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Prevention of the Polycystic Ovarian Phenotype and Characterization of Ovulatory Capacity in the Estrogen Receptor- Knockout Mouse

Receptor Biology Section (J.F.C., J.L., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Laboratory of Reproductive Biology (D.O.B.), Department of Cell Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Departments of Obstetrics and Gynecology and Cell Biology (D.W.S.), Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, MD B3–02, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov

	Abstract

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Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor- (ER) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-ß (ERß) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ER in the hypothalamic-pituitary axis of the ER-knockout (ERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the ERKO cystic ovarian phenotype. To investigate a direct role for ER within the ovary, immature ERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature ERKO female was reduced compared with age-matched wild-type (14.5 ± 2.9 vs. 40.6 ± 2.6 oocytes/animal, respectively); however, oocytes collected from the ERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and ERKO females. In addition, ovaries from immature superovulated ERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the ERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERß, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature ERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the ERKO compared with wild-type. These studies indicate that treatment of ERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ER within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the ERKO suggests an intraovarian role for ER in follicular development and ovulation.

	Introduction

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IN 1940, PENCHARZ (1) and Williams (2) independently reported a direct and specific ability of estrogens to increase ovarian weight in the hypophysectomized rat. Since this time, several intraovarian actions of estradiol have been postulated to be essential to normal follicular development and ovarian function, including: 1) induction of increased levels of its own receptor (3); 2) stimulation of DNA synthesis and proliferation (4, 5, 6, 7, 8); 3) modulation of the number and size of intercellular gap junctions (9); and 4) an attenuation of apoptosis and follicular atresia (10). Estradiol is also reported to augment the actions of FSH on granulosa cells, resulting in the maintenance of FSH-receptor (FSH-R) levels (6, 11, 12, 13) and the acquisition of LH-receptor (LH-R) (6, 14, 15, 16, 17), an event critical to successful ovulation. In addition to the intraovarian effects, the endocrine actions of estradiol in the hypothalamic-pituitary axis are also critical to ovarian function and fertility. Gonadotropin synthesis and secretion from the anterior pituitary are at least partially regulated by gonadal steroids via classical feedback mechanisms acting at both hypothalamic and pituitary sites (reviewed in Ref. 18). Several studies have demonstrated the ability of estrogen to suppress transcription of the gonadotropin subunit genes as well as the synthesis and secretion of the dimeric hormones (18). Therefore, ovarian function appears to depend on a multitude of both endocrine and local actions of estradiol that act in concert with the pituitary gonadotropins to provide for proper steroid production and folliculogenesis.

The majority of documented biological actions of estradiol are mediated via the estrogen receptor (ER), a class I member of the thyroid/steroid hormone receptor superfamily of ligand-inducible transcription factors (reviewed in Ref. 19). Previous studies have reported the presence of high-affinity and specific estrogen binding sites in the ovary of the rodent as well as several other species (3, 20, 21). However, two forms of nuclear ER are now known to exist, the well described ER, and the newly discovered ERß. Several studies have demonstrated the presence of the respective mRNAs for both ER and ERß in the ovaries of the mouse (22, 23), rat (24, 25), cow (26), and human (27, 28). Furthermore, studies employing in situ hybridization (24, 25) and immunohistochemistry (26, 29) indicate a distinct expression pattern for the two ERs in the ovary, in which ER is highly expressed in the interstitial/thecal compartment, whereas ERß appears confined to the granulosa cells of growing follicles.

The dissimilar distribution of the two ERs between and within the components of the hypothalamic-pituitary-ovarian axis suggests that separate functions are fulfilled by each. Previous in vivo investigations of the intraovarian actions of estrogen have relied on the use of estrogen antagonists or inhibitors of estrogen synthesis. However, the large amounts of estradiol synthesized in the ovary often attenuate the ability of these methods to completely block local estrogen action and thereby complicate the interpretation of experimental results. Therefore, the estrogen receptor- knockout (ERKO) mouse, previously described as homozygous for a targeted disruption of the ER gene (30, 31), provides a unique tool to clarify the role of each receptor in ovarian function. The conclusions of several past studies concerning the role of ER in the adult female were confirmed in the initial descriptions of the ERKO mouse, including estrogen insensitivity of the reproductive tract (30, 31) and hypothalamic-pituitary axis (32). The ERKO females exhibit ovaries characterized by the presence of multiple hemorrhagic and cystic follicles with no evidence of spontaneous ovulation (30, 33). This phenotype occurs despite a relatively normal expression pattern for the ERß gene in the ovaries of the ERKO mice (23, 33). In the ERKO female, disruption of the negative feedback actions of estradiol in the hypothalamic-pituitary axis results in elevated levels of the gonadotropin subunit mRNAs in the pituitary (32) and in serum LH (34). Therefore, the ERKO ovarian phenotype may be the result of a lack of ER-mediated action either within the ovary and/or at the level of the hypothalamic-pituitary axis. Herein, we describe studies demonstrating the prevention of the adult ERKO ovarian phenotype when pituitary influence is reduced through the use of a GnRH antagonist. We also describe the ability of the immature ERKO to ovulate and exhibit a relatively normal regulation of several genes critical to folliculogenesis and ovulation when stimulated with exogenous gonadotropins.

	Materials and Methods

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GnRH antagonist treatment
All procedures involving animals were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. To assess the role the chronically elevated LH may play on the development of the polycystic phenotype of the adult ERKO ovary, wild-type and ERKO females were treated with a GnRH antagonist. Each treatment consisted of a single sc injection of 0.15 ml volume of the GnRH antagonist, Antide (Sigma, St. Louis, MO) (35), suspended in vehicle (20% propylene glycol in 0.85% saline). Animals were single-housed and maintained under controlled lighting (12-h light, 12-h dark). Treatments consisted of either vehicle or Antide at 15 µg, 30 µg, or 60 µg between 1200–1300 h every 48 h. Treatments began when the animals were 28 days of age, before sexual maturation and the onset of the ERKO ovarian phenotype (33) and carried out through to 53 days of age, for a total of 12 treatments. Each group consisted of at least four animals/genotype/dose (except for ERKO-vehicle = 3). Body weights were monitored throughout the study with each group showing similar age-related increases regardless of genotype or treatment (body weight, g; 28 days = 11.9 ± 0.3; 53 days = 18.3 ± 0.2). Animals were killed at 53 days of age, 18–24 h after the final treatment. Serum was processed from blood collected from the descending aorta. One ovary was immediately snap frozen on dry ice for later RNA extraction and the other was fixed in 10% buffered formalin at 4 C for 6–8 h and then transferred to 70% ethanol at 4 C. For histological analysis, fixed tissues were paraffin embedded, sectioned at 5 µm and stained with hematoxylin and eosin according to standard histological procedures.

Superovulation with exogenous gonadotropins
Superovulation assays were carried out on wild-type and ERKO females at both immature (28 days) and peripubertal (42 days) ages. Each trial (immature = 5; peripubertal = 2) consisted of a single sc injection with 2.2 IU PMSG (Sigma) followed 48–52 h later with 3.2 IU human CG (hCG) (Sigma). The animals were then killed 16–20 h after the hCG injection and the ovaries and oviduct removed to M-2 medium (Specialty Media, Lavallette, NJ) supplemented with 0.3% hyaluronidase (Sigma). The oocyte/cumulus mass was surgically extracted from the oviduct and the oocytes were counted after enzymatic disassociation from the surrounding cumulus. The ovary/oviduct was then fixed in 10% buffered formalin and prepared for histological analysis as described above.

In vitro fertilization assays
For in vitro fertilization assays, immature (28 days) control (wild-type or heterozygous) and ERKO females were superovulated as described above. Oocyte/cumulus masses were collected from the oviducts in M2 medium (Specialty Media) under paraffin oil, pooled according to genotype, and treated with 0.3% hyaluronidase to remove the cumulus cells. Cumulus-free oocytes were washed through 2 drops of M2 and transferred to 200 µl droplets of M16 medium (Specialty Media) with BSA (20 mg/ml) under oil and incubated at 37 C in 5% CO₂. Epididymal sperm were collected from 2 CD-1 males (at least 10 wks of age) per experiment in 500 µl M2 media. The sperm were diluted 1:10 in M16 medium with BSA (20 mg/ml) under oil for capacitation at 37 C for 90 min. Oocytes were inseminated with an equal number of motile sperm in a final droplet volume of 125–200 µl at a final concentration of 1 x 10⁶ sperm/ml. After 8–10 h of incubation at 37 C, the oocytes were collected and washed through three drops of M2 medium, fixed in 2.5% glutaraldehyde, transferred to slides, and stored in 3.7% formaldehyde overnight. The oocytes were dehydrated with 95% ethanol, stained with acetolacmoid (36), and scored as fertilized if two pronuclei and a sperm tail were present within the vitellus. Three separate trials involving animals of each genotype were carried out.

RNA isolation and ribonuclease protection assay (RPA)
When the mice were killed, tissues were quickly removed and snap frozen on dry ice, followed by storage at -70 C until processing. All ovaries were trimmed of oviduct and surrounding tissue before freezing. Total RNA was isolated using TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. The concentration of the final preparations were calculated from an A₂₆₀ reading using a Beckman Coulter, Inc. DU-640 UV spectrophotometer. An aliquot of all RNA preparations was then analyzed on a 1% agarose gel to ensure integrity before further analysis.

We have previously described the generation and use of the antisense riboprobes employed to detect the mRNAs for the mouse ERß (23), LH-R, FSH-R, and PR (33) genes. The antisense riboprobe for PGS-2 mRNA corresponded to bp 175–591 of the mouse PGS-2 cDNA (GenBank no. M64291) and was transcribed from a clone generated via PCR amplification of the fragment from wild-type mouse uterine RNA and cloned into the pCR-Script SK- phagemid (Stratagene Cloning Systems, La Jolla, CA). The antisense riboprobe for the cyclin-D2 mRNA was subcloned from the complete mouse cyclin-D2 cDNA kindly provided by Dr. Charles Sherr, St. Judes Hospital (37), and corresponded to bp 506–864 (GenBank accesion no. M83749). The riboprobe for the mouse P450_scc mRNA has been previously characterized (38) and was kindly provided by Dr. Keith Parker, University of Texas-Southwestern. A riboprobe for the mouse cyclophilin mRNA was generated from the pTRI-cyclophilin template (Ambion, Inc. Austin, TX) and included in all assays for normalization between samples. The expected size (nt) of the respective protected fragments were as follows: cyc = 103, cyclin-D2 = 359, ERß = 262, LH-R = 236, P450_scc = 558, PGS-2 = 416, PR = 365. RPAs were carried out on 2 µg total RNA from the ovaries of each individual animal using the Hybspeed RPA reagents (Ambion, Inc.) according to the manufacturer’s protocol. For the superovulation studies, a total of eight animals were used for each genotype/treatment/time point (except for the wild-type-4h hCG = 7 animals) and were assayed individually for each marker. Riboprobes were generated and labeled by incorporation of ³²P-CTP (Amersham Pharmacia Biotech, Piscataway, NJ) using the Maxiscript Kit (Ambion, Inc.) according to the manufacturer’s protocol. All samples were assayed simultaneously with the same preparation of radio-labeled riboprobe. Hybridization times ranged from 1–1.5 h. All RPA samples were fractionated by electrophoresis on a 1.5 mm thick 6% bis-acrylamide/8.3 M urea/1x Tris-borate gel (National Diagnostics, Atlanta, GA). All gels were fixed and dried in an Easy Breeze Gel Dryer (Amersham Pharmacia Biotech), exposed to a phosphorimager screen and analyzed with a Storm 860 and accompanying ImageQuant Software (Molecular Dynamics, Inc., Sunnyvale, CA), followed by exposure to x-ray film.

Serum LH and progesterone RIAs
Serum was processed from whole blood collected from the descending aorta when mice were killed and stored at -70 C until analysis. Serum progesterone was assayed on duplicate 25 µl aliquots per animal using the Active Progesterone RIA (Diagnostics Systems Laboratories, Inc., Webster, TX) according to the manufacturer’s protocol. To assess the effectiveness of the GnRH antagonist treatment regimen, duplicate 100 µl aliquots of serum from each animal were measured for LH content by RIA as previously described using materials supplied from the NIDDK (39). These assays have been previously shown to accurately measure mouse LH (40, 41). Samples that exhibited undetectable levels of LH were assigned the lower limit of the assay, 0.1 ng/ml.

Statistical analysis
All data sets were tested for homoscedasticity using Levine’s test. In cases where data were found to be heteroscedastic, it was log-transformed before further statistical analysis. Where appropriate, the Student’s t test was used. In all other cases, a two-way ANOVA followed by the Bonferroni/Dunn posthoc test was used. All statistical analysis was carried out using Statview 4.0 Software (Abacus Concepts, Berkeley, CA).

	Results

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GnRH antagonist treatment
The consistent presence of multiple hemorrhagic and cystic follicles in the ovaries of the adult ERKO female has become a hallmark phenotype of this model. We have previously described this phenotype as one that becomes apparent with the onset of sexual maturity, coinciding with the commencement of increased tonic levels of serum LH (33). The lack of ER in the hypothalamic-pituitary axis of the ERKO has resulted in a disruption of the normal feedback mechanisms of estradiol required for regulation of LH synthesis and secretion (32, 34). Therefore, ERKO females exhibit chronically elevated levels of serum LH that are severalfold higher than their wild-type counterparts, beginning as early as 25 days of age (Fig. 1). To determine the extent to which the ERKO ovarian phenotype was due to persistent stimulation by the heightened LH levels, animals were administered a GnRH antagonist over the period during which the phenotype is known to develop and worsen. As described in Materials and Methods, treatments consisted of either vehicle, or 15, 30, or 60 µg GnRH antagonist (Antide) per 48 h, beginning at the age of 28 days and carried out until 53 days of age.

View larger version (35K): [in this window] [in a new window]   Figure 1. The efficacy of prolonged GnRH antagonist treatment in reducing serum LH in ERKO females. Shown is the average LH (ng/ml) (± SEM) for wild-type or ERKO females when killed at 53 days of age, following treatment every 48 h with the indicated dose of GnRH antagonist (Antide) for 24 days (n = 4 for all groups, except ERKO-vehicle, n = 3). Also shown is the average serum LH (± SEM) in immature (25 days) wild-type and ERKO females, n = 12 and 9, respectively. ***, P < 0.001 when compared with vehicle of the same genotype; §§§, P < 0.001 when genotypes are compared within the same treatment or age group. For the 25-day group, Student’s t test was used; for Antide treatments, two-way ANOVA followed by Bonferroni/Dunn posthoc test was used.

Comparative ovarian histology from representative wild-type and ERKO females at 53 days of age, following 24 days of treatment with vehicle is shown in Fig. 2, A and B. The wild-type ovary exhibits indications of normal ovarian function, including the presence of several follicles at various stages of maturation, a corpus luteum, and a recently ovulated oocyte in the oviduct. In contrast, the ERKO ovary illustrates the expected and previously described phenotype, i.e. few preantral and small antral follicles, several large and hemorrhagic cystic follicles, and a lack of corpora lutea. Also shown in Fig. 2 are representative ovaries from wild-type (C) and ERKO (D) females following treatment with the GnRH antagonist at 60 µg/48 h for 24 days. The ability of the GnRH antagonist to significantly reduce the serum LH levels (Fig. 1) and to concurrently prevent the onset of the ERKO polycystic phenotype is obvious when the ovarian morphology of the representative treated ERKO animal (Fig. 2D) is compared with that of the vehicle-treated ERKO (Fig. 2B). The ovaries of the ERKO females treated with the highest dose of the GnRH antagonist appeared more like those of the similarly treated wild-type females and strikingly different than those of the vehicle-treated ERKOs (compare Fig. 2, B, C, and D). A complete lack of the characteristic multiple cystic follicles was observed in all of the ovaries of the ERKO females treated with the highest dose of GnRH antagonist. Ovaries from ERKO females treated with 15 or 30 µg Antide per 48 h exhibited phenotypes that were intermediate of those treated with vehicle or 60 µg Antide (data not shown), indicating a physiological dose-response correlation with the level of circulating LH (Fig. 1).

View larger version (102K): [in this window] [in a new window]   Figure 2. Prevention of the ERKO ovarian phenotype and effect on gonadotropin receptor mRNA levels after prolonged treatment with a GnRH antagonist. Age matched wild-type and ERKO females were treated with increasing doses of a GnRH antagonist (Antide) every 48 h from the age of 28–53 days. Shown is histology from a representative ovary of the following treatment groups at 53 d: (A) wild-type-vehicle; (B) ERKO-vehicle; (C) wild-type 60 µg Antide/48 h; and (D) ERKO 60 µg Antide/48 h. A normal functioning ovary is represented in the wild-type-vehicle panel (A), exhibiting the various stages of folliculogenesis, multiple corpora lutea, and a recently ovulated ova within the oviduct. The ERKO-vehicle (B) illustrates the characteristic ovarian phenotype of the ERKO adult ovary, exhibiting a lack of preovulatory follicles or corpora lutea and the presence of multiple cystic follicles. This phenotype was prevented when ERKO (D) females were treated with a GnRH antagonist beginning before puberty and carried out through to adulthood, producing an ovary that looks more like that of a similarly treated wild-type (C). A phenotype that did become apparent in the GnRH antagonist-treated ERKO ovaries (D, inset) was a less dense interstitial/thecal cell compartment when compared with the similarly treated wild-type ovaries (C, inset). Also shown is the effect of increasing doses of the GnRH antagonist on the ovarian level of mRNAs for (E) FSH receptor (FSH-R) and (F) LH receptor (LH-R) when assayed by ribonuclease protection assay. Each bar represents the mean (± SEM), expressed as the % cyclophilin mRNA (n = 3 for all groups). Two-way ANOVA followed by Bonferroni/Dunn posthoc test was used. No significant differences in the FSH-R mRNA levels were observed. Statistical differences observed in the LH-R mRNA levels were as follows: ***, P < 0.001 when compared with vehicle of the same genotype; §§§, P < 0.001 when genotypes are compared within the same treatment group.

An observed biochemical effect of the Antide treatment also found to be unique to the ERKO ovary was a drastic and dose-dependent reduction in the LH-R mRNA levels when assayed by RPA (Fig. 2F). At the highest Antide dose, the level of LH-R mRNA in the ERKO ovaries was significantly reduced (P < 0.001) compared with the level observed in the ovaries of similarly treated wild-type as well as the vehicle-treated ERKO females. In contrast, RPAs for FSH-R mRNA indicated that levels remained relatively stable in the two genotypes over the increasing doses of GnRH antagonist (Fig. 2E).

Ovulation and in vitro fertilization assays
We have previously described the anovulatory phenotype of the adult (>100 days old) ERKO female, including an inability to ovulate when treated with exogenous gonadotropins (33). We therefore set out to determine if ovulation was possible in immature (28 days) and peripubertal (42 days) ERKO females, before the onset of the cumulative effects of the elevated LH described above. As described in Materials and Methods, superovulation consisted of a single sc injection with PMSG at 1300–1500 h, followed 48–52 h later with a single sc injection of hCG. As shown in Table 1, both immature and peripubertal ERKO females were able to successfully ovulate, although the number of ova collected from oviducts after superovulation was significantly reduced (immature = P < 0.001; peripubertal = P < 0.05) compared with that collected from age-matched controls. Even the greatest number of oocytes collected from a single ERKO female was below the wild-type average in each age group (Table 1A). Three of the 15 immature and 1 of the 9 peripubertal ERKO females tested did not appear to ovulate at all. The ERKO ova collected exhibited no apparent defect in their ability to undergo successful in vitro fertilization when using wild-type sperm, with a combined success rate of 59.7% and 47.6% in wild-type and ERKO, respectively (Table 1B).

View this table: [in this window] [in a new window]   Table 1. A. Oocyte yield after superovulation of wild-type and ERKO mice

View larger version (69K): [in this window] [in a new window]   Figure 3. Ovarian histology in the immature wild-type and ERKO female after superovulation. Immature wild-type and ERKO females were induced to superovulate by treatment with exogenous PMSG and human CG (hCG) as described in Materials and Methods. Shown are representative ovarian sections from a (A) wild-type and (B) ERKO females at the time of oocyte collection, approximately 20 h after hCG treatment. As shown in the wild-type ovary (A), superovulation resulted in multiple corpora lutea (CL) that appear fully differentiated. However, the ERKO ovary (B) is characterized by the distinct presence of multiple large, preovulatory but unruptured follicles (indicated by arrows). Several of the unruptured follicle of the ERKO ovary possessed granulosa cells that show no obvious signs of luteinization, whereas other follicles have successfully undergone terminal differentiation into a corpus luteum (CL).

Gonadotropin induction of ovulatory markers in the ERKO ovary

View larger version (41K): [in this window] [in a new window]   Figure 4. RPA for markers of follicular maturation in immature wild-type and ERKO ovary after superovulation. A, Representative autoradiographs from RPAs for mRNAs encoding estrogen receptor-ß (ERß), LH receptor (LH-R), P450 side-chain cleavage (P450scc) enzyme, and cyclin-D2 on individual ovarian samples during the indicated time points of the superovulation treatment, i.e. PMSG and human CG (hCG) as described in Materials and Methods. RPAs for cyclophilin (cyc) were included for normalization between samples. Shown are representative assays from wild-type and ERKO ovaries treated with either vehicle; PMSG = 48–52 h after PMSG treatment; +hCG-4 h = 48–52 h PMSG treatment followed by 4 h hCG treatment; +hCG-20 h = 48–52 h PMSG treatment followed by 20 h hCG treatment. B, Quantitative analysis of the RPAs represented as the mean (± SEM) at each treatment point for each genotype, expressed as the % cyclophilin mRNA. For each genotype and time point, n = 8 animals; with exception of wild-type-hCG-4 h, n = 7 animals. Two-way ANOVA followed by Bonferroni/Dunn posthoc test revealed statistical differences as follows: **, P < 0.01; ***, P < 0.001 when compared with vehicle of the same genotype; §§§, P < 0.001 when genotypes are compared within the same treatment group.

View larger version (21K): [in this window] [in a new window]   Figure 5. RPA for markers of ovulation in immature wild-type and ERKO ovary after superovulation. A, Representative autoradiographs from RPAs for mRNAs encoding prostaglandin synthase-2 (PGS-2) and progesterone receptor (PR) on individual ovarian samples during the indicated time points of the superovulation treatment, i.e. PMSG and human CG (hCG) as described in Materials and Methods. Shown are representative assays from wild-type and ERKO ovaries treated with either vehicle; PMSG = 48–52 h after PMSG treatment; +hCG-4 h = 48–52 h PMSG treatment followed by 4 h hCG treatment; +hCG-20 h = 48–52 h PMSG treatment followed by 20 h hCG treatment. Cyclophilin (cyc) is shown for normalization purposes. B, Quantitative representation of the RPAs for PGS-2 and PR during the above treatment time points. Each bar represents the mean (± SEM) at each treatment point for each genotype, expressed as the % cyclophilin mRNA. For each genotype and time point, n = 8 animals; with exception of wild-type hCG-4 h, n = 7 animals. Two-way ANOVA followed by Bonferroni/Dunn posthoc test revealed statistical differences as follows: ***, P < 0.001 when compared with vehicle of the same genotype; §§, P < 0.01 when genotypes are compared within the same treatment group.

View larger version (28K): [in this window] [in a new window]   Figure 6. Serum progesterone in immature wild-type and ERKO females after superovulation. Duplicate samples were assayed from individual wild-type and ERKO females killed at the following time-points during the superovulation treatment: vehicle; 48–52 h PMSG + 4 h hCG; and 48–52 h PMSG + 20 h hCG. Shown is the average serum progesterone (ng/ml) (± SEM). Sample size for each group was: wild-type vehicle, 15; ERKO vehicle, 10; wild-type 4 h hCG, 11; ERKO 4 h hCG, 10; wild-type 20 h hCG, 10; ERKO 20 h hCG, 8. Two-way ANOVA with Bonferonni/Dunn posthoc test, ***, P < 0.001 when comparing to vehicle within the same genotype. There is no statistical difference between genotypes within each time-point.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Reduction of serum LH results in a prevention of the ERKO ovarian phenotype
Both components of the hypothalamic-pituitary axis are known to express significant levels of ER (48, 49). Accordingly, disruption of the ER gene results in a loss of the negative regulatory effects of estradiol in the hypothalamic-pituitary axis, as reflected by the elevated levels of pituitary gonadotropin subunit mRNAs (32) and serum LH in the adult ERKO female (Fig. 1). As presented here, the results of prolonged treatment with a GnRH antagonist in the ERKO female indicate that: 1) increased serum LH in the ERKO female is primarily due to the loss of ER mediated actions in the hypothalamus required for proper regulation of GnRH synthesis and/or secretion; and 2) the ERKO ovarian phenotype of multiple hemorrhagic follicular cysts is the result of chronic hyperstimulation by heightened serum LH levels.

Experimental support for the abnormally high levels of serum LH as the primary cause of the ERKO ovarian phenotype may be drawn from a number of studies. Investigations involving prolonged treatment with antiestrogens over a period of at least 28 days to 6 months have produced an ovarian phenotype that is similar to that of the ERKO in both mice (50, 51) and rats (52). However, such studies using estrogen antagonists are complicated by the possible inhibition of estrogen action at both the ovarian and hypothalamic-pituitary level, as well as an inability to discriminate between ER and ERß mediated actions. Nonetheless, these studies reported that an ERKO-like ovarian phenotype was produced only after chronic treatment with those antiestrogens that crossed the blood-brain barrier and concurrently produced elevated serum LH levels, e.g. ZM-189,154 (52) and EM-800 (50, 51). In contrast, treatments with tamoxifen had no effect on serum LH levels and in turn did not produce an ovarian phenotype similar to the ERKO or to those females treated with ZM-189,154 or EM-800 (50, 51, 52). Additionally, Risma et al. (53, 54) provided definitive evidence of the effects of chronic LH exposure on the ovary via the generation of transgenic mice that overexpress an LHß transgene in the pituitary. These transgenic females possess a 15-fold increase in physiological levels of LH and exhibit an ovarian phenotype almost indistinguishable from that of the adult ERKO female at the morphological level.

The similarity between the ovarian phenotypes described in the above studies with our observations in the ERKO suggest that ER is not locally involved in the development of ovarian cysts induced by hypergonadotropism. However, there are descriptions of at least two models in which serum LH is chronically elevated but do not exhibit an ovarian phenotype similar to the ERKO or those induced by antiestrogens or transgenics as described above. Female mice that are homozygous for a targeted disruption of the FSHß-subunit gene exhibit an approximate 5-fold increase in serum LH but do not show indications of enlarged cystic follicles in the ovary (55), indicating a role for FSH action in this process as well. Bogovich has also provided supporting evidence of the need for FSH along with prolonged exposure to LH to induce follicular cysts in the rat ovary (56). Another contrasting phenotype is exhibited by the aromatase-knockout (ArKO) mice, which are homozygous for a targeted disruption of the P450_arom gene, and therefore lack the capacity to synthesize estradiol (57). Although both serum LH and FSH are elevated in the ArKO female, folliculogenesis is arrested at the antral stage and no ERKO-like cystic structures are reported (57). It is possible that a lack of estradiol synthesis has negated the intraovarian functions of ERß in the granulosa cells of the ArKO, and thereby disrupted the pathways conducive to development of the LH-induced ovarian cysts. Therefore, although the ERKO phenotype may be triggered by hyperstimulation of the follicles by LH, it is likely influenced and possibly dependent upon the actions of both FSH and ERß within the ovary. The preservation of estradiol synthesis (31), normal serum FSH levels (34), elevated ovarian levels of FSH-R and LH-R mRNAs (33), and a normal ovarian expression pattern for ERß (23, 33) in the ERKO female provides further support for this hypothesis.

A unique effect of the GnRH antagonist observed in the ERKO ovary was a significant dose-dependent reduction in LH-R mRNA levels (Fig. 1F). A possible cause of the drastic reduction in LH-R mRNA levels may be the apparent decreased cell population in the theca/interstitial compartments observed in the treated ERKO ovaries (Fig. 1D). In light of previous reports of GnRH mRNA (58), GnRH receptors (59), and GnRH regulation of LH-R mRNA and binding-sites (60, 61, 62) in the rodent ovary, a direct ovarian effect of the GnRH antagonist must also be considered. However, previous studies have shown that removal of gonadotropins via hypophysectomy results in a drastic reduction in LH-R expression in both the theca/interstitial and granulosa cell compartments of the mouse ovary (42); this effect could be reversed with treatments of exogenous LH (42) or FSH + estradiol (17). Therefore, the effect of the Antide in reducing the LH-R mRNA levels in the ERKO ovary suggest that regulation of the LH-R gene in the thecal/interstitial cells of the normal ovary may be via both ER/estradiol and LH-R/LH mediated actions. A decrease in LH-R mRNA due to lowered serum LH may not be observed in the wild-type ovary because of the possible compensating role of ER in the interstitial/thecal cells. In contrast, decreasing levels of circulating LH due to the effects of the GnRH antagonist in the context of a loss of ER action may result in the significant reduction in LH-R mRNA observed in the ERKO ovary. Because the RPA used in this study was carried out on RNA from whole ovarian tissue, future studies to localize the decreases in LH-R gene expression among the functional ovarian compartments in the ERKO ovary are warranted.

Gonadotropin induction of ovulation and follicular markers in the ERKO
Although the ovaries of the immature ERKO female do not appear morphologically different than those of age-matched wild-type littermates (33), a significant reduction in the ovulatory capacity of the immature ERKO female when superovulated with exogenous gonadotropins was observed. Similar phenotypes have been described in mice after targeted disruption of the genes for ERß (44), PR (46), PGS-2 (47), and cyclin-D2 (45). Another similarity between the ERKO and those models possessing disruptions of the genes for ERß (44), PR (46), or cyclin-D2 (63), is the presence of multiple preovulatory yet unruptured follicles after superovulation. However, quantitative RPAs for these transcripts, as well as those for LH-R and P450_scc, throughout the time-course of the superovulation treatment indicate no overall genotypic differences in the expression pattern of these genes. Therefore, the cause of the reduced ovulatory capacity observed in the immature ERKO female remains unclear. It is possible that the ER may play a regulatory role at the translational level for one or more of the above transcripts; however, this is doubtful because the successful ovulation of some ova in the ERKO suggests the presence of the required gene products. Disruption of the ER gene may have also resulted in the loss of ER/ERß cooperative functions that may be necessary for proper communication both within and between the thecal and granulosa cell compartments of the maturing follicle.

A likely contributing factor to the reduced ovulatory efficiency in the immature ERKO may be the elevated levels of serum LH that exist in these females as early as 25 days of age (Fig. 1), the approximate age at which the superovulation experiments were carried out. Although the immature ERKO ovary has not yet manifested the cumulative effects of the chronic LH stimulation, i.e. the multiple cystic follicles, it is likely that aberrations in ovarian biochemistry already exist. One indication of such abnormal signaling may be the significantly elevated levels of LH-R mRNA detected in the immature ERKO ovary compared with age-matched wild-type females (P < 0.001; Fig. 4B). We have previously described the persistence of increased LH-R mRNA levels in both the thecal and granulosa cell compartments of the ERKO ovary during adulthood and the manifestation of the polycystic phenotype (33).

Although the studies described herein provide no obvious explanation for the reduced ovulatory capacity exhibited by the ERKO ovary, these data do indicate that several biochemical processes that occur during gonadotropin stimulation in the ovary remain intact in the absence of functional ER. These include the PMSG-induced up-regulation of the LH-R gene and simultaneous down-regulation of the ERß gene. The latter is in agreement with that previously reported in the rat ovary by Byers et al. (25). Furthermore, we have provided conclusive evidence that the characteristic rapid and transient induction of the PR gene in granulosa cells after a single hCG injection (64) can occur in the absence of ER. These data support the conclusions of others that induction of the PR gene in the ovary is a rapid response to the increasing intracellular cAMP levels induced by the LH-surge (64, 65, 66). Cyclin-D2, another critical component of follicular maturation, has also been shown to be gonadotropin as well as estrogen regulated in the granulosa cells of the rat ovary (63). Although direct estrogen challenges of the ERKO ovary were not carried out in the current study, our data confirms that gonadotropin induction of the cyclin-D2 gene can occur in the absence of ER. Therefore, the data presented here indicate that any estrogen actions required for full expression of the PR and cyclin-D2 genes, as well as PGS-2, LH-R, and P450_scc expression in the ovulatory follicle are independent of ER action.

It is worth noting that although the ERKO female is able to ovulate only at younger ages and only with the administration of pharmacological levels of gonadotropins, the yielded ova are competent and able to undergo successful fertilization at a rate similar to that exhibited by control oocytes. Previous reports of ER mRNA in the mouse oocyte suggested a direct role for ER in oocyte function (67). Several studies have also provided evidence of biochemical communication between the oocyte and surrounding granulosa cells (68, 69), presumably involving the presence of gap junctions that may be partially estrogen regulated (9). Nonetheless, the loss of ER mediated action in the mouse ovary does not appear to result in any alterations in the oocyte that inhibit fertilization.

In summary, we have provided definitive evidence that the hallmark phenotype of multiple cystic follicles in the ovaries of adult ERKO females is due to chronic stimulation by heightened levels of serum LH, and therefore an effect secondary to the lack of ER in the hypothalamic-pituitary axis rather than the direct loss of receptor in the ovary. Furthermore, when superovulated before the onset of the cumulative ovarian phenotype, the immature ERKO female is responsive to exogenous gonadotropins in terms of successful ovulation and yield of competent oocytes. However, the average number of ova yielded from the immature ERKO females was lower than that from their wild-type counterparts, yet no marked dysregulation of several genes known to be critical to folliculogenesis and ovulation was observed. These data, viewed in combination with the significant ovulatory defects reported in the ERß-knockout mouse (44), strongly suggest that ERß may be the predominant ER in ovarian physiology or that the combined actions of ER and ERß are required. In summary, although a critical role for ER in ovarian function appears to involve the hypothalamic-pituitary regulation of LH synthesis and secretion, the inefficient ovulatory capacity of the ERKO female suggests an intraovarian role for this receptor as well.

	Acknowledgments

 
The authors are grateful to several individuals for their assistance and advice during these studies. We would like to especially acknowledge Mariana Yates and Linwood Koonce for their assistance in breeding and genotyping the animals; Sylvia Curtis Hewitt and April E. Chester for their donation of the mouse PGS-2 vector; Eugenia Goulding for advice in the superovulation experiments; Drs. John Nilson and Ruth Keri for the advice in the design of the GnRH antagonist experiments; Dr. Ralph Cooper and laboratory for the serum LH assays; Dr. Joseph Haseman for statistical analysis; and Drs. Barbara Davis and Wayne Bocchinfuso for the editorial comments.

	Footnotes

 
¹ Current address: Department of Biology, University of South Florida, Tampa, Florida 33620.

Received April 26, 1999.

	References

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