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. 2008 May 22;149(9):4452–4461. doi: 10.1210/en.2008-0441

G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

Cheng Wang 1, Eric R Prossnitz 1, Shyamal K Roy 1
PMCID: PMC2553386  PMID: 18499747

Abstract

Estradiol-17β (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation.

THE MECHANISMS OF primordial follicle formation and their subsequent entry in folliculogenesis in mammals are largely unknown. Estradiol-17β (E2) treatment in vivo or in vitro significantly increases the percentage of primordial follicles in the hamster ovary (1). E2 has been shown to affect the formation and development of primordial follicles in many species. Nuclear estrogen receptor-α gene (estrogen receptor-α) and estrogen receptor-β gene (estrogen receptor-β) are expressed in human (2,3), baboon (4), and hamster (5) fetal ovaries and developing rodent ovaries (6,7,8,9,10). Aromatase is expressed in human (3) and hamster (1) fetal ovaries and the rising levels of E2 during gestation attenuate oocyte apoptosis (11). Suppression of E2 production during the second half of gestation in baboons results in a marked decrease in the number of primordial follicles in the fetal ovaries (12). Furthermore, aromatase knockout mice are infertile with a decreased primordial and primary follicle pool size by 10 wk of age (13,14). However, whether E2 action in developing ovaries, especially during the formation of primordial follicles, is mediated by a transmembrane estrogen receptor, such as G protein-coupled receptor 30 (GPR30) (15,16), remains unknown.

Recently GPR30 has been implicated as a possible transmembrane mediator of E2 action. Stable transfection of GPR30 into HEK239 cells, which lack the orphan G protein-coupled receptor, results in [3H]E2 binding to cell membranes, and whereas knockdown of GPR30 with small interfering (si) RNA attenuates E2 binding (15). Furthermore, GPR30 has been shown to mediate estrogen action in various tumor cell lines (15,17,18,19,20). Using adult hamsters as the experimental model, we have demonstrated for the first time that GPR30 is expressed in ovarian granulosa and theca cells, and the expression is regulated by FSH and LH (21). Whether E stimulation of primordial follicle formation requires GPR30 is unclear. The objectives of the present study were to examine whether GPR30 was expressed in ovarian cells during the critical window of primordial follicle formation, and whether GPR30 could mediate the rapid action of estrogen in neonatal ovary cells and was required for the formation of primordial follicles.

Materials and Methods

E2 was purchased from Steraloids, Inc. (Newport, RI); DMEM and other cell culture medium were from Invitrogen (Carlsbad, CA). Pluronic F127, fura-2AM, Ribogreen RNA quantification kit, and Alexa-conjugated second antibodies were from Molecular Probes, Inc. (Eugene, OR). RNeasy minikit was from QIAGEN Inc. (Valencia, CA). GPR30 antibody was described previously (17,21); second antibodies for Western blotting chemiluminescence were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Enhanced chemiluminescence advance Western blotting detection kit was from GE Healthcare (Buckinghamshire, UK). Optitran nitrocellulose membrane was from Schleicher & Schuell Bioscience (Dassel, Germany). Metafectane transfection reagent was from Biontex Laboratories GmbH (Martinsried/Planegg, Germany). PCR chemicals were from Roche Molecular Biochemicals (Indianapolis, IN), Amersham Pharmacia Biotech Boehringer (Piscataway, NJ), and Promega Corp. (Madison, WI). Charcoal/dextran-treated fetal bovine serum (FBS) was from HyClone Laboratories Inc. (Logan, UT), and E2-BSA and E2-BSA-fluorescein isothiocyanate (FITC) were from Sigma Chemical Co. (St. Louis, MO). All other chemicals were purchased from Sigma, Fisher Scientific (Pittsburgh, PA), or United States Biochemicals (Cleveland, OH).

Female golden hamsters (90–100 g, Charles River Laboratories, Inc., Wilmington, MA) were housed according to the U.S. Department of Agriculture and Institutional Animal Care and Use Committee guidelines under controlled climatic condition with 14-h light, 10-h dark cycle. The use of hamsters in this study was approved by the Institutional Animal Care and Use Committee. Females with at least three consecutive estrous cycles were mated with males on the evening of proestrus, and the presence of sperm in the vaginal smear the next morning was considered d 1 of pregnancy.

Measurement of GPR30 mRNA levels in perinatal ovaries

RNA was isolated from ovaries from embryonic day (E) 13 through postnatal day (P) 15 hamsters and quantified as described previously (21,22). For real-time quantification, in vitro transcribed GPR30 mRNA was used as standards in parallel with the sample RNA for obtaining true quantitative values. PCR primers and probes were designed from hamster GPR30 cDNA sequence (accession no. DQ237895) (21) and synthesized in the Eppley Molecular Biology Core (University of Nebraska Medical Center). The sequences of forward and reverse primers and the probe for real-time PCR quantification of GPR30 were 5′-TTCCGCACCAAGCACCAT-3′, 5′-AGCCACTGCACCTCTCTGACA-3′, and 6-FAM-5′-CGTGCACCTGCG GCACAC TG-3′-BLACKHOLE, respectively. The values were presented as femtogram GPR30 mRNA per microgram total RNA. The specificity of changes in GPR30 mRNA expression during ovarian development was verified by measuring the levels of actin mRNA in the same samples, and the results were presented as nanograms of actin mRNA per microgram total RNA. We used actin mRNA because the expression of this gene was not affected during ovarian development or by hormonal manipulations (5).

Determination of GPR30 protein expression in perinatal ovaries

The ovarian homogenate was prepared from E13 through P15 ovaries, and GPR30 protein was immunoblotted as described previously (21). The levels of tubulin were also determined to verify the specificity of GPR30 expression during ovarian development. Each group had at least three replicates of samples collected from three different animals. To verify the specificity of the GPR30 antibody in Western blotting, P8 ovarian homogenate was gel fractionated and transferred to the nitrocellulose membrane similar to other samples. The membrane was probed with the GPR30 antibody that was preneutralized with the antigen peptide followed by chemiluminescence signal recording. The membrane was stripped and then reprobed with the antitubulin antibody and chemiluminescence signal was recorded.

The spatiotemporal expression of GPR30 protein was examined using paraformaldehyde fixed neonatal hamster ovary sections as described previously for adult hamster ovaries (21). The images were captured by a Qimaging digital camera (Qimaging, Surrey, British Columbia, Canada) and Openlab (Improvision, Waltham, MA) image analysis software. The exposure time of the camera was set for subtracting background fluorescence that was present in sections incubated with the nonimmune IgG of the host species. GPR30 immunosignal was merged with the nuclear signal to determine the cellular site of protein expression.

Subcellular localization of the GPR30 protein in neonatal ovarian cells in vitro

Because GPR30 had been reported in both the plasma membrane (23) as well as in the endoplasmic reticulum (17) of breast cancer cells, we sought to examine the location of GPR30 in neonatal ovary cells by immunofluorescence colocalization. P6 ovary cells were dispersed and cultured on coverslips with DMEM containing 10% steroid-free FBS for 48 h. After attachment, cells were washed with ice-cold PBS, fixed in freshly prepared ice-cold 4% paraformaldehyde in PBS (pH 7.4) for 10 min, blocked with 10% normal donkey serum containing 0.1% Triton X-100 for 60 min, and then probed with antibodies against GPR30 and N-cadherin (membrane marker) or KDEL (Lys-Asp-Glu-Leu) [endoplasmic reticulum (ER) marker]. The fluorescence signal was captured using a epifluorescence microscope (Leica, Québec, Canada), a Qimaging Retiga Exi 1394 digital camera, and Openlab software.

Detection of E2-BSA binding to the ER in neonatal ovarian cells in vitro

P6 ovary cells were dispersed as described previously, incubated with 1 μm E2-BSA-FITC at 37 C for 5 min, rinsed thoroughly to remove any unbound ligand, and analyzed by epifluorescence microscopy. The fluorescence signal was quantified using National Institutes of Health image analysis software (Bethesda, MD). The data were expressed as the mean OD per pixel ± sem of samples from at least three ovaries from three different animals after subtracting nonspecific fluorescence signal. The specificity of binding was verified by exposing ovarian cells to 100 μm of E2 or E2-BSA for 30 min before E2-BSA-FITC exposure for 5 min.

To determine the colocalization of E2-BSA-FITC binding sites and GPR30 protein, P6 ovarian cells were cultured in 10% steroid-free FBS in DMEM for 48 h. After attachment, cells were cultured in serum-free DMEM for 24 h and then exposed to 1 μm E2-BSA-FITC for 5 min followed by thorough rinsing with PBS to remove excess ligand. Cells were then exposed to 0.1% trypan blue in saline for 5 min at room temperature and examined for dye inclusion as an index of damaged plasma membrane. Cells were then washed in ice-cold PBS, fixed for 10 min in ice-cold freshly prepared 4% paraformaldehyde and processed for immunofluorescence localization of GPR30 as described previously. The E2-BSA-FITC, GPR30, and nuclear fluorescence was merged.

In a parallel experiment, P6 ovarian cells were cultured in 10% steroid-free FBS in DMEM for 48 h. After attachment, cells were transfected with 100 nm GPR30 siRNA or nontargeting siControl RNA for 6 h as described previously for growth differentiation factor-9 (22). Cells were washed with antibiotic-free DMEM three times and then cultured with DMEM containing 10% steroid-free FBS for another 48 h. One group of cells was collected to determine the GPR30 protein levels with immunoblotting as described previously, whereas another group of cells was exposed to 1 μm E2-BSA-FITC to detect the membrane E2 binding by immunofluorescence localization.

Measurement of the rapid action of E2 on neonatal hamster ovarian cells

The existence of a rapid action of E2 in neonatal ovarian cells was determined by culturing P6 ovarian cells on coverslips for 48 h in 10% steroid-free FBS as described previously. After attachment, the medium was replaced with phenol red-free DMEM containing 1% solution of (100 ng/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenium 107 μg/ml linoleic acid, and 25 μg/ml BSA (1,24,25) and cultured for 24 h. Cells were then placed in Ringer’s solution and loaded with 7 μm fura-2-AM in 20% Pluronic acid and incubated for 30 min at 37 C. The coverslips were washed with Ringer’s solution to remove excess fura 2-AM and incubated for 15 min to allow the hydrolysis of fura 2-AM into its active-dye form, fura 2. Each coverslip was inserted into the cuvette of a double-beam spectrofluorometer (Photon Technology International, Princeton, NJ) for fluorescence recording. Treatment with E2-BSA or 1 μm E2 was carried out sequentially by adding the appropriate concentrations of each substance into the cuvettes in Ringer’s solution with ample washing between exposures. The excitation wavelength was alternated between 340 and 380 nm, and the emission fluorescence was recorded at 510 nm. The fluorescence ratio, which represents the relative intracellular free calcium (Ca2+) concentration signal, was calculated as F340:F380.

Effect of FSH in vivo and in vitro on GPR30 expression in the neonatal hamster ovary

The FSH regulation of GPR30 expression during the formation of primordial follicles was examined by sc injecting pregnant hamsters with 200 μl of an anti-FSH serum on the 12th day of gestation as reported previously (26). Control animals received an equal volume of normal rabbit serum. Female P1 pups received a sc injection of 20 IU equine chorionic gonadotropin (eCG) or saline at 0900 h. Ovaries were collected on P8 and processed for immunofluorescence localization of GPR30 protein as described earlier.

The in vitro effect of FSH was examined by transfecting E15 ovaries with a control or GPR30 siRNA as described previously (22), and culturing ovaries for 9 d in the absence or presence of 1 ng/ml ovine FSH-20 (National Institutes of Health). Ovaries were processed for immunofluorescence localization of GPR30.

Effect of GPR30 siRNA on E2-stimulated formation and development of primordial follicles in neonatal hamster ovary

E15 ovaries were cultured and transfected with control or GPR30 siRNA as described for previous experiment. Ovaries were cultured for a total of 9 d in the absence or presence of 3.6 nm of E2 or 1 μm of E2-BSA with or without 100 nm ICI 182,780. The specificity of GPR30 siRNA effect was examined by real-time RT-PCR and Western blot analysis, respectively, of the levels of GPR30, estrogen receptor-α, and estrogen receptor-β mRNA and protein in cultured ovaries. The percentage of primordial follicles was determined by morphometric analysis as described previously (1,22,24,25). The effect of E2 was tested on GPR30 siRNA-treated ovaries because E2 was the natural ligand in vivo.

Data analysis

Cultures and immunofluorescence localization were repeated at least three times using ovaries from different animals. Ovaries from untreated and E2 or siRNA-treated groups were cultured in parallel. Ovaries from E15 embryos of each pregnant hamster were pooled to obtain one sample for each embryonic age. There were three pregnant hamsters for each embryonic age to obtain three samples. RNA and protein samples for each postnatal day were prepared from ovaries pooled from three litters, and there were at least three samples for each fetal and postnatal day. All quantitative data were analyzed by one-way ANOVA with Student-Newman-Keuls post hoc test using InStat statistical analysis software (GraphPad Software, San Diego, CA). The level of significance was P < 0.05.

Results

Expression of GPR30 mRNA and protein during perinatal hamster ovary development

GPR30 mRNA levels decreased from E13 through P2 and thereafter started increasing from P3 and peaked on P6 followed by a decline through P12 (Fig. 1A). However, ovarian actin levels remained unchanged throughout indicating the specificity of developmental expression of GPR30 mRNA (Fig. 1B). Consistent with the mRNA levels, GPR30 protein levels decreased significantly between E13 and P2 and then increased significantly by P3 and again by P7 (Fig. 1B), coinciding with the formation of primordial follicles on P8 (24,26). Thereafter GPR30 protein levels decreased to P3 levels by P10 and remained steady (Fig. 1B). Ovarian levels of tubulin did not show any apparent change (Fig. 1B, middle panel). Although the presence of tubulin signal (Fig. 1B, inset, lower panel) confirmed the loading of sample, no GPR30 signal (Fig. 1B, inset, top panel) could be observed for P8 ovary sample when GPR30 antibody was preneutralized with the antigen peptide, indicating the specificity of the antibody.

Figure 1.

Figure 1

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GPR30 mRNA and protein levels in hamster ovaries during perinatal development. A, Real-time RT-PCR quantification of ovarian GPR30 and actin mRNA levels on each day of development. Actin mRNA levels were presented as nanograms per microgram RNA. B, Ovary lysate for each day of development was analyzed by Western immunoblotting for GPR30 (top panel) and tubulin (middle panel). The intensity (OD) of chemiluminescence signal was presented relative to the OD of tubulin signal (bottom panel). Box inset, Verification of the specificity of the GPR30 antibody in Western blotting. Nitrocellulose membrane containing gel fractionated P8 ovary homogenate was probed with the GPR30 antibody that was preneutralized with the antigen peptide (top panel). After recording the chemiluminescence signal, the membrane was stripped and reprobed with the tubulin antibody (bottom panel). Each bar represented a mean ± sem of at least three samples. Bars with a different letter, P < 0.05; bars with a same letter, P > 0.05.

Localization of GPR30 protein in neonatal hamster ovaries during the formation of primordial follicles

On E13, GPR30 immunosignal was mainly located in the somatic cells, whereas very low expression was present in some oocytes in the clusters (egg nests) (Fig. 2A). GPR30 expression in somatic cells increased with development, but appreciable increase was observed by P6 when cells in large patches expressed GPR30 (Fig. 2B). Occasionally few oocytes in the egg nests showed discrete GPR30 expression (Fig. 2B). On P8, GPR30 expression in the interstitial cells surrounding primordial follicle structures increased markedly, whereas immature granulosa cells showed modest immunoreactivity (Fig. 2C). Previously we reported that the first cohort of primordial follicles also appeared on P8 (22,26). Although the overall GPR30 expression in interstitial cells declined appreciably by P15, granulosa cells adjacent to the basal lamina showed intense GPR30 immunosignal at the basal surface (Fig. 2D). Furthermore, discrete GPR30 expression was observed at random in granulosa cells inside the follicles (Fig. 2D).

Figure 2.

Figure 2

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Immunofluorescence localization of GPR30 protein in hamster ovaries collected on E15 (A), P6 (B), P8 (C), and P15 (D). GPR30 immunosignal is green, whereas red indicates nuclei. S, Somatic cells; O, oocytes; OC, oocyte clusters; GC, granulosa cells; IC, interstitial cells; S0, primordial follicles; S1, primary follicles; S2, follicles with two layers of GC (stage 2); S, follicles with five to six layers of GC. Bar, 10 μm.

Subcellular localization of the GPR30 in neonatal ovarian cells in vitro

In cultured P6 cells, GPR30 immunoreactivity was located in the perinulear area (Fig. 3A). KDEL, a marker for the ER, was also localized at the perinuclear area (Fig. 3B). GPR30 immunosignal colocalized with KDEL staining and extended outward (Fig. 3C). No overlap of GPR30 and N-cadherin immunosiganls could be detected in cells (data not shown), suggesting that under this experimental condition, GPR30 could not be located in the plasma membrane.

Figure 3.

Figure 3

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Localization of GPR30 in cultured P6 ovarian cells. A, GPR30. B, KDEL for ER. C, Merged images. Note that GPR30 was also present outside the ER. Bar, 10 μm.

Binding of E2 to GPR30 in P6 hamster ovarian cells

To determine whether a specific binding of E2-BSA existed in ovarian cells, P6 ovarian cells were exposed to FITC-conjugated E2-BSA in culture. The binding of E2-BSA-FITC occurred in the perinuclear area of the freshly dispersed P6 ovarian cells within 5 min of exposure (Fig. 4A), and the binding could be effectively displaced with a preexposure to 100 μm E2 or E2-BSA (Fig. 4, B–D). A colocalization analysis of E2-BSA-FITC binding and GPR30 immunofluorescence in P6 ovarian cells revealed that two immunosignals overlapped almost completely (Figs. 5, A–C), suggesting that E2-BSA indeed bound to GPR30 located at the perinuclear area of ovarian cells. It was noteworthy that despite the impermeable nature of the E2-BSA-FITC, the ligand gained access to intracellular binding sites within a very short time. All cells excluded trypan blue indicating that the cells’ plasma membrane was not compromised with E2-BSA-FITC exposure (data not shown).

Figure 4.

Figure 4

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Binding of E2-BSA-FITC to P6 hamster ovarian cells. Cells were mechanically dispersed from ovaries, washed, and immediately exposed to E2-BSA-FITC (A), E2-BSA-FITC + E2-BSA (B), or E2-BSA-FITC + E2 (C) as indicated in the experimental design. Bar, 10 μm. D, FITC signal was digitized and expressed as OD per pixel. Both E2-BSA and E2 significantly attenuated E2-BSA-FITC binding to ovarian cells. Con, Control. Bars with different letter, P < 0.05; bars with same letter, P > 0.05.

Figure 5.

Figure 5

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Binding of E2-BSA-FITC to GPR30 in P6 ovarian cells cultured for 96 h. A, Cells exposed to E2-BSA-FITC. B, Immunofluorescence localization of GPR30. C, Merged images. E2-BSA-FITC bound to GPR30 located at the perinuclear area of ovarian cells. Bar, 10 μm.

The binding of E2-BSA to ovarian GPR30 was further validated by siRNA knockdown of GPR30 in P6 ovarian cells cultured for 96 h. Distinct E2-BSA-FITC binding was observed in cells exposed to nontargetting siControl RNA (Fig. 6, A and B). Perinuclear E2-BSA-FITC binding was also evident in MCF-7 cells, which served as a positive control for membrane estrogen receptor (Fig. 6C). GPR30 siRNA significantly knocked down GPR30 protein levels in cultured P6 ovarian cells without affecting the levels of tubulin (Fig. 6D), and the decrease coincided with a marked attenuation of E2-BSA-FITC binding (Fig. 6, E and F).

Figure 6.

Figure 6

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siRNA knockdown of GPR30 protein and E2-BSA-FITC binding in P6 ovarian cells cultured for 96 h. A, Binding of E2-BSA-FITC to P6 ovarian cells transfected with nontargetting siControl RNA. B, DIC image of cells in A. C, Perinuclear E2-BSA-FITC binding to MCF7 breast cancer cells. D, Western blot showing GPR30 protein levels in cells exposed to siControl or GPR30 siRNA. The top panel shows a blot of two samples each of control and GPR30 siRNA-treated cells probed with the GPR30 antibody, the middle panel shows the levels of tubulin, and the bottom panel shows the OD of GPR30 protein relative to tubulin. Con, Control. Each bar represented a mean OD ± sem of three samples. Bars with different letter, P < 0.05. E, Binding of E2-BSA-FITC to P6 ovarian cells transfected with GPR30 siRNA. F, DIC image of the cell in E. Bar, 10 μm.

Functionally E2-BSA stimulated intracellular Ca2+ mobilization in cultured P6 ovarian cells, which was mimicked by E2 (Fig. 7), thus validating the existence of a mediator of the rapid action of E2 in neonatal ovarian cells.

Figure 7.

Figure 7

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Effect of E2 or E2-BSA on intracellular Ca2+ release in P6 ovarian cells in vitro. Both E2 and E2-BSA caused an increase in intracellular Ca2+.

FSH regulation of GPR30 expression in neonatal ovaries in vivo and in vitro

Previously we demonstrated that in utero exposure of E12 hamsters to an FSH-antiserum resulted in a significant reduction in primordial follicle formation, which could be reversed by a single injection of eCG on P1 (26). Using the similar experimental strategy, we examined whether GPR30 expression during perinatal ovary development was regulated by FSH. Compared with control P8 ovaries (Fig. 2C), FSH antiserum exposure resulted in a marked reduction in GPR30 expression on P8 (Fig. 8A). However, a single injection of eCG on P1 reversed the inhibitory effect of the antiserum on GPR30 expression (Fig. 8B), and the immunosignal was comparable with untreated P8 ovaries (Fig. 2C). No GPR30 immunosignal could be detected in a P8 ovary section when the GPR30 antibody was preneutralized with excess antigen (Fig. 8C), thus verifying the specificity of the antibody. Similarly, compared with basal levels of GPR30 expression in E15 ovaries cultured for 9 d (Fig. 8D), FSH exposure resulted in a significant up-regulation of GPR30 protein (Fig. 8E), which was completely attenuated by a preexposure to GPR30 siRNA (Fig. 8F). Furthermore, siRNA exposure clearly decreased the basal GPR30 expression as well (Fig. 8F).

Figure 8.

Figure 8

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GPR30 immunofluorescence in sections of P8 ovaries (A–C) and E15 ovaries cultured for 9 d in vitro (D–F). A and B, Sections of P8 hamster ovaries exposed in utero to an FSH-antiserum on E12 and then treated on P1 with either BSA-saline (A) or eCG (B). C, A section of a P8 hamster ovary exposed to the GPR30 antibody, which was preneutralized with the antigen peptide. D, Ovaries were transfected with a nontargetting siControl RNA. Ovaries were cultured with 1 ng/ml ovine FSH-20 (E) or transfected with the GPR30 siRNA (F) and then cultured with FSH. Images are representatives of three ovaries for each group. For the sake of brevity, no attempt was made to label follicles in images D–F. Bar, 10 μm. S, Somatic cells; GC, granulosa cells; S0, primordial follicles; IC, interstitial cells; O, oocytes.

Effect of GPR30 siRNA on E2-stimulated formation of primordial follicle

To delineate whether GPR30 would mediate at least part of the E2 action on the formation of primordial follicles, we used siRNA targeting GPR30. GPR30 siRNA markedly reduced GPR30 protein level in E15 ovaries cultured for 9 d; however, the siRNA did not affect the levels of estrogen receptor-α, estrogen receptor-β protein, or tubulin protein (Fig. 9A). Approximately 9% primordial follicles formed in E15 ovaries cultured for 9 d without any hormonal stimulus (Fig. 9B). E2 treatment not only significantly stimulated the formation of primordial follicles but also activated their development into the primary stage (Fig. 9B), which never occurred in ovaries cultured without E2 or FSH. E2-BSA mimicked the effect of E2 (9B). ICI182,780 did not affect the basal or E2-BSA-stimulated primordial follicle development, but it blocked the stimulatory effect of E2 (Fig. 9B). In contrast, either E2 or E2-BSA failed to stimulate primordial follicle formation in GPR30 siRNA-exposed ovaries (Fig. 9B). The GPR30 siRNA also attenuated primordial follicle formation under basal condition and synergized with ICI182,780 to further reduce the formation of primordial follicles (Fig. 9B).

Figure 9.

Figure 9

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The formation of primordial follicles in E15 ovaries transfected with control or GPR30 siRNA, and cultured for 9 d with or without E2 or E2-BSA in the absence or presence of ICI182,780 (ICI). A, Western blot detection of the GPR30, estrogen receptor (ER)-α and ERβ in 9-d cultured ovaries transfected with a control or GPR30 siRNA. The Western blots represent membranes probed with the ERα, ERβ, GPR30, or tubulin antibody; the bar graph represents the OD of GPR30 protein relative to that of tubulin ± sem of three separate experiments. B, Percentage of follicles relative to the oocytes. Each bar represents a mean ± sem of three ovaries. Bars with a different letter, P < 0.05; bars with the same letter, P > 0.05.

Discussion

The results of the present study provide the first evidence that a membrane estrogen receptor, GPR30, is expressed in perinatal hamster ovary cells, mediates the rapid action of E2 in ovarian cells, and plays an important role in mediating the effect of E2 on ovarian primordial follicle formation and development. Furthermore, we also demonstrated that ovarian expression of GPR30 is regulated by FSH. One of the novel findings is that the intracellular actions of nuclear estrogen receptors and GPR30 seem to converge downstream of ligand receptor interaction to bring about the folliculotropic effect of E2. The effect of E2 on early folliculogenesis has been demonstrated in many species including the hamster (1,9,12,14,27,28,29). Evidence has accumulated to suggest that in addition to nuclear receptors, E2-modulation of cell functions involves membrane estrogen receptors, such as estrogen receptor-α (30) and GPR30 (15,17,19,31). Recently we reported that GPR30 is expressed in adult hamster ovarian cells and the expression is regulated by FSH (1). GPR30 has been implicated in triggering a broad range of rapid E2 action at the plasma membrane (15,17,32,33). Consistent with the report published by Revankar et al. (17), our data indicate that GPR30 protein is associated with the ER and mediates rapid E2 action, such as intracellular Ca2+ mobilization in neonatal ovary cells. The ER location of GPR30 may be relevant to the rapid action of E2 because the ER plays a pivotal role in the control of Ca2+ storage and signaling (34). GPR30 has also been localized in the Golgi apparatus of oxytocin neurons but not on the cell surface (35). However, the presence of GPR30 in the plasma membrane of GPR30 overexpressing HEK-293 cells and SKBR3 breast cancer cells has been reported recently (23). The data need to be interpreted cautiously because overexpression of the receptor protein may alter the physiological distribution of the protein within cells. The cellular exclusion of trypan blue suggests that cell membrane remains intact in the presence of E2-BSA-FITC in the culture. Therefore, the binding of E2-BSA-FITC to the ER membrane of P6 ovarian cells within 5 min emphasizes a rapid endocytosis-mediated receptor-ligand entry in cells and suggests that GPR30 may shuttle rapidly from the plasma membrane to the ER membrane; hence, the conventional immunofluorescence approach becomes inadequate for its detection. This conjecture is supported by the finding that appreciable GPR30 is located outside the ER, which may reflect the protein in transit. Furthermore, our preliminary data indicate that phenylarsine oxide, a blocker of endocytosis, causes E2-BSA-FITC to stay at the plasma membrane. Further studies are in progress.

The unique developmental expression of GPR30 mRNA and protein coinciding with the formation of primordial follicles provides a strong circumstantial evidence that this membrane estrogen receptor plays an important role in the differentiation of somatic cells into immature granulosa cells leading to the formation of primordial follicles. The increased expression of GPR30 on P8 coincides with the increase in serum E2 (1). Furthermore, CYP19A is expressed in neonatal hamster ovary cells, which are capable of producing E2 from testosterone (1). The evidence for the functional importance of GPR30 in primordial follicle formation comes from the results of siRNA knockdown. The decline in GPR30 expression after P8 may indicate a stable basal expression once the cell assembly around the oocytes has been completed, and follicular development has been established. It can be conjectured that GPR30 in somatic cells and the oocytes may mediate E2 action on somatic cell proliferation and differentiation and facilitates somatic cell and oocyte communication, leading to the differentiation of granulosa cells and the formation and development of primordial follicles. In contrast to the results presented herein, GPR30 null mice do not display any abnormal reproductive phenotype and are fertile (36). The role of estrogen on mouse primordial follicle formation or development is not clear at present; however, the present results clearly demonstrate that both classical and membrane estrogen receptors are required for mediating the action of estrogen on primordial follicle formation in another rodent species. It is possible that similar to classical estrogen receptors, GPR30 may have a limited role in early folliculogenesis in mice. Deletion of both estrogen receptor-α and -β genes in mice also does not affect the formation and development of primordial follicles (37,38), even though such deletion is expected to eliminate the membrane form of the receptor.

Whereas the expression of GPR30 and its role as a membrane estrogen receptor have been demonstrated in various cell lines, the hormonal regulation of GPR30 expression in tissues is poorly understood, and virtually nothing is known about its regulation in neonatal ovarian cells. In a solitary report, Ahola et al. (39) reported that progesterone stimulates GPR30 mRNA levels in MCF-7 breast cancer cells; however, despite being directly regulated by progesterone GPR30 appears not to be the primary target gene for progesterone because gene expression occurs with a lag time between 8 and 18 h (39). Furthermore, no progestin response element can be located in the promoter or regulatory site of GPR30 gene (39), but an activator protein-1 site is located in the GPR30 promoter site (39). The decrease in GPR30 expression in FSH antiserum-treated animals and its reversal by eCG and siRNA knockdown of FSH-mediated GPR30 expression in vitro provide strong evidence that FSH regulates GPR30 expression in neonatal ovarian cells and corroborate our previous findings that FSH action is important for primordial follicle formation, at least in the hamster (26). Both FSH and LH up-regulate GPR30 protein levels in adult hamster ovaries as well (21). FSH up-regulates nuclear estrogen receptor expression coinciding with the formation of primordial follicles (5), and FSH stimulates the production of E2 by neonatal hamster ovaries (1). Therefore, it appears that FSH-mediated primordial follicle development may involve E2 action via GPR30 as well. Although siRNA knockdown of GPR30 has a stronger suppressive effect on primordial follicle formation, the inhibition of E2 action by ICI182,780 suggests that both forms of receptors may be necessary for the differentiation of somatic cells into granulosa cells. The effect of E2 can be on the oocytes, granulosa cells, or both because both cell types in the hamster ovary possess dual forms of estrogen receptors (5,21). However, it is apparent that the effect of one form of receptor depends on the functionality of the other because specific inactivation of either form of the receptor blocks E2-mediated primordial follicle formation. Furthermore, the synergism between GPR30 siRNA and ICI182,780 also suggests that at least part of the downstream mechanisms of nuclear estrogen receptor action require E2 activation of GPR30.

In conclusion, this is the first report of a unique expression of GPR30 in the perinatal ovary during the formation of the first cohort of primordial follicles and the involvement of an intracellular membrane receptor mediating E2 action in ovarian cells. The study also documents that FSH regulates the expression of GPR30 in ovarian cells, which is essential for mediating the action of E2 on primordial follicle formation. Furthermore, the ER location of GPR30 suggests that a rapid endocytotic process may exist for ligand-receptor transport from the plasma membrane. Finally, one of the most novel findings is that GPR30 mediates a part of the E2 action on ovarian cells, and GPR30 activity is essential for the functionality of the classic estrogen receptors during the formation of primordial follicles.

Footnotes

This work was supported by Grant R01-HD38468 from the National Institute of Child Health and Human Development, a grant from the Leland and Dorothy Olson Foundation to the Department of Obstetrics-Gynecology, University of Nebraska Medical Center (to S.K.R.), and Grant R01-CA118743 from the National Cancer Institute (to E.P.). C.W. was the recipient of a Lalor Foundation Postdoctoral fellowship.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 22, 2008

Abbreviations: Ca2+, Free calcium; E, embryonic day; E2, estradiol-17β; eCG, equine chorionic gonadotropin; ER, endoplasmic reticulum; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GPR30, G protein-coupled receptor 30; P, postnatal day; si, small interfering.

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