In the following text, evidence will be displayed that follicular "expansion," oocyte maturation, fertilization, and, at least, initial embryonic cleavage are possible in a local microenvironment variably devoid of estrogenic support. Special note, however, must be made of the use of the term follicular "expansion" as distinct from the term follicular "development" or "growth." The reasons for these distinctions draw on the realization that sonographic evidence of antral formation cannot be equated with the cytodifferentiative and mitotic events associated with "true" follicular maturation, since sonography technology, although a great tool, is a nonmechanistic, nonfunctional method of evaluation of ovarian follicular cell differentiation and function. As such, the following studies suggest that suboptimally primed follicles may fail to respond to the subsequent ovulatory trigger. Consequently, follicular "expansion" in the face of a substantial hypoestrogenic environment may not be compatible with a successful ovulatory cascade. Indeed, the simple accumulation of follicular fluid and hence the formation of a sonographically detectable antrum does not necessarily imply appropriate granulosa cell proliferation and differentiation, nor does it attest to the corresponding alterations occurring at the level of the theca-interstitial cell compartment. Consequently, in the interest of conservatism as well as accuracy, the term follicular "expansion" is best used to emphasize that the conclusions rely heavily on sonographic technology.

B. Lessons learned from the 17{alpha}-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{alpha}-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{alpha}-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. 9Go). Follicular development was monitored by vaginal sonography.



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Figure 9. Superovulation paradigm was applied to a female subject with virtually complete 17{alpha} hydroxylase/17–20 lyase deficiency. [Derived from (262 ).]

 
Throughout ovulation induction, serum 17ß-estradiol levels as well as serum androstenedione levels remained undetectable. As expected, the circulating levels of LH also remained quite low at 3.5 IU/liter. The circulating levels of FSH, however, increased progressively in keeping with the exogenously provided hFSH injections (Fig. 10Go). Despite the backdrop of profound hypoestrogenism, sonographic monitoring revealed progressive follicular "expansion," the largest follicle exceeding 18 mm in diameter (Fig. 11Go). 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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Figure 10. Circulating levels of estradiol (E2), progesterone (Po), LH, and FSH in the course of superovulation as applied to a female subject with virtually complete 17{alpha}-hydroxylase/17–20 lyase deficiency. [Derived from (262 ).]

 


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Figure 11. Sonographically determined ovarian follicular size in the course of superovulation as applied to a female subject with virtually complete 17{alpha}-hydroxylase/17–20 lyase deficiency. [Derived from (262 ).]

 
Strikingly, follicular fluid 17ß-estradiol concentrations in the patient under study were 103–104 times lower than those encountered in control follicles (30, 22, and 272 pg/ml vs. 0.36–1.96 µg/ml). Intrafollicular testosterone levels were similarly reduced 100–1,000 times relative to those encountered in controls. To eliminate possible non-17ß-estradiol estrogenic substances in the follicular fluid, the authors set out to assay the "total estrogen-active" materials using a recently developed RRA (263). The latter is based on the competitive inhibition of binding of radiolabeled 17ß-estradiol to rat uterine receptors. Using this approach, the authors confirmed the intrafollicular environment of the index subject to be extremely hypoestrogenic with follicular fluid values of 0.15 and 0.23 nmol/liter (normal range 1,140–2,400) of ER binding equivalent (EBE), a level similar to that documented in the serum of castrate women (264).

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{alpha}-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{alpha}-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. 12Go). 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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Figure 12. Circulating levels of 17ß-estradiol (E2) as assessed by RIA over a 10-day period during which ovulation induction was carried out either with highly purified urinary hFSH (pure FSH) or partially purified urinary hFSH + hLH (hMG). [Reproduced with permission from B. Couzinet et al.: J Clin Endocrinol Metab 66:552–556, 1988 (276 ). © The Endocrine Society.]

 
It must be pointed out, however, that none of the subjects under study were LH free. Indeed, some of the subjects displayed minute levels of LH in extracted and concentrated urinary samples. Moreover, there was a finite, albeit minute, amount of exogenous LH activity even in the purified FSH preparation. This LH "contamination" notwithstanding, 17ß-estradiol levels were significantly lower than normally encountered. Even though follicular "expansion" was demonstrated by ultrasound in all women regardless of the plasma estrogen level, note was made of the fact that the circulating levels of 17ß-estradiol at the time of hCG administration were lower in the three women who did not ovulate as compared with their ovulatory counterparts (18, 34, and 114 vs. a mean of 207 pg/ml). As such, these observations might indicate that the health and functional capacity of the follicle might be related to the intrafollicular level of estrogens.

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. 13Go). 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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Figure 13. Recombinant hFSH and hCG dose regimen administered to a patient with isolated gonadotropin deficiency. Serum FSH and LH levels are shown in the upper two panels. Diameters of separate follicles as determined by vaginal sonography for both ovaries, and sonographic estimation of endometrial thickness are shown in the lower two panels. [Reproduced with permission from D.C.J.M. Schoot et al.: J Clin Endocrinol Metab 74:1471–1473, 1992 (277 ). © The Endocrine Society.]

 
As expected, treatment with the recombinant hFSH led to an increase in the circulating levels of FSH to 8.5 IU/liter, serum LH levels remaining low at 0.09 to 0.38 IU/liter (Fig. 13Go). Serum 17ß-estradiol levels (Fig. 14Go) peaked at only 64 pg/ml on day 15 (day of maximal follicular size), endometrial thickness increasing by only 2 mm during treatment (Fig. 13Go). 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. 14Go). 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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Figure 14. Left panel, Circulating levels of estradiol as measured in a patient with isolated gonadotropin deficiency after the administration of recombinant hFSH before the day on which ovarian follicles reach their maximum size (day 17). As a reference, the circulating levels of daily estradiol (mean ± 2 SD) are shown for seven normally cycling women up to the day of the LH peak. Right panel, Intrafollicular concentrations of 17ß-estradiol and androstenedione in three separate follicles (13, 15, and 18 mm diameter) obtained from a patient with isolated gonadotropin deficiency after the administration of recombinant hFSH. Median 17ß-estradiol and androstenedione concentrations in small (3–9 mm) follicles (n = 118), and large (13–24 mm) follicles (n = 7) are shown. [Reproduced with permission from D.C.J.M. Schoot et al.: J Clin Endocrinol Metab 74:1471–1473, 1992 (277 ). © The Endocrine Society.]

 
As such, this study demonstrates that follicular "expansion" is possible in the face of an 17ß-estradiol-poor (but not 17ß-estradiol-free) intrafollicular microenvironment. The above notwithstanding, the preceding study does not shed light on the ovulatory capacity of "expanded"/17ß-estradiol-poor follicles, nor is any insight derived as to the functional adequacy of the oocytes in question.

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. 9Go. 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{alpha} 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.


    VIII. The Primate/Human Ovary as a Site of Estrogen Action
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
Unlike the nonprimate, far less information is available on the direct effects of estrogen on the primate/human ovary. In large measure, the relative paucity of information as it relates to the primate reflects the complexity and expense associated with this experimental model. The inherent limitations imposed on human investigation account, if only in part, for the limited amount of information pertaining to the human ovary. The above notwithstanding, this section attempts to summarize key observations relative to the primate/human ovary secured under either in vitro or in vivo circumstances.

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{alpha}-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.


    IX. Estrogen Reception and Action: The Nonclassical Alternative(s)
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
When determining the status of "classical" ovarian ERs, consideration must be given to the possible involvement of estrogens (if any) by way of alternative (and admittedly hypothetical) mechanism(s). Traditional theory has held that steroids function via binding to nuclear receptors with subsequent activation of gene transcription and protein synthesis. However, the possibility does exist that estrogens act via nonclassical mechanisms in the ovary. Such a model might involve nonclassical nuclear ERs, novel forms of nonnuclear (possibly membranous) ERs, other "orphan" receptors of the steroid/thyroid receptor superfamily, or non-receptor- mediated events.

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-{alpha} (TGF-{alpha}) (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{alpha} mediates the short-term effects of estrogen on eNOS activity (351, 352). In cells lacking ER{alpha} 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. 15Go). 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.



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Figure 15. Spatial dynamics of intracellular free calcium concentration ([Ca2+]i) elevations induced by estradiol in a maturing oocyte. The [Ca2+]i changes are in pseudocolor according to the scale bar, where the lowest values are coded black. A–D, Rising phase of initial [Ca2+]i increase after estradiol addition to a zona-free oocyte. The time interval between successive images displayed in this series is 4 sec (every fourth image from a series of recordings taken at 1-sec intervals is shown). E–H, Rising phase of the first of the series of secondary [Ca2+]i elevations after the initial estradiol-induced [Ca2+]i increase. The time interval between successive images displayed in this series is 1 sec. [Reproduced from J. Tesarik and C. Mendonza: J Clin Endocrinol Metab 80:1438–1443, 1992 (354 ). © The Endocrine Society.]

 
A study by Razandi et al. (355) demonstrated membrane and nuclear-associated estrogen binding sites after the transfection of Chinese hamster ovary (CHO) cells with either ER{alpha} or ERß cDNA. The nuclear estrogen-binding component was attributed to nuclear localization of both ER{alpha} and ERß. Competitive binding studies of the nuclear and membrane fractions revealed very similar dissociation constants. Estrogen binding to ER{alpha} 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
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
It was the purpose of this review to critically evaluate the role of estrogen in primate/human follicular development as compared with other species. This section attempts to answer some of the key questions raised:

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{alpha} in granulosa cells of human origin (233) or in granulosa cells of primate origin (236). Another study (234), however, suggested the presence of ER{alpha} 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{alpha}-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{alpha} 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{alpha}-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{alpha} transcripts in the human oocyte. Revelli et al. (237), however, failed to document ER{alpha} 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).


    XI. Directions for Future Research
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 
Future research must settle the question of whether or not the primate/human granulosa cell or oocyte are, in fact, sites of estrogen reception. Studies of granulosa cells and of oocyte ER mRNA expression need be repeated in human/primate models. In this context, strong emphasis could be placed on surgically derived human ovarian material for which the confounding variable of vaginal mucosal contamination is a nonissue. This material could be subjected to intense immunohistochemical staining (using variant-specific antibodies) and to careful molecular probing using a battery of complementary and therefore confirmatory techniques. In addition, a significant effort must be invested in the probing for ERß transcripts and in the detection of the corresponding protein by means of specific antisera. In so doing, one should be able to establish whether or not the primate/human ovary is in fact a site of estrogen reception and to establish the relevant preponderance of the relevant ER subtypes.

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{alpha} 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.


    Acknowledgments
 
The authors would like to acknowledge the technical support provided by Ms. Denise Roseweg, Ms. Cookie Smajda, Ms. Linda Elder, Ms. Andrea Raposa, and Ms. Michelle S. Lewandowski. The authors also wish to acknowledge the significant contributions of Dr. Richard L. Stouffer, Ph.D. (Oregon Regional Primate Research Facilities) and Dr. Gilbert S. Greenwald, Ph.D. (The University of Kansas Medical Center), who kindly agreed to thoroughly and enthusiastically review and improve the manuscript.


    Footnotes
 
Address reprint requests to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, 546 Chipeta Way Suite 310, Salt Lake City, Utah 84108. E-mail: eadashi{at}hsc.utah.edu

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.). Back


    References
 Top
 Abstract
 I. Introduction
 II. 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. Summary
 XI. Directions for Future...
 References
 

  1. Hsueh AJ, Adashi EY, Jones PB, Welsh TH 1984 Hormonal regulation of the differentiation of cultured granulosa cells. Endocr Rev 5:76–127[Abstract/Free Full Text]
  2. Drummond AE, Findlay JK 1999 The role in estrogen folliculogenesis. Mol Cell Endocrinol 151:57–64[CrossRef][Medline]
  3. Josso N 1996 Sexual Differentiation. In: Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive Endocrinology, Surgery and Technology, ed 1. Lippincott-Raven, Philadlphia, pp 59–75
  4. Topper YL, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1056[Free Full Text]
  5. Kleinberg DL, Niemann W, Flamm E, Cooper P, Babitsky G, Valenski Q 1985 Primate mammary development. Effects of hypophysectomy, prolactin inhibition, and growth hormone administration. J Clin Invest 75:1943–1950
  6. Hotchkiss J, Knobil E 1996 The hypothalamic pulse generator. The reproductive core. In: Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive Endocrinology, Surgery and Technology, ed. 1. Lippincott-Raven, Philadelphia, pp 123–163
  7. Navot D, Laufer N, Kopolovic J, Rabinowitz R, Birkenfeld A, Lewin A, Granat M, Margalioth EJ, Schenaker JG 1986 Artificially induced endometrial cycles and establishment of pregnancies in the absence of ovaries. N Engl J Med 314:806–811[Abstract]
  8. Lessey BA, Killam AP, Metzxger DA, Haney AF, Greene GL, McCarty KS 1988 Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metal 67:334–340[Abstract/Free Full Text]
  9. Dey SK 1996 Implantation. In: Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive Endocrinology, Surgery and Technology, ed 1. Lippincott-Raven, Philadelphia, pp 421–435
  10. Barrett-Connor E 1993 Estrogen and estrogen-progestogen replacement: therapy and cardiovascular diseases. Am J Med 95:40S–43S
  11. (PEPI) TPEPI 1995 Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women: the postmenopausal estrogen/progestin interventions trial. JAMA 273:199–208[Abstract/Free Full Text]
  12. Turner RT, Riggs BL, Spelsberg TC 1994 Skeletal effects of estrogens. Endocr Rev 15:275–300[Abstract/Free Full Text]
  13. Richelson LS, Wahner HW, Melton LJ, Riggs BL 1994 Relative contributions of aging and estrogen deficiency to postmenopausal bone loss. N Engl J Med 311:1273–1275[Abstract]
  14. Halbreich U 1990 Gonadal hormones and antihormones, serotonin, and mood. Psychopharmacol Bull 26:291–295[Medline]
  15. Phillips S, Sherwin B 1992 Effects of estrogen on surgically menopausal women. Psychoneuroendocrinology 17:485–495[CrossRef][Medline]
  16. Richards JS 1975 Estradiol receptor content in rat granulosa cells during follicular development: modification by estradiol and gonadotropins. Endocrinology 97:1174–1184[Abstract/Free Full Text]
  17. Kim I, Shaha C, Greenwald GS 1984 A species difference between hamster and rat in the effects of oestrogens on growth of large preantral follicles. J Reprod Fertil 72:179–185[Abstract/Free Full Text]
  18. Richards JS 1974 Estradiol binding to rat corpora lutea during pregnancy. Endocrinology 95:1046–1053[Abstract/Free Full Text]
  19. Saiduddin S, Zassenhaus HP 1977 Estradiol 17-ß receptors in the immature rat ovary. Steroids 29:197–213[CrossRef][Medline]
  20. Arakawa S, Iyo M, Ohkawa R, Kambegawa A, Okinaga S, Arai K 1989 Steroid hormone receptors in the uterus and ovary of immature rats treated with gonadotropins. Endocrinol Jpn 36: 219–228
  21. Hutz RJ, Gold DA, Dierschke DJ 1987 Diminished steroidogenic response of hamster granulosa cells to estrogen in vitro. Cell Tissue Res 248:531–534[CrossRef][Medline]
  22. Kim I, Greenwald GS 1987 Effect of estrogens on follicular development and ovarian and uterine estrogen receptors in the immature rabbit, guinea pig and mouse. Endocrinol Jpn 34:871–878[Medline]
  23. Stumpf WE 1969 Nuclear concentration of 3H-estradiol in target tissues. Dry-mount autoradiography of vagina, oviduct, ovary, testis, mammary tumor, liver and adrenal. Endocrinology 85:31–37[Abstract/Free Full Text]
  24. Hillier SG, Saunders PT, White R, Parker MG 1989 Oestrogen receptor mRNA and a related RNA transcript in mouse ovaries. J Mol Endocrinol 2:39–45[Abstract/Free Full Text]
  25. Wu TC, Wang L, Wan YJ 1992 Expression of estrogen receptor gene in mouse oocyte and during embryogenesis. Mol Reprod Dev 33:407–412[CrossRef][Medline]
  26. Clemens JW, Richards JAS 1994 Analysis of estrogen receptor mRNA in rat granulosa cells during differentiation. In: Eppig JJ (ed) Frontiers in Ovarian Research. Tenth Ovarian Workshop. Serono Symposia USA, Norwell, MA, p 19
  27. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G, Waterfield M, Chambon P 1986 Cloning of the human oestrogen receptor cDNA. J Steroid Biochem 24:77–83[CrossRef][Medline]
  28. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–30[Abstract/Free Full Text]
  29. Mosselman S, Polman J, Dijkema R 1996 ERß identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  30. Katzenellenbogen BS, Korach KS 1997 Editorial: a new actor in the estrogen receptor drama—enter ER-ß. Endocrinology 138:861–862[Free Full Text]
  31. Kuiper GG, Gustafsson JA 1997 The novel estrogen receptor-ß subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS Lett 410:87–90[CrossRef][Medline]
  32. Gustafsson JA 1997 Estrogen receptor ß—getting in on the action? Nat Med 3:493–494[CrossRef][Medline]
  33. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S 1997 Human estrogen receptor ß binds DNA in a manner similar to and dimerizes with estrogen receptor {alpha}. J Biol Chem 272:25832–25838[Abstract/Free Full Text]
  34. Chu S, Fuller PJ 1997 Identification of a splice variant of the rat estrogen receptor ß gene. Mol Cell Endocrinol 132:195–199[CrossRef][Medline]
  35. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson JA, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  36. Cowley SM, Hoare S, Mosselman S, Parker MG 1997 Estrogen receptors {alpha} and ß form heterodimers on DNA. J Biol Chem 272:19858–19862[Abstract/Free Full Text]
  37. Pennisi E 1997 Differing roles found for estrogen’s two receptors. Science 277:1439[Free Full Text]
  38. Tong W, Perkins R, Xing L, Welsh WJ, Sheehan DM 1997 QSAR models for binding of estrogenic compounds to estrogen receptor {alpha} and ß subtypes. Endocrinology 138:4022–4025[Abstract/Free Full Text]
  39. Leygue E, Dotzlaw H, Lu B, Glor C, Watson PH, Murphy LC 1998 Estrogen receptor ß: mine is longer than yours? J Clin Endocrinol Metab 83:3754–3755[Free Full Text]
  40. Chaidarun SS, Swearingen B, Alexander JM 1998 Differential expression of estrogen receptor-ß (ERß) in human pituitary tumors: functional interactions with ER{alpha} and a tumor-specific splice variant. J Clin Endocrinol Metab 83:3308–3315[Abstract/Free Full Text]
  41. Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, Su JL, Kliewer SA, Lehmann JM, Willson TM 1998 Cloning and characterization of human estrogen receptor ß isoforms. Biochem Biophys Res Commun 247:75–78[CrossRef][Medline]
  42. Chaidarun SS, Klibanski A, Alexander JM 1997 Tumor-specific expression of alternatively spliced estrogen receptor messenger ribonucleic acid variants in human pituitary adenomas. J Clin Endocrinol Metab 82:1058–1065[Abstract/Free Full Text]
  43. Murphy LC, Dotzlaw H, Leygue E, Douglas D, Coutts A, Watson PH 1997 Estrogen receptor variants and mutations. J Steroid Biochem Mol Biol 62:363–372[CrossRef][Medline]
  44. Rosenkranz K, Hinney A, Ziegler A, Hermann H, Fichter M, Mayer H, Siegfried W, Young JK, Remschmidt H, Hebebrand J 1998 Systematic mutation screening of the estrogen receptor ß gene in probands of different weight extremes: identification of several genetic variants. J Clin Endocrinol Metab 83:4524–4527[Abstract/Free Full Text]
  45. Hanstein B, Liu H, Yancisin MC, Brown M 1999 Functional analysis of a novel estrogen receptor-ß isoform. Mol Endocrinol 13:129–137[Abstract/Free Full Text]
  46. Sun J, Meyers MJ, Fink BE, Rajendran R, Katzenellenbogen JA, Katzenellenbogen BS 1999 Novel ligands that function as selective estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-ß. Endocrinology 140:800–804[Abstract/Free Full Text]
  47. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353–365[Abstract/Free Full Text]
  48. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA 1997 Mouse estrogen receptor ß forms estrogen response element-binding heterodimers with estrogen receptor {alpha}. Mol Endocrinol 11:1486–1496[Abstract/Free Full Text]
  49. Hileman SM, Handa RJ, Jackson GL 1999 Distribution of estrogen receptor-ß messenger ribonucleic acid in the male sheep hypothalamus. Biol Reprod 60:1279–84[Abstract/Free Full Text]
  50. Rosenfeld CS, Yuan X, Manikkam M, Calder MD, Garverick HA, Lubahn DB 1999 Cloning, sequencing, and localization of bovine estrogen receptor-ß within the ovarian follicle. Biol Reprod 60:691–697[Abstract/Free Full Text]
  51. Wu WX, Ma XH, Smith GC, Nathanielsz PW 2000 Differential distribution of ERalpha and ERbeta mRNA in intrauterine tissues of the pregnant rhesus monkey. Am J Physiol Cell Physiol 278:C190–198
  52. Pelletier G, Labrie C, Labrie F 2000 Localization of oestrogen receptor {alpha}, oestrogen receptor ß and androgen receptors in the rat reproductive organs. J Endocrinol 165:359–370[Abstract]
  53. Mowa CN, Iwanaga T 2000 Developmental changes of the oestrogen receptor-{alpha} and –ß mRNAs in the female reproductive organ of the rat—an analysis by in situ hybridization. J Endocrinol 167:363–369[Abstract]
  54. Mowa CN, Iwanaga T 2000 Differential distribution of oestrogen receptor-{alpha} and -ß mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. J Endocrinol 165:59–66[Abstract]
  55. Jefferson WN, Couse JF, Banks EP, Korach KS, Newbold RR 2000 Expression of estrogen receptor beta is developmentally regulated in reproductive tissues of male and female mice. Biol Reprod 62:310–317[Abstract/Free Full Text]
  56. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER{alpha}-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
  57. Pelletier G, Luu-The V, Charbonneau A, Labrie F 1999 Cellular localization of estrogen receptor beta messenger ribonucleic acid in cynomolgus monkey reproductive organs. Biol Reprod 61:1249–1255[Abstract/Free Full Text]
  58. Saunders PT, Millar MR, Williams K, Macpherson S, Harkiss D, Anderson RA, Orr B, Groome NP, Scobie G, Fraser HM 2000 Differential expression of estrogen receptor-{alpha} and -ß and androgen receptor in the ovaries of marmosets and humans. Biol Reprod 63:1098–1105[Abstract/Free Full Text]
  59. Brandenberger AW, Tee MK, Jaffe RB 1998 Estrogen receptor {alpha} (ER-{alpha}) and ß (ER-ß) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-ß in neoplastic tissues. J Clin Endocrinol Metab 83:1025–1028[Abstract/Free Full Text]
  60. Hillier SG, Anderson RA, Williams AR, Tetsuka M 1998 Expression of oestrogen receptor {alpha} and ß in cultured human ovarian surface epithelial cells. Mol Hum Reprod 4:811–815[Abstract/Free Full Text]
  61. Saunders PT, Maguire SM, Gaughan J, Millar MR 1997 Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 154:R13–16
  62. Bao B, Kumar N, Karp RM, Garverick HA, Sundaram K 2000 Estrogen receptor-ß expression in relation to the expression of luteinizing hormone receptor and cytochrome P450 enzymes in rat ovarian follicles. Biol Reprod 63:1747–1755[Abstract/Free Full Text]
  63. Drummond A, Baillie AJ, Findlay JK 1999 Ovarian estrogen receptor {alpha} and ß mRNA expression: impact of development and estrogen. Mol Cell Endocrinol 149:153–161[CrossRef][Medline]
  64. Wang H, Eriksson H, Sahlin L 2000 Estrogen receptors {alpha} and ß in the female reproductive tract of the rat during the estrous cycle. Biol Reprod 63:1331–1340[Abstract/Free Full Text]
  65. Shughrue PJ, Lane MV, Scrimo PJ, Merchenthaler I 1998 Comparative distribution of estrogen receptor-{alpha} (ER-{alpha}) and ß (ER-ß) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 63:498–504[CrossRef][Medline]
  66. Sharma SC, Clemens JW, Pisarska MD, Richards JS 1999 Expression and function of estrogen receptor subtypes in granulosa cells: regulation by estradiol and forskolin. Endocrinology 140:4320–4334[Abstract/Free Full Text]
  67. Byers M, Kuiper GG, Gustafsson JA, Park-Sarge O-K 1997 Estrogen receptor-ß mRNA expression in rat ovary: down-regulation by gonadotropins. Mol Endocrinol 11:172–182[Abstract/Free Full Text]
  68. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  69. Telleria CM, Zhong L, Deb S, Srivastava RK, Park KS, Sugino N, Park-Sarge OK, Gibori G 1998 Differential expression of the estrogen receptors {alpha} and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 139:2432–2442[Abstract/Free Full Text]
  70. Fitzpatrick SL, Funkhouser JM, Sindoni DM, Stevis PE, Deecher DC, Bapat AR, Merchenthaler I, Frail DE 1999 Expression of estrogen receptor-ß protein in rodent ovary. Endocrinology 140:2581–2591[Abstract/Free Full Text]
  71. Petersen DN, Tkalcevic GT, Koza-Taylor PH, Turi TG, Brown TA 1998 Identification of estrogen receptor ß2, a functional variant of estrogen receptor ß expressed in normal rat tissues. Endocrinology 139:1082–1092[Abstract/Free Full Text]
  72. Hiroi H, Inoue S, Watanabe T, Goto W, Orimo A, Momoeda M, Tsutsumi O, Taketani Y, Muramatsu M 1999 Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus. J Mol Endocrinol 22:37–44[Abstract]
  73. Sar M, Welsch F 1999 Differential expression of estrogen receptor-ß and estrogen receptor-{alpha} in the rat ovary. Endocrinology 140: 963–971
  74. Veldhuis JD 1981 Interactions among endocrine control systems in the regulation of ovarian function. Clin Biochem 14:252–258[CrossRef][Medline]
  75. Kawashima M, Greenwald GS 1993 Comparison of follicular estrogen receptors in rat, hamster, and pig. Biol Reprod 48:172–179[Abstract]
  76. Pencharz RI 1940 Effects of estrogens and androgens alone and in combination with chorionic gonadotropin on the ovary of the hypophysectomized rats. Science 91:554–555[Free Full Text]
  77. Williams PC 1940 Effects of stilbestrol on the ovaries of hypophysectomized rats. Nature 145:388–389
  78. Simpson ME, Evans HM, Frankel-Conrat HL, Li CH 1941 Synergism of estrogens with pituitary gonadotropins in hypophysectomized rats. Endocrinology 28:37–41[Abstract/Free Full Text]
  79. Williams PC 1944 Ovarian stimulation by oestrogens: effects in immature hypophysectomized rats. Proc R Soc Lond B 132:189–199
  80. Williams PC 1945 Studies of the biological action of serum gonadotrophin. J Endocrinol 4:131–136
  81. Hisaw FL 1947 Development of the graafian follicle and ovulation. Physiol Rev 27:95–119[Free Full Text]
  82. Paesi FJA 1952 The effect of small doses of oestrogen on the ovary of the immature rat. Acta Endocrinol (Copenh) 11:251–268
  83. Payne RW, Hellbaum AA 1955 The effect of estrogens on the ovary of the hypophysectomized rat. Endocrinology 57:193–199
  84. Payne RW, Runser RH 1958 The influence of estrogen and androgen on the ovarian response of hypophysectomized immature rats to gonadotropins. Endocrinology 62:313–321
  85. Payne RW, Runser RH 1959 Quantitative response of the rat ovary to pituitary gonadotropin as modified by estrogen. Endocrinology 65:383–388
  86. Payne RW, Runser RH, Hagans JA, Morrison RD 1959 Assay of follicle-stimulating hormone in the hypophysectomized estrogen-treated immature female rat. Endocrinology 65:389–394
  87. Meyer JE, Bradbury JT 1960 Influence of stilbestrol on the immature rat ovary and its response to gonadotrophin. Endocrinology 66:121–128[Abstract/Free Full Text]
  88. Bradbury JT 1961 Direct action of estrogen on the ovary of the immature rat. Endocrinology 68:115–120[Abstract/Free Full Text]
  89. Smith BD, Bradbury JT 1963 Ovarian response to gonadotropins after pretreatment with diethylstilbestrol. Am J Physiol 204:1023–1027[Abstract/Free Full Text]
  90. DeWit JE 1953 The effect of oestradiol monobenzoate on follicles of various sizes in the ovary of the hypophysectomized rat. Acta Endocrinol (Copenh) 12:123–139
  91. Ingram DL 1959 The effect of oestrogen on the atresia of ovarian follicles. J Endocrinol 19:123–125
  92. Ingram DL 1959 The effect of gonadotrophins and oestrogen on ovarian atresia in the immature rat. J Endocrinol 19:117–122
  93. Goldenberg RL, Vaitukaitis JL, Ross GT 1972 Estrogen and follicle stimulating hormone interactions on follicle growth in rats. Endocrinology 90:1492–1498[Abstract/Free Full Text]
  94. Harman SM, Louvet JP, Ross GT 1975 Interactions of estrogen and gonadotropins in follicular atresia. Endocrinology 96:1145–1152[Abstract/Free Full Text]
  95. Rao MC, Midgley AR, Richards JS 1978 Hormonal regulation of ovarian cellular proliferation. Cell 14:71–78[CrossRef][Medline]
  96. Ross GT, Hillier SG 1979 Experimental aspects of follicular maturation. Eur J Obstet Gynecol Reprod Biol 9:169–174[Medline]
  97. Billig H, Furuta I, Hsueh AJ 1993 Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133: 2204–2212
  98. Sadrkhanloo R, Hofeditz C, Erickson GF 1987 Evidence for widespread atresia in the hypophysectomized estrogen-treated rat. Endocrinology 120:146–155[Abstract/Free Full Text]
  99. Merk FB, Botticelli CR, Albright JT 1972 An intercellular response to estrogen by granulosa cells in the rat ovary: an electron microscope study. Endocrinology 90:992–1007[Abstract/Free Full Text]
  100. Burghardt RC, Anderson E 1981 Hormonal modulation of gap junctions in rat ovarian follicles. Cell Tissue Res 214:181–193[CrossRef][Medline]
  101. Richards JS, Jonassen JA, Rolfes AI, Korsey KA, Reichert LE 1979 Adenosine 3',5'-monophosphate luteinizing hormone receptor, and progesterone during granulosa cell differentiation: Effects of estradiol and follicle stimulating hormone. Endocrinology 104: 765–773
  102. Jonassen JA, Bose K, Richards JS 1982 Enhancement and desensitization of hormone-responsive adenylate cyclase in granulosa cells of preantral and antral ovarian follicles: effects of estradiol and follicle-stimulating hormone. Endocrinology 111:74–79[Abstract/Free Full Text]
  103. Robker RL, Richards JS 1998 Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12:924–940[Abstract/Free Full Text]
  104. Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley AR, Reichert LE 1976 Ovarian follicular development in the rat: hormone receptor regulation by estradiol, FSH, and LH. Endocrinology 99:1562–1570[Abstract/Free Full Text]
  105. Kessel B, Liu YX, Jia XC, Hsueh AJ 1985 Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells. Biol Reprod 32:1038–1050[Abstract]
  106. Adashi EY, Hsueh AJ 1982 Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells. J Biol Chem 259:6077–6088
  107. Daniel SA, Armstrong DT 1983 Involvement of estrogens in the regulation of granulosa cell aromatase activity. J Physiol Pharmacol 61:507–511
  108. Welsh TJ, Shuang LZ, Hsueh AJ 1983 Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells. Endocrinology 112:1916–1924[Abstract/Free Full Text]
  109. Richards JS, Rolfes AI 1980 Hormonal regulation of cyclic AMP-binding to specific receptor proteins in rat ovarian follicles: characterization by photoaffinity labeling. J Biol Chem 255:5481–5489[Free Full Text]
  110. Kent J 1973 Some effects of steroids on the immature mouse ovary in vitro. J Reprod Fertil 34:297–303[Abstract/Free Full Text]
  111. Halling A, Forsberg JG 1990 Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life. J Reprod Fertil 88:399–404[Abstract/Free Full Text]
  112. Wang X-N, Greenwald GS 1993 Synergistic effects of steroids with FSH on folliculogenesis, steroidogenesis and FSH- and hCG- receptors in hypophysectomized mice. J Reprod Fertil 99:403–413[Abstract/Free Full Text]
  113. Chiras DD, Greenwald GS 1978 Effects of steroids and gonadotropins on follicular development in the hypophysectomized hamster. Am J Anat 152:307–319[CrossRef][Medline]
  114. Hunter R 1980 Control of ovulation and induction of superovulation. In: Physiology and Technology of Reproduction in Female Domestic Animals. Anonymous Academic Press, London
  115. Stoklosowa S, Gregoraszczuk E, Channing CP 1982 Estrogen and progesterone secreton by isolated cultured porcine thecal and granulosa cells. Biol Reprod 26:943–952[CrossRef][Medline]
  116. Foxcroft GR, Hunter MG, Grant SA 1989 The physiology of follicular maturation in the pig. Acta Physiol Pol 40:53–63[Medline]
  117. Haney A, Schomberg D 1978 Steroidal modulation of progesterone secretion by granulosa cells from large porcine follicles: a role for androgens and estrogens in controlling steroidogenesis. Biol Reprod 19:242–248[Abstract]
  118. Veldhuis JD, Klase PA, Hammond JM 1980 Divergent effects of prolactin upon steroidogenesis by porcine granulosa cells in vitro: influence of cytodifferentiation. Endocrinology 107:42–46[Abstract/Free Full Text]
  119. Veldhuis JD, Hammond JM 1980 Oestrogens regulate divergent effects of prolactin in the ovary. Nature 284:262–264[CrossRef][Medline]
  120. Sites CK, Kessel B, LaBarbera AR 1996 Adhesion proteins increase cellular attachment; follicle-stimulating hormone receptors, and progesterone production in cultured porcine granulosa cells. Proc Soc Exp Biol Med 212:78–83[CrossRef][Medline]
  121. May JV 1993 Porcine granulosa and theca cell culture: bioassay applications for factors modulating cell proliferation and differentiation. Methods Toxicol 38:295–319
  122. Hsu CJ, Hammond JM 1987 Gonadotropins and estradiol stimulates immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro. Endocrinology 120:198–203[Abstract/Free Full Text]
  123. Hammond JM, Baranao LS, Skleris D, Knight AB, Romanus JA, Rechler MM 1985 Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology 117:2553–2557[Abstract/Free Full Text]
  124. Hsu C, Hammond JM 1987 Concomitant effects of growth hormone on secretion of insulin-like growth factor I and progesterone by cultured porcine granulosa cells. Endocrinology 121:1343–1348[Abstract/Free Full Text]
  125. Samaras SE, Canning SF, Barber JA, Simmen FA, Hammond JM 1996 Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells. Endocrinology 137:4657–4664[Abstract]
  126. Deguchi J, Maruo T, Masuo H, Mochizuki M 1996 Tumor necrosis factor {alpha} regulates the proliferative activity and differentiated function of granulosa cells: in vivo study with a porcine model. Nippon Sanka Fujinka Gakkai Zasshi 48:1043–1050[Medline]
  127. Morbeck DE, Flowers WL, Britt JH 1993 Response of porcine granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and epidermal growth factor in vitro. J Reprod Fertil 99:577–584[Abstract/Free Full Text]
  128. Zhang Q, Bagnell CA 1993 Relaxin stimulation of porcine granulosa cell deoxyribonucleic acid synthesis in vitro: interactions with insulin and insulin-like growth factor I. Endocrinology 132:1643–1650[Abstract/Free Full Text]
  129. Kubota T, Kamada S, Taguchi M, Aso T 1994 Autocrine/paracrine function of transforming growth factor ß 1 in porcine granulosa cells. Hum Reprod 9:2118–2122[Abstract/Free Full Text]
  130. Maruo T, Hiramatsu S, Otani T, Hayashi M, Mochizuki M 1992 Increase in the expression of thyroid hormone receptors in porcine granulosa cells early in follicular maturation. Acta Endocrinol (Copenh) 127:152–160[Abstract/Free Full Text]
  131. Guthrie HD, Bolt DJ, Cooper BS 1993 Changes in follicular estradiol-17ß, progesterone and inhibin immunoactivity in healthy and atretic follicles during preovulatory maturation in the pig. Domest Anim Endocrinol 10:127–140[CrossRef][Medline]
  132. Machelon V, Nome F, Salesse R 1994 Comparative IL-6 effects on FSH and hCG-induced functions in porcine granulosa cell cultures. Cell Mol Biol 40:373–380[Medline]
  133. Sirotkin AV, Nitray J 1994 Effects of prolactin on estrogen, cAMP and oxytocin secretion by porcine granulosa cell in vitro. Reprod Nutr Dev 34:141–148
  134. Kamada S, Blackmore PF, Oehninger S, Gordon K, Hodgen GD 1994 Existence of P2-purinoceptors on human and porcine granulosa cells. J Clin Endocrinol Metab 78:650–656[Abstract]
  135. Samaras SE, Hammond JM 1995 Insulin-like growth factor binding protein-3 inhibits porcine granulosa cell function in vitro. Am J Physiol 268:E1057–E1064
  136. Conley AJ, Howard HJ, Slanger WD, Ford JJ 1994 Steroidogenesis in the preovulatory porcine follicle. Biol Reprod 51:655–661[Abstract]
  137. Urban RJ, Veldhuis JD 1992 Endocrine control of steroidogenesis in granulosa cells. Oxf Rev Reprod Biol 14:225–262[Medline]
  138. Veldhuis JD, Klase PA, Hammond JM 1981 Sex steroids modulate prolactin action in spontaneously luteinizing porcine granulosa cell in vitro. Endocrinology 108:1463–1468[Abstract/Free Full Text]
  139. Veldhuis JD, Klase PA, Hammond JM 1981 Direct actions of 17ß estradiol on progesterone production by highly differentiated porcine granulosa cells in vitro. II. Regulatory interactions of estradiol with luteinizing hormone and cyclic nucleotides. Endocrinology 109:433–442[Abstract/Free Full Text]
  140. Veldhuis JD, Klase PA, Strauss III JF, Hammond JM 1982 Facilitative interactions between estradiol and luteinizing hormone in the regulation of progesterone production by cultured swine granulosa cells: relation to cellular cholesterol metabolism. Endocrinology 111:441–447[Abstract/Free Full Text]
  141. Veldhuis JD, Klase PA, Strauss III JF, Hammond JM 1982 The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells. Endocrinology 111:144–151[Abstract/Free Full Text]
  142. Veldhuis JD, Klase PA, Sandow BA, Kolp LA 1983 Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and hCG. J Clin Endocrinol Metab 57:87–94[Abstract/Free Full Text]
  143. Veldhuis JD, Gwynne JT, Klase PA, Strauss III JF, Demers LM 1984 Role of estradiol as a biological amplifier of gonadotropin action in the ovary: in vitro studies using swine granulosa cells and homologous lipoproteins. Endocrinology 114:2312–2322[Abstract/Free Full Text]
  144. Veldhuis JD, Gwynne JT 1985 Estrogen regulates low-density lipoprotein metabolism by cultured swine granulosa cells. Endocrinology 117:1321–1327[Abstract/Free Full Text]
  145. Veldhuis JD 1986 Mechanisms subserving the bipotential actions of estrogen on ovarian cells: studies with a selective anti-estrogen, LY156758, and the sparingly metabolizable estrogen agonist, moxestrol. J Steroid Biochem 24:977–982[CrossRef][Medline]
  146. Veldhuis JD, Rodgers RJ, Furlanetto RW 1986 Synergistic actions of estradiol and the insulin-like growth factor, somatomedin C, on swine ovarian (granulosa) cells. Endocrinology 119:530–538[Abstract/Free Full Text]
  147. Veldhuis JD, Rodgers RJ, Dee A, Simpson E 1986 The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage of cytochrome P-450 and adrenodoxin in ovarian cells. J Biol Chem 261:2499–2502[Abstract/Free Full Text]
  148. Veldhuis JD, Rodgers RJ, Hewlett EL 1988 Actions of cyclic AMP on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis. Mol Endocrinol 2:499–506[Abstract/Free Full Text]
  149. Urban RJ, Garmey JC, Shupnik MA, Veldhuis JD 1991 Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme concentrations in primary cultures of porcine granulosa cells. Endocrinology 128:2000–2007[Abstract/Free Full Text]
  150. Garmey JC, Day RN, Day KH, Veldhuis JD 1993 Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II (IGF-II): in vitro studies with swine granulosa cells. Endocrinology 133:800–808[Abstract/Free Full Text]
  151. Winters TA, Hanten JA, Veldhuis JD 1998 In situ amplification of the cytochrome P450 cholesterol side chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-I and FSH alone or in concert. Endocrine 9:57–63[CrossRef][Medline]
  152. LaVoie H, Day RN, Garmey JC, Veldhuis JD 1999 Concerted regulation of low density lipoprotein receptor gene expression by FSH and IGF-I in porcine granulosa cells: promoter activation, mRNA stability, and sterol feedback. Endocrinology 140:178–186[Abstract/Free Full Text]
  153. Urban RJ, Garmey JC, Shupnik MA, Veldhuis JD 1990 Insulin-like growth factor type I increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells. Endocrinology 127:2481–2488[Abstract/Free Full Text]
  154. Veldhuis JD, Hammond JM 1979 Role of ornithine decarboxylase in granulosa cell replication and steroidogenesis in vitro. Biochem Biophys Res Commun 91:770–777[Medline]
  155. Veldhuis JD 1985 Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells: II Relation of estradiol’s stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells. Endocrinology 117:1076–1083[Abstract/Free Full Text]
  156. Ainsworth L, Tsang BK, Downey BR, Marus GL, Armstrong DT 1996 The PMSG/hCG-treated prepubertal gilt as a model for the study of the actions of pituitary and ovarian hormones on follicular cell differentiation. In: Roland R, Van Hul EV, Hillier SG, McNatty KP, Shoemaker J (eds) Follicular Maturation and Ovulation: Excerpta Medica International Congress Series. Excerpta Medica, Amsterdam, The Netherlands, pp 997–1102
  157. Morley P, Khalil MW, Calaresu FR, Armstrong DT 1989 Catecholestrogens inhibit basal and luteinizing hormone-stimulated androgen production by porcine thecal cells. Biol Reprod 41:446–453[Abstract]
  158. Guthrie HD, Barber JA, Leighton JD, Hammond JM 1994 Steroidogenic cytochrome P450 enzyme messenger ribonucleic acids and follicular fluid steroids in individual follicles during preovulatory maturation in the pig. Biol Reprod 51:465–471[Abstract]
  159. Kim SH, Cho KW, Lim SH, Hwang YH, Ryu H, Oh SH, Seul KH, Jeong GB, Yoon S 1992 Presence and release of immunoreactive atrial natriuretic peptide in granulosa cells of the pig ovarian follicle. Regul Pept 42:153–162[CrossRef][Medline]
  160. Samaras SE, Hagen DR, Bryan KA, Mondschein JS, Canning SF, Hammond JM 1994 Effects of growth hormone and gonadotropin on the insulin-like growth factor system in the porcine ovary. Biol Reprod 50:178–186[Abstract]
  161. Flores JA, Winters TA, Veldhuis JD 1996 Nature of endothelin binding in the porcine ovary. Endocrinology 136:5014–5019[Abstract]
  162. Kubota T, Kamada S, Aso T 1994 Endothelin-1 as a local ovarian regulator in porcine granulosa cells. Horm Res 41:29–35
  163. Itahana K, Morikazu Y, Takeya T 1996 Differential expression of four connexin genes, Cx-26, Cx-30.3, Cx-32, and Cx-43, in the porcine ovarian follicle. Endocrinology 137:5036–5044[Abstract]
  164. Fujiwara H, Haeda M, Honda T, Yamada S, Ueda M, Kanzaki H, Suginami H, Mori T 1996 Granulosa cells express integrin {alpha}6: possible involvement of integrin {alpha}6 in folliculogenesis. Horm Res 46:24–30
  165. Lee VY, Britt JH, Dunbar BS 1996 Localization of laminin proteins during early follicular development in pig and rabbit ovaries. J Reprod Fertil 108:115–122[Abstract/Free Full Text]
  166. Cutler RE, Maizels ET, Hunzicker-Dunn M 1994 Protein kinase-C in the rat ovary: estrogen regulation and localization. Endocrinology 135:1669–1678[Abstract]
  167. Veldhuis JD, Demers LM 1986 An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis: studies in cultured swine granulosa cells. Biochem J 239:505–511[Medline]
  168. Veldhuis JD, Hewlett EL 1985 Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary. Biochem Biophys Res Commun 131:1168–1174[CrossRef][Medline]
  169. Wheeler MB, Veldhuis JD 1987 Catalytic and receptor binding properties of the calcium-sensitive phospholipid-dependent protein kinase (protein kinase C) in swine luteal cytosol. Mol Cell Endocrinol 50:123–129[CrossRef][Medline]
  170. Veldhuis JD, Yoshida K, DuBois W, Fields MJ 1987 Maitotoxin stimulates steroid and peptide hormone secretion by swine luteal tissue. Am J Physiol 252:E8–E12
  171. Veldhuis JD, Rodgers RJ 1987 Mechanism subserving the steroidogenic synergism between FSH and the insulin-like growth factor, IGF-I (somatomedin C): alterations in cellular sterol metabolism in swine granulosa cells. J Biol Chem 262:7658–7664[Abstract/Free Full Text]
  172. Veldhuis JD, Demers LW 1987 Activation of protein kinase C is coupled to prostaglandin F2-{alpha} synthesis in the ovary: studies in cultured swine granulosa cells. Mol Cell Endocrinol 49:249–254[CrossRef][Medline]
  173. Veldhuis JD, Demers LM 1987 Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells. Prostaglandins 33:819–829[CrossRef][Medline]
  174. Wheeler MB, Veldhuis JD 1988 Interactions of protein kinase C with receptor and non-receptor mediated cyclic AMP generation in swine granulosa cells. Mol Cell Endocrinol 59:195–203[CrossRef][Medline]
  175. Wheeler MB, Veldhuis JD 1989 Facilitative actions of the protein kinase C effector system on hormonally stimulated cyclic adenosine 3',5'-monophosphate production by swine luteal cells. Endocrinology 125:2414–2420[Abstract/Free Full Text]
  176. Flores JA, Veldhuis JD, Leong DA 1990 Follicle stimulating hormone evokes an increase in intracellular free calcium ion concentrations in single ovarian (granulosa) cells. Endocrinology 127:3172–3179[Abstract/Free Full Text]
  177. Flores JA, Leong DA, Veldhuis JD 1992 Is the calcium signal induced by FSH on swine granulosa cells mediated by adenosine 3',5'-cyclic monophosphate-dependent protein kinase A? Endocrinology 130:1862–1866[Abstract/Free Full Text]
  178. Flores JA, Garmey JC, Nestler JE, Veldhuis JD 1993 Sites of inhibition of steroidogenesis by activation of protein kinase C in ovarian (granulosa) cells. Endocrinology 132:1983–1990[Abstract/Free Full Text]
  179. Dufau ML, Veldhuis JD, Fraioli F, Johnson ML, Beitins IZ 1983 Mode of secretion of bioactive luteinizing hormone in man. J Clin Endocrinol Metab 57:993–1000[Abstract/Free Full Text]
  180. Benoit AM, Veldhuis JD 1995 Differential effects of pulsatile vs. continuous FSH delivery on the production of cAMP and the accumulation of cytochrome P450 cholesterol side-chain cleavage enzyme (SCC) mRNA in perifused porcine granulosa cells. Biol Reprod 53:46–54[Abstract]
  181. Maitra A, LaVoie HA, Day RN, Garmey JC, Veldhuis JD 1995 Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and IGF-I: synergism with FSH or protein kinase A agonist. Endocrinology 136:5111–5117[Abstract]
  182. Hatey F, Mulsant P, Bonnet A, Benne F, Gasser F 1995 Protein kinase C inhibition of in vitro FSH-induced differentiation in pig granulosa cells. Mol Cell Endocrinol 107:9–16[CrossRef][Medline]
  183. Balasubramanian K, LaVoie HA, Garmey JC, Stocco DM, Veldhuis JD 1997 Regulation of porcine granulosa cell steroidogenic acute regulatory protein (Star) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist. Endocrinology 138:433–439[Abstract/Free Full Text]
  184. Veldhuis JD 1987 Prostaglandin F-2-{alpha} initiates polyphosphatidylinositol hydrolysis and membrane translocation of protein kinase C in swine ovarian cells. Biochem Biophys Res Commun 149: 112–117
  185. Guthrie HD, Cooper BS, Welch GR, Zakaria AD, Johnson LA 1995 Atresia in follicles grown after ovulation in the pig: measurement of increased apoptosis in granulosa cells and reduced follicular fluid estradiol-17ß. Biol Reprod 52:920–927[Abstract]
  186. Tosser-Klopp G, Benne F, Bonnet A, Mulsant P, Gasser F, Hatey F 1997 A first catalog of genes involved in pig ovarian follicular differentiation. Mamm Genome 8:250–254[CrossRef][Medline]
  187. Roy SK, Greenwald GS 1996 Methods of separation and in vitro culture of pre-antral follicles from mammalian ovaries. Hum Reprod Update 2:236–245[Abstract/Free Full Text]
  188. LaVoie HA, Garmey JC, Veldhuis JD 1999 Mechanisms of insulin-like growth factor I augmentation of follicle-stimulating hormone-induced porcine steroidogenic acute regulatory protein gene promoter activity in granulosa cells. Endocrinology 140:146–153[Abstract/Free Full Text]
  189. Leighton JK, Grimes RW, Canning S, Hammond JM 1993 Expression of the IGF system in primary and immortalized porcine ovarian granulosa cells. Mol Cell Endocrinol 97:29–35[CrossRef][Medline]
  190. Sites CK, Patterson K, Jamison CS, Degen SJ, LaBarbera AR 1994 Follicle-stimulating hormone (FSH) increases FSH receptor messenger ribonucleic acid while decreasing FSH binding in cultured porcine granulosa cells. Endocrinology 134:11–17[Abstract/Free Full Text]
  191. Veldhuis JD 1987 Mechanisms subserving hormone action in the ovary: role of calcium ions as assessed by steady-state calcium exchange in cultured swine granulosa cells. Endocrinology 120:445–449[Abstract/Free Full Text]
  192. Kwan I, Farookhi R, Huynh HT, Murphy BD, Turner JD, Downey BR 1996 Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2). Mol Reprod Dev 45:299–307[CrossRef][Medline]
  193. Conley AJ, Ford SP 1989 Direct luteotrophic effect of oestradiol-17ß on pig corpora lutea. J Reprod Fertil 87:125–131[Abstract/Free Full Text]
  194. Ford SP, Magness RR, Farley DB, Van Orden DE 1982 Local and systemic effects of intrauterine estradiol-17ß on luteal function of nonpregnant sows. J Anim Sci 55:657–664
  195. Kraeling RR, Barb CR, Davis BJ 1975 Prostaglandin-induced regression of porcine corpora lutea maintained by estrogen. Prostaglandins 9:459–462[CrossRef][Medline]
  196. Garbers DL, First NL 1969 The effects of injected oestradiol-17ß, progesterone and dietary ICI 33828 on ovarian and pituitary functions in the sow and gilt. J Reprod Fertil 20:451–464[Abstract/Free Full Text]
  197. Gardner ML, First NL, Casida LE 1963 Effect of exogenous estrogens on corpus luteum maintenance in gilts. J Anim Sci 22:132–134[Abstract/Free Full Text]
  198. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331:1056–1061[Abstract/Free Full Text]
  199. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166[Abstract/Free Full Text]
  200. Korach KS 1994 Insights from the study of animals lacking functional estrogen receptor. Science 266:1524–1527[Abstract/Free Full Text]
  201. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS 1995 Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–1454[Abstract/Free Full Text]
  202. Das SK, Taylor JA, Korach KS, Paria BC, Dey SK, Lubahn DB 1997 Estrogenic responses in estrogen receptor-alpha deficient mice reveal a distinct estrogen signaling pathway. Proc Natl Acad Sci USA 94:12786–12791[Abstract/Free Full Text]
  203. Cooke PS, Buchanan DL, Lubahn DB, Cunha GR 1998 Mechanism of estrogen action: lessons from the estrogen receptor-{alpha}-knockout mouse. Biol Reprod 59:470–475[Free Full Text]
  204. Curtis SW, Clark J, Myers P, Korach KS 1999 Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor {alpha} knockout mouse uterus. Proc Natl Acad Sci USA 96:3646–3651[Abstract/Free Full Text]
  205. Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar M, Mayo KE, Korach KS 1999 Targeted disruption of the estrogen receptor-{alpha} gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology 140:2733–2744[Abstract/Free Full Text]
  206. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson J-A, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
  207. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417[Abstract/Free Full Text]
  208. Rosenfeld CS, Murray AA, Simmer G, Hufford MG, Smith MF, Spears N, Lubahn DB 2000 Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-{alpha} knockout mice. Biol Reprod 62:599–605[Abstract/Free Full Text]
  209. Dukes M, Chester R, Yarwood L, Wakeling AE 1994 Effects of a non-steroidal pure antioestrogen, ZM 189,154, on oestrogen target organs of the rat including bones. J Endocrinol 141:335–341[Abstract/Free Full Text]
  210. Sourla A, Luo S, Labrie C, Belanger A, Labrie F 1997 Morphological changes induced by 6-month treatment of intact and ovariectomized mice with tamoxifen and the pure antiestrogen EM-800. Endocrinology 138:5605–5617[Abstract/Free Full Text]
  211. Luo S, Martel C, Sourla A, Gauthier S, Merand Y, Belanger A, Labrie C, Labrie F 1997 Comparative effects of 28-day treatment with the new anti-estrogen EM-800 and tamoxifen on estrogen-sensitive parameters in intact mice. Int J Cancer 73:381–391[CrossRef][Medline]
  212. Risma KA, Clay CM, Nett TM, Wagner T, Yun J, Nilson JH 1995 Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci USA 92:1322–1326[Abstract/Free Full Text]
  213. Risma KA, Hirshfield AN, Nilson JH 1997 Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology. Endocrinology 138:3540–3547[Abstract/Free Full Text]
  214. Couse JF, Bunch DO, Lindzey J, Schomberg DW, Korach KS 1999 Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse. Endocrinology 140:5855–5865[Abstract/Free Full Text]
  215. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
  216. Fisher CR, Graves KH, Parlow AF, Simpson ER 1998 Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc Natl Acad Sci USA 95:6965–6970[Abstract/Free Full Text]
  217. Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN 1998 Molecular mechanisms of ovulation and luteinization. Mol Cell Endocrinol 145:47–54[CrossRef][Medline]
  218. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M 2000 Effect of single and compound knockouts of estrogen receptors {alpha} (ER{alpha}) and ß (ERß) on mouse reproductive phenotypes. Development 127:4277–4291[Abstract]
  219. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen receptors {alpha} and ß. Science 286:2328–2331[Abstract/Free Full Text]
  220. Simpson ER 1998 Genetic mutations resulting in estrogen insufficiency in the male. Mol Cell Endocrinol 145:55–59[CrossRef][Medline]
  221. Honda S, Harada N, Ito S, Takagi Y, Maeda S 1998 Disruption of sexual behavior in male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene. Biochem Biophys Res Commun 252: 445–449
  222. Robertson KM, O’Donnell L, Jones ME, Meachem SJ, Boon WC, Fisher CR, Graves KH, McLachlan RI, Simpson ER 1999 Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene. Proc Natl Acad Sci USA 96:7986–7991[Abstract/Free Full Text]
  223. Jones ME, Simpson ER 2000 Oestrogens in male reproduction. Baillieres Clin Endocrinol Metab 14:505–516
  224. Jones ME, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto J, Oz OK, Leury BJ, Robertson KM, Yao S, Simpson ER2000 Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc Natl Acad Sci USA 97:12735–12740
  225. Nemoto Y, Toda K, Ono M, Fujikawa-Adachi K, Saibara T, Onishi S, Enzan H, Okada T, Shizuta Y 2000 Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice. J Clin Invest 105:1819–1825[Medline]
  226. Oz OK, Zerwekh JE, Fisher C, Graves K, Nanu L, Millsaps R, Simpson ER 2000 Bone has a sexually dimorphic response to aromatase deficiency. J Bone Miner Res 15:507–514[CrossRef][Medline]
  227. Agarwal VR, Sinton CM, Liang C, Fisher C, German DC, Simpson ER 2000 Upregulation of estrogen receptors in the forebrain of aromatase knockout (ArKO) mice. Mol Cell Endocrinol 162:9–16[CrossRef][Medline]
  228. Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG, Jones ME, Simpson ER, Findlay JK 2000 An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene. Endocrinology 141:2614–2623[Abstract/Free Full Text]
  229. Miyaura C, Toda K, Inada M, Ohshiba T, Matsumoto C, Okada T, Ito M, Shizuta Y, Ito A 2001 Sex- and age-related response to aromatase deficiency in bone. Biochem Biophys Res Commun 280:1062–1068[CrossRef][Medline]
  230. Toda K, Saibara T, Okada T, Onishi S, Shizuta Y 2001 A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19). J Endocrinol 168:217–220[Abstract]
  231. Simpson ER 2000 Genetic mutations resulting in loss of aromatase activity in humans and mice. J Soc Gynecol Invest 7:S18–21
  232. Billiar RB, Loukides JA, Miller MM 1992 Evidence of the estrogen receptor in the ovary of the baboon (Papio anubis). J Clin Endocrinol Metab 75:1159–1165[Abstract]
  233. Wu TC, Wang L, Wan Y-JY 1993 Detection of estrogen receptor messenger ribonucleic acid in human oocytes and cumulus-oocyte complexes using reverse transcriptase-polymerase chain reaction. Fertil Steril 59:54–59[Medline]
  234. Hurst BS, Zilberstein M, Chou JY, Litman B, Stephens J, Leslie KK 1995 Estrogen receptors are present in human granulosa cells. J Clin Endocrinol Metab 80:229–232[Abstract]
  235. Hurst BS, Leslie KK 1997 Uses of plasmid technologies. Mol Hum Reprod 3:643–645[Abstract/Free Full Text]
  236. Chandrasekher YA, Melner MH, Nagalla SR, Stouffer RL 1994 Progesterone receptor, but not estradiol receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and the corpus luteum in rhesus monkeys. Endocrinology 135:307–314[Abstract]
  237. Revelli A, Pacchioni D, Cassoni P, Bussolati G, Massobrio M 1996 In situ hybridization study of messenger RNA for estrogen receptor and immunohistochemical detection of estrogen and progesterone receptors in the human ovary. Gynecol Endocrinol 10:177–186[Medline]
  238. Brandenberger AW, Tee MK, Lee JY, Chao V, Jaffe RB 1997 Tissue distribution of estrogen receptors {alpha} (ER-{alpha}) and ß (ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 82:3509–3512[Abstract/Free Full Text]
  239. Enmark E, Pelto-Huikko P, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjold M, Gustafsson J-A 1997 Human estrogen receptor ß-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 82:4258–4265[Abstract/Free Full Text]
  240. Pau CY, Pau KY, Spies HG 1998 Putative estrogen receptor ß and {alpha} mRNA expression in male and female rhesus macaques. Mol Cell Endocrinol 146:59–68[CrossRef][Medline]
  241. Chaffin CL, Stouffer RL, Duffy DM 1999 Gonadotropin and steroid regulation of steriod receptor and aromatic hydrocarbon receptor mRNA in macaque granulosa cells during the periovulatory interval. Endocrinology 140:4753–4760[Abstract/Free Full Text]
  242. Chiang CH, Cheng KW, Igarashi S, Nathwani PS, Leung PC 2000 Hormonal regulation of estrogen receptor alpha and beta gene expression in human granulosa-luteal cells in vitro. J Clin Endocrinol Metab 85:3828–3839[Abstract/Free Full Text]
  243. Flouriot G, Griffin C, Kenealy M, Sonntag-Buck V, Gannon F 1998 Differentially expressed messenger RNA isoforms of the human estrogen receptor-{alpha} gene are generated by alternative splicing and promoter usage. Mol Endocrinol 12:1939–1954[Abstract/Free Full Text]
  244. Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, M M 1998 Molecular cloning and characterization of human estrogen receptor ßcx: a potential inhibitor of estrogen action in human. Nucleic Acids Res 26:3505–3512[Abstract/Free Full Text]
  245. Misao R, Nakanishi Y, Sun WS, Fujimoto J, Iwagaki S, Hirose R, Tamaya T 1999 Expression of oestrogen receptor {alpha} and ß mRNA in corpus luteum of human subjects. Mol Hum Reprod 5:17–21[Abstract/Free Full Text]
  246. Hild-Petito S, Stouffer RL, Brenner RM 1988 Immunohistochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 123:2896–2905[Abstract/Free Full Text]
  247. Greene GL, Nolan C, Engler JP, Jensen EV 1980 Monoclonal antibodies to human estrogen receptor. Proc Natl Acad Sci USA 77:5115–5119[Abstract/Free Full Text]
  248. Linstedt AD, West NB, Brenner RM 1986 Analysis of monomeric-dimeric states of the estrogen receptor with monoclonal antiestrophillins. J Steroid Biochem 24:677–686[CrossRef][Medline]
  249. Iwai T, Nanbu Y, Taii S, Fujii S, Mori T 1990 Immunohistochemical localization of estrogen receptors and progesterone receptors in the human ovary throughout the menstrual cycle. Virchows Arch 412:369–375
  250. Iwai T, Fujii S, Nanbu Y, Nonogaki H, Konishi I, Mori T, Okamura H 1991 Effect of human chorionic gonadotropin on the expression of progesterone receptors and estrogen receptors in rabbit ovarian granulosa cells and the uterus. Endocrinology 129:1840–1848[Abstract/Free Full Text]
  251. Horie K, Takakura K, Fujiwara H, Suginami LS, Liao S, Mori T 1992 Immunohistochemical localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to oestrogen and progesterone receptor expression. Hum Reprod 7: 184–190
  252. Hutz RJ, Wagner N, Krause P, Fisher C, Syed N, Dierschke DJ, Monniaux D, Tomanek M 1993 Localization of estrogen receptors in rhesus monkey ovary. Am J Primatol 31:299–309[CrossRef]
  253. Suzuki T, Sasano H, Kimura N, Tamura M, Fukaya Yajima A, Nagura H 1994 Immunohistochemical localization of progesterone, androgen, and oestrogen receptors in the human ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes. Hum Reprod 9:1589–1595[Abstract/Free Full Text]
  254. Taylor AH, Al-Azzawi F 2000 Immunolocalisation of oestrogen receptor ß in human tissues. J Mol Endocrinol 24:145–155[Abstract]
  255. Duffy DM, Chaffin CL, Stouffer RL 2000 Expression of estrogen receptor alpha and beta in the rhesus monkey corpus luteum during the menstrual cycle: regulation by luteinizing hormone and progesterone. Endocrinology 141:1711–1717[Abstract/Free Full Text]
  256. Pelletier G, El-Alfy M 2000 Immunocytochemical localization of estrogen receptors {alpha} and ß in the human reproductive organs. J Clin Endocrinol Metab 85:4835–4840[Abstract/Free Full Text]
  257. Yanase T, Simpson ER, Waterman M 1991 17{alpha}-hydroxylase/17,20-lyase deficiency: from clinical investigation to molecular definition. Endocr Rev 12:91–108[Abstract/Free Full Text]
  258. Mallin SR 1968 Congenital adrenal hyperplasia secondary to 17-hydroxylase deficiency: two sisters with amenorrhea, hypokalemia, hypertension and cystic ovaries. Ann Intern Med 70:69–73[CrossRef]
  259. Araki S, Chikazawa K, Sekiguchi I, Yamauchi H, Motoyama M, Tamada T 1987 Arrest of follicular development in a patient with 17{alpha}-hydroxylase deficiency: folliculogenesis in association with a lack of estrogen synthesis in the ovaries. Fertil Steril 47:169–172[Medline]
  260. Nagamani M, Dinh TV 1986 17{alpha}-Hydroxylase deficiency in genetic females. A report of two cases. J Reprod Med 31:734–738[Medline]
  261. Roger M, Merceron RE, Girard F, Canlorbe P, Dehennin L, Konopka P, Seneze J, Toublanc JE 1982 Dexamethasone-suppressible hypercorticosteronism in 46XX subjects with ambiguous genitalia and ovarian cysts. Partial defect of 17{alpha}-hydroxyase or 17,20-desmolase. Horm Res 16:23[Medline]
  262. Rabinovici J, Blankstein J, Goldman B, Rudak E, Dor Y, Pariente C, Geir A, Lunenfeld B, Mashiac S 1989 IVF and primary embryonic cleavage are possible in 17{alpha}-hydroxylase deficiency despite extremely low intrafollicular 17ß E2. J Clin Encrinol Metab 68:693–697[Abstract/Free Full Text]
  263. Geier A, Lunenfeld B, Pariente C, Kotev-Emeth S, Shadmi A, Kokia E, Blankstein J 1987 Estrogen receptor binding material in blood of patients following clomiphene citrate administration: determination by radioreceptor assay. Fertil Steril 47:778–784[Medline]
  264. Pariente C, Rabinovici J, Lunenfeld B, Rudak E, Dor J, Mashiach S, Levran D, Blankstein J, Geier A 1990 Steroid secretion by granulosa cells isolated from a woman with 17{alpha}-hydroxylase deficiency. J Clin Endocrinol Metab 71:984–987[Abstract/Free Full Text]
  265. Zelinski-Wooten MB, Hess DL, Wolf DP, Stouffer RL 1994 Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fertil Steril 61:1147–1155[Medline]
  266. Mannaerts B, Uilenbroek J, Schot P, DeLeeuw R 1994 Folliculogenesis in hypophysectomized rats after treatment with recombinant human follicle-stimulating hormone. Biol Reprod 51:72–81[Abstract]
  267. Zelinski-Wooten MB, Hess DL, Baughman WO, Molskness TA, Wolf DP, Stouffer RL 1993 Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in Rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function. J Clin Endocrinol Metab 76:988–995[Abstract]
  268. Selvaraj N, Shetty G, Vijayalakshmi K, Bhatnagar AS, Moudgal NR 1994 Effect of blocking oegrogen synthesis with a new generation aromatase inhibitor CGS 16949A on follicular maturation induced by pregnant mare serum gonadotrophin in the immature rat. J Endocrinol 142:563–570[Abstract/Free Full Text]
  269. Selvaraj J, Bhatnagar AS, Moudgal NR 1995 Aromatase inhibition during follicular phase in Bonnet monkeys: is there a role for estrogen in follicular maturation in the primate? Endocrine 3: 245–249
  270. Shetty G, Krishnamurthy H, Krishnamurthy HN, Bhatnagar AS, Moudgal RN 1997 Effect of estrogen deprivation on the reproductive physiology of male and female primates. J Steroid Biochem Mol Biol 61:157–166[CrossRef][Medline]
  271. Ito Y, Fisher CR, Conte FA, Grambach MM, Simpson ER 1993 Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries. Proc Natl Acad Sci USA 90:11673–11677[Abstract/Free Full Text]
  272. Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER 1994 A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom). J Clin Endocrinol Metab 78:1287–1292[Abstract]
  273. Morishima A, Grumbach MM, Simpson ER, Fisher C, Quin K 1995 Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J Clin Endocrinol Metab 80:3689–3698[Abstract]
  274. Mullis PE, Yoshimura N, Kuhlmann B, Lippuner K, Jaeger P, Harada H 1997 Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood. J Clin Endocrinol Metab 82:1739–1745[Abstract/Free Full Text]
  275. Bulun SE 1996 Aromatase deficiency in women and men: would you have predicted the phenotypes? J Clin Endocrinol Metab 81:867–71[CrossRef][Medline]
  276. Couzinet B, Lestrat N, Brailly S, Forest M, Schaison G 1988 Stimulation of ovarian follicular maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab 66:552–556[Abstract/Free Full Text]
  277. Schoot DCJM, Herjan JT, Bennink C, Mannaerts B, Lamberts SWJ, Bouchard P, Fauser BC 1992 Human recombinant FSH induced growth of preovulatory follicles without concomitant increase in androgen and estrogen biosynthesis in a woman with isolated GT deficiency. J Clin Endocrinol Metab 74:1471–1473[Abstract]
  278. Mannaerts B, Leeuw R, VanRavestein JG, Wezenbeek P, Schuurs A, Kloosterboer H 1991 Comparative in vitro and in vivo studies on the biological characteristics of human recombinant follicle-stimulating hormone. Endocrinology 129:2623–2630[Abstract/Free Full Text]
  279. Shoham Z, Mannaerts B, Insler V, Coelingh HB 1993 Induction of follicular growth using recombinant human follicle-stimulating hormone in two volunteer women with hypogonadotropic hypogonadism. Fertil Steril 59:738–742[Medline]
  280. Shoham Z, Balen A, Patel A, Jacobs HS 1991 Results of ovulation induction using human menopausal gonadotropins or purified follicle-stimulating hormone in hypogonadotropic hypogonadism patients. Fertil Steril 56:1048–1053[Medline]
  281. Schoot BCJM, Harlin J, Shoham Z, Mannaerts BMJL, Lahlou N, Bouchard P, Coelingh HJTB, Faiser BCJM 1994 Recombinant human follicle-stimulating hormone and ovarian response in gonadotrophin-deficient women. Hum Reprod 9:1237–1242[Abstract/Free Full Text]
  282. Balasch J, Miro F, Burzaco I, Casamitjana R, Civico S, Ballesca JL, Puerto B, Vanrell JA 1995 The role of luteinizing hormone in human follicle development and oocyte fertility: evidence from in-vitro fertilization in a woman with long-standing hypogonadotrophic hypogonadism and using recombinant human follicle stimulating hormone. Hum Reprod 10:1678–1683[Abstract/Free Full Text]
  283. Lunenfeld B, Haviv F, Insler V 1996 Gonadotropin-releasing hormone analogs in perspective: a promise fulfilled. In: Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive Endocrinology, Surgery and Technology, ed 1. Lippincott-Raven, Philadelphia, pp 1649–1662
  284. Ben-Chetrit A, Gotlieb L, Wong PY, Casper RF 1996 Ovarian response to recombinant human follicle-stimulating hormone in luteinizing hormone-depleted women: examination of the two cell, two gonadotropin theory. Fertil Steril 65:711–717[Medline]
  285. Kakar SS, Musgrove LC, Devor DC, Sellers JC, Neill JD 1992 Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Biochem Biophys Res Commun 189:289–295[CrossRef][Medline]
  286. Nathwani PS, Kang SK, Cheng KW, Choi KC, Leung PC 2000 Regulation of gonadotropin-releasing hormone and its receptor gene expression by 17ß-estradiol in cultured human granulosa-luteal cells. Endocrinology 141:1754–1763[Abstract/Free Full Text]
  287. Kang SK, Tai CJ, Nathwani PS, Leung PC 2001 Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells. Endocrinology 142:182–192[Abstract/Free Full Text]
  288. Loy RA 1994 The pharmacology and potential applications of GnRH antagonists. Curr Opin Obstet Gynecol 6:262–268[Medline]
  289. Karnitis VJ, Townson DH, Friedman CI, Danforth DR 1994 Recombinant human follicle-stimulating hormone stimulates multiple follicular growth, but minimal estrogen production in gonadotropin-releasing hormone antagonist-treated monkeys: examining the role of luteinizing hormone in follicular development and steroidogenesis. J Clin Endocrinol Metab 79:91–97[Abstract]
  290. Zelinski-Wooten MB, Hutchison JS, Hess DL, Wolf DP, Stouffer RL 1995 Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys. Hum Reprod 10:1658–1666[Abstract/Free Full Text]
  291. Wang XN, Greenwald GS 1993 Human chorionic gonadotropin or human recombinant follicle-stimulating hormone (FSH)-induced ovulation and subsequent fertilization and early embryo development in hypophysectomized FSH-primed mice. Endocrinology 132:2009–2016[Abstract/Free Full Text]
  292. Koering MJ, Danforth DR, Hodgen GD 1991 Early folliculogenesis in the primate ovaries: testing the role of estrogen. Biol Reprod 45:890–897[Abstract]
  293. Koering MJ, Danforth DR, Hodgen GD 1994 Early follicle growth in the juvenile Macaca monkey ovary: the effects of estrogen priming and follicle-stimulating hormone. Biol Reprod 50:686–694[Abstract]
  294. Hutz RJ, Dierschke DJ, Wolf RC 1990 Role of estradiol in regulating ovarian follicular atresia in rhesus monkeys: a review. J Med Primatol 19:553–571[Medline]
  295. Hutz RJ, Morgan PM, Krueger GS, Durning M, Dierschke DJ 1989 Direct effect of estradiol-17ß on progesterone accumulation by ovarian granulosa cells from Rhesus monkeys. Am J Primatol 17:87–92
  296. Harlow CR, Hillier SG, Hodges JK 1986 Androgen modulation of follicle-stimulating hormone-induced granulosa cell steroidogenesis in the primate ovary. Endocrinology 119:1403–1405[Abstract/Free Full Text]
  297. Shaw HJ, Hodges JK 1992 Effects of oestradiol-17ß on FSH-stimulated steroidogenesis in cultured marmoset granulosa cells. J Endocrinol 132:123–131[Abstract/Free Full Text]
  298. Shaw HJ, Boddy SE, Hodges JK 1993 Developmental changes in marmoset granulosa cell responsiveness to insulin-like growth factor-I: interactions with follicle stimulating hormone and estradiol. Biol Reprod 49:568–576[Abstract]
  299. Hutz RJ 1989 Disparate effects of estrogens on in vitro steroidogenesis by mammalian and avian granulosa cells. Biol Reprod 40:709–713[Abstract]
  300. Zelinski-Wooten MB, Stouffer RL 1996 Steroid receptors and action in the primate follicle. Trends Endocrinol Metab 7:177–183[CrossRef][Medline]
  301. Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 254:1636–1639[Abstract/Free Full Text]
  302. Smith CL, Conneely OM, O’Malley BW 1993 Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone. Proc Natl Acad Sci USA 90:6120–6124[Abstract/Free Full Text]
  303. Ignar-Trowbridge DM, Pimentel M, Parker MG, McLachlan JA, Korach KS 1996 Peptide growth factor cross-talk with the estrogen receptor requires the A/B domain and occurs independently of protein kinase C or estradiol. Endocrinology 137:1735–1744[Abstract]
  304. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662[Abstract/Free Full Text]
  305. Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Medline]
  306. Aronica SM, Katzenellenbogen BS 1993 Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I. Mol Endocrinol 7:743–752[Abstract/Free Full Text]
  307. Gonzalez D, Bellido C, Aguilar R, Garrido-Gracia JC, Hernandez G, Alonso R, Sanchez-Criado JE 2000 Luteinizing hormone secretion elicited in a ligand-independent activation of progesterone receptor manner at pituitary level in the rat: differential effect of two selective estrogen receptor modulators. Neurosci Lett 289: 111–114
  308. Sadar MD, Gleave ME 2000 Ligand-independent activation of the androgen receptor by the differentiation agent butyrate in human prostate cancer cells. Cancer Res 60:5825–5831[Abstract/Free Full Text]
  309. Coutts AS, Leygue E, Murphy LC 1999 Variant estrogen receptor-alpha messenger RNA expression in hormone-independent human breast cancer cells. J Mol Endocrinol 23:325–336[Abstract]
  310. White R, Sjoberg M, Kalkhoven E, Parker MG 1997 Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine. EMBO J 16:1427–1435[CrossRef][Medline]
  311. Weigel NL, Zhang Y 1998 Ligand-independent activation of steroid hormone receptors. J Mol Med 76:469–479[CrossRef][Medline]
  312. Kudolo GB, Elder MG, Myatt L 1984 A novel oestrogen-binding species in rat granulosa cells. J Endocrinol 102:83–91[Abstract/Free Full Text]
  313. Kudolo GB, Elder MG, Myatt L 1984 Further characterization of the second oestrogen-binding species of the rat granulosa cell. J Endocrinol 102:93–102[Abstract/Free Full Text]
  314. Kudolo GB, Elder MG, Myatt L 1987 Oestrogen receptor in the granulosa cell during postnatal development of the rat ovary. J Endocrinol 112:333–338[Abstract/Free Full Text]
  315. Wehling M, Christ M, Thiesen K 1991 High affinity aldosterone binding to plasma membrane rich fractions from mononuclear leukocytes: is there a membrane receptor for mineralocorticoids? Biochem Biophys Res Commun 181:1306–1312[CrossRef][Medline]
  316. Wehling M, Christ M, Thiesen K 1992 Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action. Am J Physiol 263:E974–979
  317. Hua SY, Chen YZ 1989 Membrane receptor-mediated electrophysiological effects of glucocorticoid on mammalian neurons. Endocrinology 124:687–691[Abstract/Free Full Text]
  318. Trueba M, Ibarrola I, Vallejo AI, Sancho MJ, Marino A, Macarulla JM 1989 Characterization of specific binding sites for corticosterone in mouse liver plasma membrane. Membr Biochem 8:229–239[Medline]
  319. Orchinik M, Murray TF, Moore FL 1991 A corticosteroid receptor in neuronal membranes. Science 252:1848–1851[Abstract/Free Full Text]
  320. Chen YZ, Jua SY, Wang CA, Wu LG, Gu Q, Xing BR 1991 An electrophysiological study on the membrane receptor-mediated action of glucocorticoids in mammalian neurons. Neuroendocrinology 53:25–30
  321. Orchinik M, Murray TF, Franklin PH, Moore FL 1992 Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes. Proc Natl Acad Sci USA 89:3830–3834[Abstract/Free Full Text]
  322. Machelon V, Nome F, Tesarik J 1998 Nongenomic effects of androstenedione on human granulosa luteinizing cells. J Clin Endocrinol Metab 83:263–269[Abstract/Free Full Text]
  323. Benten WP, Lieberherr M, Stamm O, Wrehlke C, Guo Z, Wunderlich F 1999 Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol Biol Cell 10:3113–3123[Abstract/Free Full Text]
  324. Benten WP, Lieberherr M, Giese G, Wrehlke C, Stamm O, Sekeris CE, Mossmann H, Wunderlich F 1999 Functional testosterone receptors in plasma membranes of T cells. FASEB J 13:123–133[Abstract/Free Full Text]
  325. Baulieu EE, Godeau F, Schorderet M, Schorderet-Slatkine S 1978 Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium. Nature 275:593–598[CrossRef][Medline]
  326. Finidori-Lepicard J, Schorderet-Slatkine S, Janoune J, Baulieu EE 1981 Progesterone inhibits membrane-bound adenylate cyclase in Xenopus laevis oocytes. Nature 292:255–257[CrossRef][Medline]
  327. Finidori J, Hanoune J, Baulieu EE 1982 Adenylate cyclase in Xenopus laevis oocytes: characterization of the progesterone-sensitive, membrane-bound form. Mol Cell Endocrinol 18:211–227
  328. Baulieu EE 1983 Steroid-membrane-adenylate cyclase interactions during Xenopus laevis oocyte meiosis reinitiation: a new mechanism of steroid hormone action. Exp Clin Endocrinol 81:3–16[Medline]
  329. Baulieu EE, Schorderet-Slatkine S 1983 Steroid and peptide control mechanisms in the membrane of Xenopus laevis oocytes resuming neiotic division. Ciba Found Symp 98:137–158[Medline]
  330. Blondeau JP, Baulieu EE 1984 Progesterone receptor characterized by photoaffinity labelling in the plasma membrane of Xenopus laevis oocytes. Biochem J 219:785–792[Medline]
  331. Blondeau JP, Baulieu EE 1985 Progesterone-inhibited phosphorylation of a unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes. J Biol Chem 260:3617–3625[Abstract/Free Full Text]
  332. Grazzini E, Guillon G, Mouillac B, Zingg HH 1998 Inhibition of oxytocin receptor function by direct binding of progesterone. Nature 392:509–512[CrossRef][Medline]
  333. Rae MT, Menzies GS, Bramley TA 1998 Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes. J Endocrinol 159:413–427[Abstract]
  334. Bayaa M, Booth RA, Sheng Y, Liu XJ 2000 The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism. Proc Natl Acad Sci USA 97:12607–12612[Abstract/Free Full Text]
  335. Gu Q, Korach KS, Moss RL 1999 Rapid action of 17ß-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors. Endocrinology 140:660–666[Abstract/Free Full Text]
  336. McEwen BS 1991 Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol Sci 12:141–147[CrossRef][Medline]
  337. McEwen BS 1992 Steroid hormones: effects on brain development and function. Horm Res 37:1–10
  338. Brussaard AB, Wossink J, Lodder JC, Kits KS 2000 Progesterone-metabolite prevents protein kinase C-dependent modulation of {gamma}-aminobutyric acid type A receptors in oxytocin neurons. Proc Natl Acad Sci USA 97:3625–3630[Abstract/Free Full Text]
  339. Dong E, Matsumoto K, Uzunova V, Sugaya I, Takahata H, Nomura H, Watanabe H, Costa E, Guidotti A 2001 Brain 5{alpha}-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation. Proc Natl Acad Sci USA 98:2849–2854[Abstract/Free Full Text]
  340. Milner TA, McEwen BS, Hayashi S, Li CJ, Reagan LP, Alves SE 2001 Ultrastructural evidence that hippocampal {alpha} estrogen receptors are located at extranuclear sites. J Comp Neurol 429:355–371[CrossRef][Medline]
  341. Pietras RJ, Szego CM 1977 Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265:69–72[CrossRef][Medline]
  342. Pietras RJ, Szego CM 1979 Estrogen receptors in uterine plasma membrane. J Steroid Biochem 11:1471–1483[CrossRef][Medline]
  343. Pietras RJ, Szego CM 1980 Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes. Biochem J 191:743–760[Medline]
  344. Rambo CO, Szego CM 1983 Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds. J Cell Biol 97:679–685[Abstract/Free Full Text]
  345. Komesaroff PA, Black CVS, Westerman RA 1998 A novel, nongenomic action of estrogen on the cardiovascular system. J Clin Endocrinol Metab 83:2313–2316[Abstract/Free Full Text]
  346. Mendelsohn ME, Karas RH 1999 The protective effects of estrogen on the cardiovascular system. N Engl J Med 340:1801–1811[Free Full Text]
  347. Mendelsohn ME 2000 Mechanisms of estrogen action in the cardiovascular system. J Steroid Biochem Mol Biol 74:337–343[CrossRef][Medline]
  348. Mendelsohn ME 2000 Nongenomic ER-mediated activation of endothelial nitric oxide synthase: how does it work? What does it mean? Circ Res 87:956–960[Abstract/Free Full Text]
  349. Caulin-Glaser T, Garcia-Cardena G, Sarrel P, Sessa WC, Bender JR 1997 17 ß-estradiol regulation of human endothelial cell basal nitric oxide release, independent of cytosolic Ca2+ mobilization. Circ Res 81:885–892[Abstract/Free Full Text]
  350. Lantin-Hermoso RL, Rosenfeld CR, Yuhanna IS, German Z, Chen Z, Shaul PW 1997 Estrogen acutely stimulates nitric oxide synthase activity in fetal pulmonary artery endothelium. Am J Physiol 273:L119–L126
  351. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW 1999 Estrogen receptor {alpha} mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401–406[Medline]
  352. Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, Mendelsohn ME, Anderson RG, Shaul PW 2000 Estrogen receptor {alpha} and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87:E44–E52
  353. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL 1992 A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131:1305–1312[Abstract/Free Full Text]
  354. Tesarik J, Mendoza C 1995 Nongenomic effects of 17ß-estradiol on maturing human oocytes: relationship to oocyte developmental potential. J Clin Endocrinol Metab 80:1438–1443[Abstract]
  355. Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER{alpha} and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319[Abstract/Free Full Text]



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Home page
ReproductionHome page
E. Millesi, A. Strauss, T. Burger, I. E Hoffmann, and M. Walzl
Follicular development in European ground squirrels (Spermophilus citellus) in different phases of the annual cycle
Reproduction, August 1, 2008; 136(2): 205 - 210.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
J. L. Kipp, S. M. Kilen, T. K. Woodruff, and K. E. Mayo
Activin Regulates Estrogen Receptor Gene Expression in the Mouse Ovary
J. Biol. Chem., December 14, 2007; 282(50): 36755 - 36765.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
C. Wang, E. R. Prossnitz, and S. K. Roy
Expression of G Protein-Coupled Receptor 30 in the Hamster Ovary: Differential Regulation by Gonadotropins and Steroid Hormones
Endocrinology, October 1, 2007; 148(10): 4853 - 4864.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
J. L. Kipp, S. M. Kilen, S. Bristol-Gould, T. K. Woodruff, and K. E. Mayo
Neonatal Exposure to Estrogens Suppresses Activin Expression and Signaling in the Mouse Ovary
Endocrinology, May 1, 2007; 148(5): 1968 - 1976.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
C. Wang and S. K. Roy
Development of Primordial Follicles in the Hamster: Role of Estradiol-17{beta}
Endocrinology, April 1, 2007; 148(4): 1707 - 1716.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
W. Luo and M. C. Wiltbank
Distinct Regulation by Steroids of Messenger RNAs for FSHR and CYP19A1 in Bovine Granulosa Cells
Biol Reprod, August 1, 2006; 75(2): 217 - 225.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
D. M. Campagne
Should fertilization treatment start with reducing stress?
Hum. Reprod., July 1, 2006; 21(7): 1651 - 1658.
[Abstract] [Full Text] [PDF]


Home page
ReproductionHome page
J. L Juengel, D. A Heath, L. D Quirke, and K. P McNatty
Oestrogen receptor {alpha} and {beta}, androgen receptor and progesterone receptor mRNA and protein localisation within the developing ovary and in small growing follicles of sheep
Reproduction, January 1, 2006; 131(1): 81 - 92.
[Abstract] [Full Text] [PDF]


Home page
Mol. Endocrinol.Home page
Z. T. Ruiz-Cortes, S. Kimmins, L. Monaco, K. H. Burns, P. Sassone-Corsi, and B. D. Murphy
Estrogen Mediates Phosphorylation of Histone H3 in Ovarian Follicle and Mammary Epithelial Tumor Cells via the Mitotic Kinase, Aurora B
Mol. Endocrinol., December 1, 2005; 19(12): 2991 - 3000.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
S. A.R. Doi, M. Al-Zaid, P. A. Towers, C. J. Scott, and K. A.S. Al-Shoumer
Irregular cycles and steroid hormones in polycystic ovary syndrome
Hum. Reprod., September 1, 2005; 20(9): 2402 - 2408.
[Abstract] [Full Text] [PDF]


Home page
Mol Hum ReprodHome page
A. Can, O. Semiz, and O. Cinar
Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis
Mol. Hum. Reprod., June 1, 2005; 11(6): 389 - 396.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
P. Yang, J. Wang, Y. Shen, and S. K. Roy
Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone
Endocrinology, December 1, 2004; 145(12): 5757 - 5766.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
S.K. Goswami, T. Das, R. Chattopadhyay, V. Sawhney, J. Kumar, K. Chaudhury, B.N. Chakravarty, and S.N. Kabir
A randomized single-blind controlled trial of letrozole as a low-cost IVF protocol in women with poor ovarian response: a preliminary report
Hum. Reprod., September 1, 2004; 19(9): 2031 - 2035.
[Abstract] [Full Text] [PDF]


Home page
Hum Reprod UpdateHome page
J. D. Hennebold
Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies
Hum. Reprod. Update, May 1, 2004; 10(3): 227 - 239.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
C. Hegele-Hartung, P. Siebel, O. Peters, D. Kosemund, G. Muller, A. Hillisch, A. Walter, J. Kraetzschmar, and K.-H. Fritzemeier
Impact of isotype-selective estrogen receptor agonists on ovarian function
PNAS, April 6, 2004; 101(14): 5129 - 5134.
[Abstract] [Full Text] [PDF]


Home page
J ANIM SCIHome page
S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter
Ovarian follicular growth and atresia: The relationship between cell proliferation and survival
J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52.
[Abstract] [Full Text] [PDF]


Home page
J. Clin. Endocrinol. Metab.Home page
F. Gaytan, M. L. Barreiro, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere
Immunolocalization of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in the Cyclic Human Ovary
J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 879 - 887.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
P. Yang, A. Kriatchko, and S. K. Roy
Expression of ER-{alpha} and ER-{beta} in the Hamster Ovary: Differential Regulation by Gonadotropins and Ovarian Steroid Hormones
Endocrinology, June 1, 2002; 143(6): 2385 - 2398.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
H. A. LaVoie, D. C. DeSimone, C. Gillio-Meina, and Y. Y. Hui
Cloning and Characterization of Porcine Ovarian Estrogen Receptor {beta} Isoforms
Biol Reprod, March 1, 2002; 66(3): 616 - 623.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
Y. Ikeda, A. Nagai, M.-a. Ikeda, and S. Hayashi
Increased Expression of Mullerian-Inhibiting Substance Correlates with Inhibition of Follicular Growth in the Developing Ovary of Rats Treated with E2 Benzoate
Endocrinology, January 1, 2002; 143(1): 304 - 312.
[Abstract] [Full Text] [PDF]


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