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Departments of Obstetrics and Gynecology (C.W., S.K.R.) and Cellular and Integrative Physiology (S.K.R.), Durham Research Center, University of Nebraska Medical Center, Omaha, Nebraska 68198; and Department of Cell Biology and Physiology (E.R.P.), University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Address all correspondence and requests for reprints to: Shyamal K. Roy, Departments of Obstetrics and Gynecology and Cellular and Integrative Physiology, DRC 5013, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515. E-mail: skroy{at}unmc.edu.
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Introduction |
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) and ESR2 (also known as ERß), and their role in regulating ovarian follicular development in many species (1, 2, 9, 10, 11, 12, 13, 14). Evidence has accumulated to indicate that estrogen also affects reproductive target tissues via nongenomic mechanisms that do not involve the classic ESR (15, 16, 17). However, the identity of the membrane ESR (mESR) remains controversial. It has been suggested that the mESR is a variant of the classical ESR, such as ESR1 and ESR2 that mediate estrogen action in the target cells, such as endothelial, neuronal, and pituitary cells (18, 19, 20, 21). On the other hand, evidence has also accumulated for the presence of novel G protein-coupled mESRs, which are unrelated to nuclear ESR but mediate the nongenomic action of estrogen in many cell types (22, 23, 24, 25). Recent studies have confirmed that the G protein-coupled receptor 30 (GPR30) functions as a novel transmembrane ESR in many cancer cell lines (26, 27, 28). Thomas et al. (27) have demonstrated that in SKBR3 breast cancer cells, which lack nuclear ESR, GPR30 functions as a high-affinity, saturable, displaceable, and single-binding-site-specific receptor for estrogens. Furthermore, progesterone-induced increases and RNA interference-induced decreases in GPR30 expression in SKBR3 cells coincide with altered estradiol-17ß (E2) binding. Revankar et al. (28) have demonstrated that GPR30 specifically binds E2 and fluorescent E2 derivatives and initiates intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate. All these lines of evidence clearly establish that GPR30 is an alternative form of mESR, which exists and functions independent of the nuclear ESR. Although the presence of classical ESRs in ovarian follicles has been documented (2), evidence for the presence of mESRs in follicular cells is lacking. Preliminary studies in our laboratory suggest that E2 can rapidly phosphorylate MAPK in hamster granulosa cells (Roy, S. K., unpublished observation); therefore, a functional mESR may exist in follicular cells. Although the membrane versions of the ESR1 and ESR2 can mediate the E2 action in hamster follicular cells, the involvement of an independent mESR, such as GPR30, cannot be ruled out. Therefore, the objectives of the present studies were to determine whether hamster ovarian cells would express GPR30 mRNA and protein and whether gonadotropins and ovarian steroid hormones would regulate the expression as the first step toward understanding the role of GPR30 in follicular development.
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Materials and Methods |
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Animals and treatments
Female golden hamsters (90–100 g; Charles River Laboratories, Inc., Wilmington, MA) were housed in climate-controlled conditions with a 14-h dark, 10-h light cycle and free access to food and water according to the U.S. Department of Agriculture and Institutional Animal Care and Use Committee (IACUC) guidelines. The use of hamsters in this study was approved by the IACUC. Ovaries were obtained from hamsters with three consecutive estrous cycles at 0900 h of each day of the estrous cycle and also at 1600 h on proestrous (d 4). There were three hamsters for each day of the cycle, and the experiment was repeated twice. To obtain ovaries from hypophysectomized hamsters, females were hypophysectomized on d 1 at 0900 h (estrous) as previously described (29, 30). Ten days after HX, hamsters were injected sc with 1) 0.5% BSA in saline (vehicle control), 2) 10 µg ovine-FSH-20 twice daily for 2 d, 3) 10 µg ovine-LH-26 twice daily for 2 d, or 4) a combination of FSH and LH in 0.5% BSA in saline, and ovaries were collected 48 h after the first injection. The second group of hypophysectomized hamsters was injected sc with a single dose of 1) 100 µg E2-cipionate, 2) 500 µg progesterone, or 3) a combination of E2 and progesterone, all in U.S. pharmacopoeia-grade sesame oil. Control animals received an equal volume of sesame oil vehicle. We never observed any estrogenic effect of sesame oil in the hamster (10). There were three hamsters per group, and the entire experiment was repeated three times for reproducibility. Ovaries were collected 24 h after the injection. Ovaries from all animals were either embedded in OCT Tissue-TEK medium for frozen sections or frozen in liquid N2 for RNA and protein extraction and stored at –80 C until use.
Cloning of hamster GPR30 cDNA
RT-PCR was used for obtaining a partial hamster GPR30 cDNA clone. cDNA was amplified from total ovarian RNA according to the protocol described previously (31). The forward primer, 5'-ACATCCTCATCCTGGTGGTGAAC-3', and the reverse primer, 5'-TGGAATGACGGGCCTTGAGCG-3', corresponding to 230-1083 bases of mouse GPR30 cDNA (accession no. NM_029771) were designed using Amplify (Dr. Bill Engels, Genetics Department, University of Wisconsin) and Vector NTI primer design software version 10 (Invitrogen), and synthesized in The University of Nebraska DNA synthesis core (Eppley Institute). The PCR was done for 30 cycles with an annealing temperature of 55 C, and the cDNA was cloned into a PCR 4-TOPO plasmid vector, sequenced in the DNA sequencing core facility (University of Nebraska at Lincoln, Lincoln, NE), and blast-searched in the NIH GenBank. After confirmation, hamster GPR30 sequence was compared with the rat, mouse, and human GPR30 sequences using a Vector NTI gene analysis software version 10 [Molecular Analysis Core, University of Nebraska Medical Center (UNMC)].
Real-time RT-PCR quantification of GPR30 mRNA levels in the hamster ovary
RNA was isolated from frozen ovaries using Trizol (Invitrogen) followed by the RNeasy mini kit (QIAGEN Inc., Valencia, CA) according to the manufacturers’ instructions and previously used protocols (10, 31). The amount of RNA was quantified using the Ribogreen kit (Molecular Probes) according to the manufacturer’s protocol. For real-time quantification, mRNA standards for GPR30 and actin were used in parallel with the sample RNA to obtain true quantitative values. PCR primers and probes were designed using Primer Express software version 2.0 (Applied Biosystems, Inc., Foster City, CA) from hamster GPR30 and actin nucleic acid sequences, which were partially cloned in this study and previously (10), respectively. The forward and reverse primers and probe for GPR30 were 5'-TTCCGCACCAAGCACCAT-3', 5'-AGCCACTGCACCTCTCTGACA-3', and 6-FAM-5'-CGTGCACCTGCGGCACACTG-3'-BLACKHOLE, respectively, and for actin they were 5'-TGACCGAGCGTGGCTACAG-3', 5'-CTTCTCTTTGATGTCACGCACAAT-3', and 6-FAM-5'-TCACCACCACAGCCGAGAGGGA-3'-BLACKHOLE, respectively. Hamster GPR30 and actin mRNA were synthesized using specific cDNA and the AmpliScribe T7-Flash transcription kit and quantified using Ribogreen, and the authenticity was verified by RT-PCR using the forward and reverse primers designed specifically for real-time RT-PCR. A series of eight standards ranging from 0.1 fg to 1 ng (in 10-fold increments) of specific mRNA were reverse transcribed in duplicates along with ovarian RNA samples and used for real-time quantification of GPR30 and actin using the Opticon thermocycler (Bio-Rad, Hercules, CA). PCR amplification was carried out for 40 cycles after an initial denaturation at 95 C for 15 min to activate the HotStart Taq polymerase (QIAGEN). Each cycle of PCR consisted of 30 sec of denaturation at 94 C and 30 sec annealing at 55 C, followed by a plate reading with a final extension at the end of 40 cycles for 10 min at 72 C. The authenticity of the PCR signal was verified using reactions without RNA or RNA without RT. The values were presented as femtograms of mRNA per microgram of total RNA. The levels of actin mRNA for each sample were also presented to prove the specificity of GPR30 gene expression.
Northern hybridization detection of hamster GPR30 in the ovary
Northern hybridization of hamster GPR30 mRNA was done essentially as described (31) to verify a critical finding of the real-time RT-PCR. The membrane was exposed to a storage phosphor screen and the signal digitized in a Cyclone phosphorimager (PerkinElmer, Norwalk, CT). The approximate size of the GPR30 transcript was calculated from the relative positions of RNA size markers (Invitrogen).
Localization of GPR30 protein by fluorescence immunohistochemistry
Although the GPR30 antibody was thoroughly characterized using human cell lines (28), the specificity of detection in hamster ovarian cells was verified by neutralizing the antibody with 10-fold molar excess of the antigen peptide and using the neutralized antibody in immunofluorescence and Western blot detection of GPR30 protein. Additionally, a single band of the expected molecular weight was observed by Western blotting using this antibody.
GPR30 protein was localized in 6-µm-thick frozen sections fixed in freshly prepared ice-cold 4% paraformaldehyde in PBS (pH 7.4) as described previously (10, 32) using 1:25,000 dilution of a rabbit polyclonal anti-GPR30 antibody. The GPR30 signal was developed using a donkey antirabbit-IgG-Alexa 488, and nuclei were stained with 4',6-diamidino-2-phenylindole. The images were captured with a Leica DMR microscope (North Central Instruments, Inc., Plymouth, MN) fitted with a Retiga EX1394 digital camera (QImaging Corp., Surrey, British Columbia, Canada) and Openlab image analysis software (Improvision, Lexington, MA). The exposure time of the camera was set to subtract background fluorescence that was present in sections incubated with the nonimmune IgG of the host species. The 4',6-diamidino-2-phenylindole signal was converted to red for contrast, and the green GPR30-specific fluorescence signal was merged with the nuclear signal to determine the cellular site of protein expression. GPR30 fluorescence intensity was measured using the NIH ImageJ Image analysis software. Signal intensity (OD/pixel) was recorded from the granulosa and thecal compartments of five to 10 follicles per section from three animals, whereas the intensity for the interstitial cell compartment was recorded from three ovary sections from three animals. At least 10 areas of interest in each cell compartment were digitized to obtain the average for the compartment, which was considered as one observation (n = 1). The total number of follicles per section provided the mean for the granulosa and thecal cell compartments for that ovary. The mean and the SEM represented three ovaries (n = 3), and was calculated using the InStat statistical software (GraphPad Software, San Diego, CA). A d-1 ovary section was examined by a Zeiss confocal microscope (UNMC Imaging Core) at x630 for determining the cellular distribution of GPR30 protein.
Immunoblot detection of GPR30 protein in the ovary
The first experiment focused on determining the cellular sites of GPR30 expression in the hamster ovary. Ovaries from hamsters in diestrous (d 3 at 0900 h) were homogenized in a 50 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na-pyruvate, 2 mM Na-orthovanadate, 10% glycerol, and 10% of a protease inhibitor cocktail (Sigma) on ice using a Dounce homogenizer. The homogenate was centrifuged at 1000 x g at 4 C, and the pellet was sonicated on ice in 30 µl epidermal growth factor receptor-lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4) containing 1% Triton X-100 and the protease inhibitor cocktail and centrifuged at 26,000 x g for 20 min at 4 C. The supernatant was used as the crude nuclear fraction. The 1000 x g supernatant was centrifuged at 100,000 x g for 1 h at 4 C, and the supernatant was used as the cytosolic fraction, whereas the pellet was sonicated in 50 µl epidermal growth factor receptor-lysis buffer with Triton X-100 and protease inhibitors on ice, kept on ice for 20 min, and centrifuged at 26,000 x g at 4 C for 20 min, and the supernatant was used as the crude membrane protein. The protein concentration was determined by the micro-BCA kit (Pierce, Rockford, IL), and 5 µg membrane protein and 20 µg cytosolic and nuclear protein were fractionated in a 10% polyacrylamide gel, transferred to an Optitran nitrocellulose membrane, and probed with the same anti-GPR30 antibody that was used for immunofluorescence detection or antibodies directed against Na+-K+-ATPase 2 (membrane marker), GATA4 (nuclear marker), or glyceraldehyde-3-phosphate dehydrogenase (cytosol marker). Secondary antibody binding was detected with the enhanced chemiluminescence (ECL) Advance Western blotting detection kit (GE Healthcare) and recorded by a UVP gel documentation system (UVP, Upland, CA). The experiment was repeated three times to confirm reproducibility.
Based on the results of subcellular localization, crude membrane preparations of ovaries from cyclic hamsters and hypophysectomized hamsters with or without hormonal replacement were used for GPR30 protein expression determination by Western immunoblotting as described for experiment 1, except the intensity of the ECL signal was quantified. Because a housekeeping membrane protein was difficult to select, the proteins on the nitrocellulose membrane were stained with SyproRuby stain (Invitrogen) according to the manufacturer’s instruction and digitized using an ethidium bromide filter and the UVP gel documentation system. The OD corresponding to the total immunofluorescence signal in each lane was used for normalizing the ECL signal for GPR30 protein.
To determine GPR30 expression during ovarian follicular development, follicles at primary (stage 1) through large antral (stage 10) were isolated from diestrous (d 3, 0900 h) hamsters as described previously (33). The description of developmental stages is as follows: stages 1–4, follicles with one to four layers of granulosa cells without theca; stage 5, follicles with five to six layers of granulosa cells with a few thecal cells; stage 6, follicles with seven to eight layers of granulosa cells and a developed theca; stage 7, follicles with incipient antral cavities; and stages 8–10, follicles with increasing antrum (33). Follicles were ruptured to extract granulosa cells, and thecal shells (whenever present) were cleaned of granulosa cells manually as best as possible without the use of any enzyme to avoid any damage to the receptor protein. No attempt was made to collect the oocytes. Therefore, granulosa cell preparations represented a pure population, whereas theca preparation contained a few attached granulosa cells. Cells were washed thoroughly to remove BSA, and the crude plasma membrane was prepared as described earlier. After measuring the protein concentration, 5 µg total protein was used for Western blot detection of GPR30 as described earlier. Each group had three replicates representing three animals.
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To determine nucleic acid and protein information about hamster GPR30, an 854-bp GPR30 cDNA, which was 231 bp shorter than the full-length rat or mouse GPR30 cDNA (NM_029771, NM_133573) was cloned, and the sequence was submitted to the GenBank (accession no. DQ237895). Comparison of the nucleic acid sequence revealed that hamster GPR30 cDNA was 94.7, 95.1, and 95% similar to the corresponding segments of rat, mouse, and human GPR30 cDNA, respectively (Fig. 1
). Similarly, the amino acid sequence of hamster GPR30 was 99.3, 98.2, and 98.1% similar to that of rat, mouse, and human, respectively (Fig. 2). The partial amino acid sequence of hamster GPR30 protein contained six of the seven transmembrane-spanning domains (Transmembrane Predictor: http://www.ch.embnet.org/cgi-bim/TMPTED_for_parser). Furthermore, analysis of the amino acid sequence revealed that it contained a conserved cystine residue in each of the first two extracellular loops (Cys130, Cys205 and 207) that are believed to be necessary for the formation of intramolecular disulfide bridges to maintain the receptor structure. The hamster GPR30 protein also contained a D-R-Y triplet, an exceptionally conserved sequence among many G protein-coupled receptors (GPCRs) and believed to play a role in signal transduction (34), located in the second intracytoplasmic loop after the third transmembrane domain. NetPhos (http://www.cbs.dtu.dk/services/NetPhos/) analysis revealed the presence of four putative serine and threonine and two tyrosine phosphorylation sites. Prosite (http://www.expasy.ch/prosite/) analysis indicated the presence of a putative N-glycosylation site and four possible protein kinase C phosphorylation sites (Fig. 2) that matched closely with those in mouse GPR30 protein. The presence of a Cys352 in the last hydrophilic region might suggest a putative palmitoylation site that anchors the carboxy terminus to the inside of the cell membrane. Subcellular distribution analysis revealed that GPR30 was expressed exclusively in the membrane fraction of the hamster ovary (Fig. 2).
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). GPR30 mRNA levels declined to d 1 level by d 4 (proestrous) morning with a further decline 2 h after the preovulatory gonadotropin surge (Fig. 3A). No change in the levels of actin mRNA was evident throughout the estrous cycle (Fig. 3A), suggesting the specificity of GPR30 mRNA expression. The purpose of the Northern blot analysis was to verify the increase in GPR30 mRNA levels observed on d 3 morning by real-time RT-PCR and also to gather information about GPR30 transcripts in the hamster ovary. Four transcripts were visible in the blot (Fig. 3C) regardless of the cycle day. The approximate size of the most prominent transcript was 1.4 kb (Fig. 3C). Furthermore, the levels of the 1.4-kb transcript increased 2-fold on d 3 compared with d 1 (Fig. 3, C and D), whereas the expression levels of other three transcripts did not change significantly (Fig. 3C). These results not only confirmed the real-time data but also corroborated the protein data and implicated the 1.4-kb transcript to be biologically relevant in ovarian functions. Based on the information, no attempt was made to repeat the Northern analysis for all days of the estrous cycle.
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). However, in contrast to the mRNA levels, GPR30 protein levels increased moderately but significantly on d 2 morning, indicating the presence of additional posttranscriptional regulation.
Based on the results of the Western immunoblotting, we wanted to examine whether there was any cell-type-specific expression of GPR30 protein in the hamster ovary. Although immunofluorescence localization corroborated the immunoblot results, an estrous cycle-specific expression of the receptor protein was evident (Fig. 4, A–F). Appreciable GPR30 immunosignal was present mainly in the granulosa cells of preantral follicles on d1 at 0900 h, but modest immunosignal was also present in the theca interna cells (Fig. 4, A and E). Confocal microscopic examination confirmed the epifluorescence microscopic findings but also revealed that GPR30 immunostaining was not uniformly distributed in the granulosa cells. Furthermore, punctate immunostaining was primarily intracellular (Fig. 4B). Low but discernible expression was evident in cells of the newly formed corpora lutea (Fig. 4A), but interstitial cells showed modest expression (Fig. 4, A and E). GPR30 expression increased significantly only in the thecal cells on d 2 at 0900 h (Fig. 4E); however, receptor expression increased markedly in all cell types on d 3 at 0900 h with maximal expression in the theca (Fig. 4, C and E). Focal increased expression in the granulosa cell compartment and considerable immunostaining in the oocytes was also evident (Fig. 4C). GPR30 expression declined considerably by the morning of d 4 primarily in the granulosa and theca cells (Fig. 4E) and then further declined in all cell types by d 4 at 1600 h (Fig. 4, D and E). No GPR30-specific immunostaining was observed when sections of a d-3 ovary were exposed to the antibody preneutralized with GPR30 peptide (Fig. 4F).
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). GPR30 protein levels in the granulosa cells dropped significantly when preantral follicles transited from stage 5 to stage 6 and when theca layer started to appear (33) and then increased again with the growth of the antral follicles (Fig. 5). Thecal cells had a relatively higher level of GPR30 protein expression from the outset, and the levels increased steadily with the growth of antral follicles (Fig. 5).
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), which corresponded with a decrease in the levels of GPR30 protein (Fig. 6B). Neither the mRNA nor the protein levels were affected by E2 or progesterone treatment, either alone or in combination (Fig. 6, A and B). In contrast, treatment with FSH, LH, or both resulted in a marked decrease in GPR30 mRNA levels (Fig. 6A) that corresponded with increases in the levels of GPR30 protein (Fig. 6B). Interestingly, FSH had the strongest effect on GPR30 mRNA levels that was somewhat blunted by LH (Fig. 6B). No change in the levels of ovarian actin mRNA was evident (Fig. 6A), indicating the specificity of the hormonal effect.
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, A and E). Treatment with FSH alone resulted in the formation of antral follicles with increased GPR30 expression exclusively in the granulosa cells (Fig. 7, B and E). In contrast, whereas treatment with LH alone resulted in increased GPR30 expression only in the theca cells (Fig. 7, C and E), the level of expression in the granulosa cells increased remarkably without any change in the theca when LH was administered along with FSH (Fig. 7, D and E). It was also noteworthy that the increased expression was localized in the antral granulosa cells.
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Discussion |
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The deduced amino acid sequence from the cloned hamster GPR30 cDNA represents a significant portion of the GPR30 protein (from 78 to 361 of 375 total amino acids). This sequence has almost all of the structural features of the GPCR superfamily. The conserved cystine residues in the extracellular loops have been suggested to provide structural integrity by forming intramolecular disulfide bridges (34). The D-R-Y triplet present in the hamster GPR30 protein is an exceptionally conserved sequence among many GPCRs and believed to play a role in signal transduction (34). The presence of a Cys352 in the last hydrophilic region has been suggested to be a putative palmitoylation site that anchors the carboxy terminus to the inside of the cell membrane. However, because of the length limitation of the present sequence, we cannot detect N-glycosylation sites in the extracellular N terminus of the receptor, a feature common to many GPCRs (34). Although we cannot categorically identify the transcript variant primarily responsible for generating GPR30 protein in ovarian cells, the appreciably higher level of expression of the 1.4-kb transcript compared with the other three variants and its increased expression on d 3 corresponding to GPR30 protein suggest that the 1.4-kb transcript may be the physiologically relevant form in the ovary. The reported size of the mouse and rat GPR30 mRNA is 2.5 and 1.3 kb, respectively, and both encode an identical GPR30 protein. Four GPR30 transcript variants have been identified in the human (36, 37); however, such information about rodent GPR30 is not available in the GenBank. Human GPR30 transcript variants differ significantly at the 5'-untranslated region, primarily at the 5'-site, but are 100% identical at the reading frame and 99% identical at the 3'-untranslated region. Furthermore, all four transcripts encode an identical GPR30 protein.
Previous studies have indicated that GPR30 is expressed in the organelle membrane of cultured cells (28). Unlike cultured cells, granulosa cells within follicles present scanty cytosolic compartment; hence, a clear distinction between intracellular and plasma membrane localization of GPR30 presents a challenge. Nevertheless, the distinct punctate intracellular localization of GPR30 protein in granulosa and theca cells in the confocal image suggests a possible association with the organelle membrane, although a plasma membrane localization for some granulosa cells cannot be ruled out at present. Filardo et al. (38), using a selected group of HEK-293 cells expressing HA-GPR30 transgene, have convincingly demonstrated GPR30 localization in the plasma membrane. Furthermore, they have shown that the plasma membrane-located GPR30 is internalized via clathrin-coated pits within 5 min of E2 stimulation (38). In contrast to cells expressing a GPR30 transgene, we examine natural expression of GPR30 in ovarian cells in their native location under a dynamically changing gonadotropin and ovarian steroid hormone milieu. Therefore, we do not know whether GPR30 is expressed in the organelle membrane under natural conditions or the intracellular location of GPR30 in ovarian cells is the result of estrogen action either during the estrous cycle or after exogenous administration. Studies are in progress to determine the true site of GPR30 expression in ovarian cells. The unique follicle stage-dependent expression of GPR30 protein suggests that GPR30 may play a role in preantral follicle development. Conversely, the gradual increase in GPR30 expression in granulosa and theca cells suggests that the differentiation of follicular cells after selection may also require the nongenomic action of E2 via GPR30. The expression pattern and hormonal regulation of GPR30 in hamster follicular cells are quite different from those of classical ESR1 and ESR2 (10). These differences may indicate that estrogen regulation of follicular development and functions are regulated by both classical and membrane ESRs in a cell-type-specific and temporal manner. Although a possible contribution of membrane ESR1 in mediating the estrogen action in follicular cells cannot be ruled out, the distinct presence of GPR30 highlights the fact that estrogen action on follicular cells may be mediated by more than one form of mESR. The presence of transmembrane ESR1 and its role in the activation of ERK1 in many cell types has been demonstrated (18, 39, 40, 41). Whereas immunoblot analysis has indicated overall changes in the GPR30 protein levels, immunofluorescence quantification reveals cell types that primarily contribute to the overall changes in ovarian GPR30 protein expression. The marked decline in GPR30 expression on proestrous morning and after the gonadotropin surge suggests that GPR30 may be involved in gonadotropin-induced proliferation as well as differentiation of follicular and other ovarian cells. Because exogenously administered gonadotropins can up-regulate follicular GPR30 expression in hamsters devoid of the pituitary, the decreased expression in vivo after the preovulatory gonadotropin surge leads to the speculation that other hormones/factors may contribute to the observed decline. Cortisol may be a putative hormone modifying the gonadotropin effect in vivo because a cortisol surge has been shown to occur along with the gonadotropin surge in the hamster, and cortisol attenuates gonadotropin-induced increase in TGFß receptor mRNA levels in the hamster ovary (31). Conversely, an E2-mediated down-regulation of GPR30 protein cannot be ruled out. Serum levels of E2 increase steadily from d 2 at 0900 h through d 4 at 1500 h followed by a decline (42). The unique expression pattern of GPR30 mRNA and protein in response to steroid hormones or gonadotropins leads to the speculation that a hormone-and cell-type-specific posttranscriptional regulation may exist in hamster ovarian cells. Ovarian GPR30 mRNA and protein levels during the estrous cycle reflect the end effect of dynamically changing serum levels of ovarian steroids and pituitary gonadotropins. Serum progesterone and FSH levels are high on d 1, and the levels drop by d 2 through d 3, when serum levels of E2 increase (42, 43). FSH causes significant growth of the granulosa cell compartment that is increased further in the presence of LH (44). LH acts primarily on the theca and cannot induce preantral follicular growth by itself in hypophysectomized hamsters. Therefore, GPR30 expression in LH-treated hypophysectomized ovary remains only in the theca. Because thecal development corresponds to follicular development, steroid hormones alone in hypophysectomized condition do not cause significant thecal growth, which may explain low levels of GPR30 protein despite high levels of receptor mRNA expression. In contrast, progesterone has been shown to increase GPR30 mRNA levels in MCF7 breast cancer cells (45, 46), and the increase coincides with a proportionate decrease in cell proliferation (45). Whether progesterone plays a similar role in hypophysectomized hamsters is not known at present. Conversely, it is equally possible that GPR30 mRNA destabilization is directly proportional to protein translation, and steroid hormones and gonadotropins differentially influence the stability of GPR30 mRNA in ovarian cells. It has been demonstrated for proteins, such as histone, c-Myc, etc. that a block in translation results in mRNA stabilization (47). A 2- to 4-fold fluctuation in mRNA half-life is quite common in mammalian cells, and such changes may result in a greater than 1000-fold difference in the steady-state levels of mRNA, which ultimately determine the corresponding levels of protein (47). Furthermore, estrogen has been shown to regulate mRNA stability in many cell types (47). Apparently, a translational block exists when reproductive hormones are withdrawn by hypophysectomy, resulting in mRNA stabilization; however, steroid hormones alone cannot overcome the block. Gonadotropins, on the other hand, can overcome the block, leading to GPR30 mRNA destabilization. Because gonadotropin treatment results in follicular development and stimulation of ovarian steroidogenesis, it is possible that steroid hormone regulation of ovarian GPR30 expression requires prior action of gonadotropins. Such a mechanism will play an important role in the GPR30-mediated estrogen effect on follicular cells throughout the estrous cycle when marked changes in endogenous hormone levels are expected.
In summary, the results of the present study provide the first evidence that GPR30, a transmembrane ESR, is expressed in the granulosa and theca cells of growing follicles in the hamster ovary. Furthermore, the expression level of GPR30 mRNA and protein is follicle cell type specific and is affected by reproductive hormones. The results help us to speculate that GPR30 may be involved, at least in part, in mediating estrogen action on follicular cells.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online July 19, 2007
Abbreviations: E2, Estradiol-17ß; ECL, enhanced chemiluminescence; ESR, estrogen receptor; GPCR, G protein-coupled receptor; GPR30, G protein-coupled receptor 30; mESR, membrane ESR.
Received June 1, 2007.
Accepted for publication July 10, 2007.
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C. Wang, E. R. Prossnitz, and S. K. Roy G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary Endocrinology, September 1, 2008; 149(9): 4452 - 4461. [Abstract] [Full Text] [PDF]
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Y. Pang, J. Dong, and P. Thomas Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest Endocrinology, July 1, 2008; 149(7): 3410 - 3426. [Abstract] [Full Text] [PDF]
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T. J Heino, A. S Chagin, and L. Savendahl The novel estrogen receptor G-protein-coupled receptor 30 is expressed in human bone J. Endocrinol., May 1, 2008; 197(2): R1 - R6. [Abstract] [Full Text] [PDF]
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