Estrogen receptor-β (ERβ) is expressed in the hypothalamic-pituitary (HP) axis and gonads. ERβ-null (βERKO) female mice display defects in ovarian function and are subfertile, as characterized by fewer pregnancies and smaller litters relative to wild type (WT). We have also shown that the mean amplitude of the proestrus luteinizing hormone (LH) surge is reduced in βERKO female mice compared to WT (6 vs. 22 ng/ml). We hypothesized that the submaximal LH surges in βERKO females are due to functional defects in the ovary that result in less than sufficient estradiol (E2) production, leading to improper stimulation of the HP axis, rather than being due to defects within the HP axis itself. Ovaries from 21 day old βERKO mice were transplanted bilaterally into ovariectomized WT females (βW; n = 5) and vice versa (Wβ; n = 5); WT to WT control transplants (WW; n = 4) and mice not undergoing surgery were also included in the study (WT; n = 5; βERKO; n = 12). Seven weeks after surgery, mice were housed in close proximity to adult male mice, in a 10 h-D 14 h-L lightcycle (D = 2100 h) and estrous cycle stages were determined by daily vaginal cytology for 5 additional weeks. Serial proestrus blood samples were taken from each mouse by mandibular puncture at 1900 h, 2100 h, and 2300 h. Serum LH levels were measured using a Dissociation Enhanced Lanthanide Fluoro-Immuno-Assay (DELFIA) (lower detection level 0.03 ng/ml). Only the peak LH value from each proestrus bleed was used in the analysis. Consistent with our last study, LH surges were defined as LH > 0.43 ng/ml and ranged from 0.5 - 42 ng/ml in WT, 5 - 47 ng/ml in WW, 0.5 - 51 ng/ml in Wβ, 1 - 9 ng/ml in βW, and 0.4 - 42 ng/ml in βERKO. Means ± SE were 28 ± 11 ng/ml in WT, 28 ± 7 ng/ml in WW, 26 ± 7 ng/ml in Wβ, 7 ± 1 ng/ml in βW, and 8 ± 3 ng/ml in βERKO. These data show that the average amplitude of the LH surge was dependent on the presence of ERβ in the ovary, and was not effected by the presence or absence of ERβ in the HP axis. Based on our hypothesis that βERKO ovaries may be unable to produce the estrogen milieu needed for the LH surge to occur, we then supplemented a second group of WT and βERKO mice with 10 µg/kg E2 (n = 7 per genotype) or sesame oil (n = 7 per genotype) on the afternoon of diestrus, and collected blood samples on the following evening (proestrus) to assess LH levels. Peak LH ranged from 0.6 - 35 ng/ml in WT-oil, and 0.8 - 64 ng/ml in WT-E2; means ± SE were 13 ± 6 ng/ml in WT-oil, and 24 ± 9 ng/ml in WT-E2; medians were 8 ng/ml in WT-oil, and 25 ng/ml in WT-E2. βERKO mice were not affected by the E2 treatment (mean peak LH; oil: 4 ± 2 ng/ml vs. E2: 3 ± 1 ng/ml) suggesting that additional ovarian factors besides preovulatory E2 may be altered in βERKO mice. These data further support the need for ERβ in the ovary and less so in the HP axis in regards to the generation of the LH surge. Supported by NIEHS Intramural Research Program.

Adult estrogen receptor-α (ERα) null mice (αERKO) are anovulatory and invariably exhibit ovaries containing multiple, often hemorrhagic cystic follicles, and lack corpora lutea (CL). This phenotype does not appear until after approximately 35 d of age and worsens thereafter. In addition, αERKO ovaries exhibit aberrantly high levels of luteinizing hormone (LH)-receptor, steroidogenesis, and inhibin-A synthesis. To date, these phenotypes have been attributed largely to overstimulation of the ovaries by persistently high LH levels that result from the lack of ERαmediated negative feedback in the hypothalamopituitary axis. Indeed, treatment of young αERKO females with a GnRH antagonist prevents the ovarian phenotype. Still, the positive feedback mechanisms that provide for cyclical LH-surges are also interrupted in αERKO mice. Hence, the ovaries are never exposed to the stimuli necessary to induce ovulation and terminal differentiation of granulosa cells. Therefore, we hypothesized that a lack of cyclical LH-surges in αERKO females allows for the survival of large antral follicles that ultimately degenerate, hemorrhage and form the characteristic cysts. To test this hypothesis, we sought to prevent the ovarian phenotype by treating animals with human chorionic gonadotropin (hCG) at periodic intervals that mimic the natural LH-surge. αERKO females were injected with 10 IU hCG (n=10) or saline (n=9) every 4–5 d over a period of one month, beginning at 30 d of age. Age-matched wild type (WT; n=9) females were injected with saline as controls. Blood and ovaries were collected 3 days after the last injection or, in the case of the WT, on diestrus. Ovaries were used for histology and gene expression analyses, and plasma was prepared for assessing hormone levels. Surprisingly, regular hCG injections did not visibly abate the phenotype of hemorrhagic, cystic follicles in αERKO females. Furthermore, no discernible CL were observed in ovaries of hCG-treated αERKO females. Circulating levels of estradiol and testosterone remained elevated in hCGtreated αERKO females, suggesting further that the treatments did not induce terminal differentiation of the granulosa cells toward a luteal cell phenotype. Real-time PCR analyses revealed no effect of the hCG treatment on the abnormally high expression of LH receptor (Lhcgr), cytochrome P450 17α-hydroxylase/C17–20 lyase (Cyp17a1), cytochrome P450 aromatase (Cyp19a1), and 17β-hydroxysteroid dehydrogenase types I and III, (Hsd17b1, Hsd17b3) that is typical of αERKO ovaries. The failure of “cyclical” hCG-treatments to trigger the formation of CL was unexpected as we have previously shown that αERKO females at 28 and 42 d of age respond to a regimen of exogenous gonadotropins with ovulation and CL formation. Our current findings suggest that prior PMSG treatment may be necessary to the hCG response in αERKO females. Alternatively, the hCG-treatments may have been effective in the beginning of the experiment in the young αERKO females, but no longer one month later in the adult αERKO females that were used for tissue collection. Nonetheless, from the current data we must conclude that exogenous LH-“surges” superimposed upon αERKO females for a period of one month, do not prevent the polycystic ovarian phenotype. Supported by the Intramural Research Program of the NIH and NIEHS. (poster)

The predisposition of the testis and ovary to primarily synthesize testosterone (T) and estradiol (E2), respectively, is due to gonadal-specific cell types that differentially express the various hydroxysteroid (17beta) dehydrogenase (HSD17B) isoforms. In testes, Leydig cells rely on LH stimulation to maintain expression of the type 3 (HSD17B3) isoform, which specifically converts androstenedione to T. In ovaries, thecal interstitial (TI) cells also rely on LH to induce androgen synthesis but lack HSD17B3 and therefore secrete androgens of low biological activity. Therefore, thecal cells may possess a mechanism to repress the Leydig cell phenotype and HSD17B3 expression. E2 is known to inhibit experimentally Leydig cell function and proliferation. In the current study, we provide evidence that E2 prevents the development of functional Leydig-like cells in the murine ovary and that this action is mediated by estrogen receptor (ER) alpha. ERalpha-null (alphaERKO) female mice exhibit testis-like levels of Hsd17b3 expression in the ovaries and male-like levels of plasma T. Herein, we demonstrate that: 1) Hsd17b3 expression in alphaERKO ovaries is a primary effect of the loss of intraovarian ERalpha actions; 2) alphaERKO ovarian cells produce substantial levels of T in vitro, and this is blocked by a HSD17B3-specific inhibitor; 3) Hsd17b3 expression in alphaERKO ovaries is LH regulated and localized to the secondary interstitial (SI)/TI cells; and 4) alphaERKO SI/TI cells possess Leydig-like ultrastructural features. These data indicate that intraovarian ERalpha actions are required to repress Hsd17b3 expression in the ovary and may be important to maintaining a female phenotype in SI/TI cells.

Depending on the estrous/menstrual cycle stage in females, ovarian-derived estradiol (E2) exerts either a negative or a positive effect on the hypothalamic–pituitary axis to regulate the synthesis and secretion of pituitary gonadotropins, LH, and FSH. To study the role of estrogen receptor-α (ERα) mediating these effects, we assessed the relevant parameters in adult wild-type (WT) and ERα-null (αERKO) female mice in vivo and in primary pituitary cell cultures. The αERKO mice exhibited significantly higher plasma and pituitary LH levels relative to WT females despite possessing markedly high levels of circulating E2. In contrast, hypothalamic GnRH content and circulating FSH levels were comparable between genotypes. Ovariectomy led to increased plasma LH in WT females but no further increase in αERKO females, while plasma FSH levels increased in both genotypes. E2 treatment suppressed the high plasma LH and pituitary Lhb mRNA expression in ovariectomized WT females but had no effect in αERKO. In contrast, E2 treatments only partially suppressed plasma FSH in ovariectomized WT females, but this too was lacking in αERKO females. Therefore, negative feedback on FSH is partially E2/ERα mediated but more dependent on ovarian-derived inhibin, which was increased threefold above normal in αERKO females. Together, these data indicate that E2-mediated negative feedback is dependent on functional ERα and acts to primarily regulate LH synthesis and secretion. Studies in primary cultures of pituitary cells from WT females revealed that E2 did not suppress basal or GnRH-induced LH secretion but instead enhanced the latter response, indicating that the positive influence of E2 on gonadotropin secretion may occur at the level of the pituitary. Once again this effect was lacking in αERKO gonadotropes in culture. These data indicate that the aspects of negative and positive effects of E2 on gonadotropin secretion are ERα dependent and occur at the level of the hypothalamus and pituitary respectively.

Targeted disruption of the different ER genes has generated experimental animal models that are very useful in evaluating the distinct and cooperative roles of the two estrogen receptors, ERalpha and ERbeta, in reproductive but also non-reproductive tissues of both sexes. Phenotypic analysis has provided definitive experimental findings for estrogen receptor mediated physiological actions, involving ERalpha in uterine, mammary gland and neuroendocrine sites. ERbeta is involved most dramatically in the ovary as is ERalpha. More detailed studies in combination with tissue specific or inducible ER knock outs will be important for future research.

Data indicate that estrogen-dependent and -independent pathways are involved in the teratogenic/carcinogenic syndrome that follows developmental exposure to 17beta-estradiol or diethylstilbestrol (DES), a synthetic estrogen. However, the exact role and extent to which each pathway contributes to the resulting pathology remain unknown. We employed the alphaERKO mouse, which lacks estrogen receptor-alpha (ERalpha), to discern the role of ERalpha and estrogen signaling in mediating the effects of neonatal DES exposure. The alphaERKO provides the potential to expose DES actions mediated by the second known ER, ERbeta, and those that are ER-independent. Wild-type and alphaERKO females were treated with vehicle or DES (2 microg/pup/day for Days 1-5) and terminated after 5 days and 2, 4, 8, 12, and 20 months for biochemical and histomorphological analyses. Assays for uterine expression of the genes Hoxa10, Hoxa11, and Wnt7a shortly after treatment indicated significant decreases in DES-treated wild-type but no effect in the alphaERKO. In contrast, the DES effect on uterine expression of Wnt4 and Wnt5a was preserved in both genotypes, suggesting a developmental role for ERbeta. Adult alphaERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure exhibited in treated wild-type animals, including atrophy, decreased weight, smooth muscle disorganization, and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent vaginal cornification. Therefore, the lack of DES effects on gene expression and tissue differentiation in the alphaERKO provides unequivocal evidence of an obligatory role for ERalpha in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract.