0013-7227/92/1303-1296$03.00/O Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 3 Printed
Society
Hormonal Regulation of Follicle-Stimulating Receptor Messenger Ribonucleic Acid Levels Rat Granulosa Cells* JONATHAN
L. TILLYt,
PHILIP
S. LAPOLTS,
AND
AARON
in U.S.A
Hormone in Cultured
J. W. HSUEH
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
epidermal growth factor (EGF; 0.3-10 rig/ml), basic fibroblast growth factor (bFGF; l-30 rig/ml), or insulin-like growth factorI (IGF-I; l-30 rig/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 rig/ml) on its own receptor mRNA levels. Similarly, GnRH (IO1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (IDW, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-tr-Phe’,p-pC1-Phez,&Trp~6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitorv actions of EGF. bFGF. and GnRH. Thus. the use of cultured
ABSTRACT. The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2&y culture period. However, inclusion of FSH (30 rig/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (l-100 rig/ml) increased FSH receptor mRNA levels in a dose-dependent manner (EDm, 4.5 rig/ml), with a maximal 5.9 + 0.7-fold increase observed in response to 30 rig/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 PM), suggesting the involvement of CAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with
rat” granulosa
cells’
provides
a model
systdm
to analyze
the
hormonal regulation of FSH receptor gene expression in the ovary. (Endocrinology 130: 1296-1302, 1992)
T
HE REQUIREMENT for FSH in the differentiation and maturation of ovarian follicles has been well established (for review, see Refs. l-4). The actions of FSH are mediated through specific receptors, located exclusively on the granulosa cell plasma membrane, which are functionally coupled via membrane-associated G-proteins to the adenyl cyclase-CAMP-generating system. Previous studies have shown that FSH increases the levels of its own receptor (1, 5), although prolonged exposure to FSH may also down-regulate the FSH receptor and the responsiveness of granulosa cells to subsequent FSH challenge (6, 7). Estradiol alone does not
appear to alter the distribution, number, or affinity of FSH-binding sites within the rat ovary (1, 8); however, estrogens do synergize with FSH in vivo to increase the number of FSH receptors per granulosa cell through a process involving both induction and maintenance (1,5). Recent cloning of the rat FSH receptor cDNA has provided data indicating that this protein belongs to the G-protein-coupled receptor family, with the characteristic seven-transmembrane-spanning region (9). Additionally, evaluation of cDNA-derived amino acid sequences indicates a high homology of the FSH receptor to LH/ CG (10-13) and TSH (14, 15) receptors. However, little information is available concerning the hormonal regulation of FSH receptor gene expression in the ovary. In light of the limited and conflicting data available regarding the hormonal regulation of FSH-binding sites within the ovary, the ability to study changes in FSH receptor mRNA levels would provide a clearer insight into the regulation of FSH receptor expression. Using a specific cRNA probe for the FSH receptor nucleotide sequence,
Received September 9,199l. Address all correspondence and requests for reprints to: Dr. Aaron J.-W. Hsueh, Division of Reproductive Bioloav, Department of Gynecology and Obstetrics, Stanford University School of Medicine,-300 Pasteur Drive. Stanford. California 94305-5317. * This work was supported by NIH Grant HD-23273. 7 Postdoctoral Fellow, supported by a grant from the Lalor Foundation. $ Postdoctoral Fellow, supported by National Research Service Award HD-07252. 1296
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FSH RECEPTOR mRNA IN GRANULOSA we analyzed FSH receptor mRNA transcripts within cultured rat granulosa cells to elucidate the hormonal regulation of ovarian FSH receptor gene activation. Materials
and Methods
Hormones Ovine FSH (NIH FSH S17; 20 x NIH FSH Sl U/mg) was a gift from the National Hormone and Pituitary Distribution Program (Baltimore, MD). Receptor grade murine EGF and human insulin-like growth factor-I (IGF-I) were obtained from Collaborative Research, Inc. (Waltham, MA), whereas recombinant basic fibroblast growth factor (bFGF) was generously provided by Dr. Andreas Sommer (Synergen, Boulder, CO). GnRH and the GnRH antagonist, [Ac-D-Phe’,D-pCl-Phe*,DTrp3B6]GnRH, were provided by Dr. N. C. Ling (Whittier Institute, La Jolla, CA). Forskolin was obtained from Calbiochem (La Jolla, CA). Granulosa cell culture Granulosa cells were obtained from immature 26-day-old Sprague-Dawley rats (Johnson Laboratories, Bridgeview, IL) implanted with diethylstilbestrol capsules 4 days earlier, as previously described (16). After estimation of cell number and viability with trypan blue exclusion, 7 x lo5 viable cells were pipetted into 12 x 75-mm polypropylene tubes and cultured in 0.5 ml McCoy’s 5a medium (Gibco, Santa Clara, CA) at 37 C in a humidified 95% air-5% CO, atmosphere. For culture, the McCoy’s 5a medium was supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin sulfate, and 100 nM androstenedione in the absence and presence of different hormones. Preparation of nucleic acid probes A cRNA probe for bases 621-1031 of the rat FSH receptor nucleotide sequence (9) was synthesized using a 410-basepair (bp) cDNA template obtained by the reverse transcriptionpolymerase chain reaction (17), essentially as previously described (17-19). This stretch of FSH receptor cDNA has low (48%) sequence homology with the rat LH receptor (10) and, under the stringent hybridization conditions used in the present study, the FSH receptor probe does not cross-hybridize with LH receptor mRNA (17). For normalization of FSH receptor mRNA data, a 1.6-kilobase (kb) cDNA probe for human 28s rRNA (BumHI-EcoRI fragment pAaz) (20, 21) was prepared using the random priming method (22). Sequence analysis of the 1.6-kb human ribosomal (r) RNA gene fragment used in the present studies predicts 89% nucleotide homology to a corresponding conserved sequence in the rat 28s rRNA gene (23, 24). RNA isolation and analysis After culture, supernatant was removed from culture tubes with a glass Pasteur pipette, and the remaining cell pellet was immediately snap-frozen in a dry ice-ethanol bath and stored at -70 C. Total RNA was then extracted from the cultured cells using the Nonidet P-40 method (25) and quantitated by measuring the absorbance of samples at 260 nm. For Northern
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blot analysis, 5 pg total RNA from the various treatment groups were fractionated on denaturing agarose gels and subsequently transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) by overnight capillary electrophoresis with 20 x sodium chloride-sodium citrate (SSC). For slot blot analysis, RNA samples were denatured in the presence of 4 M formaldehyde for 15 min at 60 C and quickly chilled on ice. Samples were adjusted to 6 x SSC and applied directly to nitroceilulose membranes using a slot blot apparatus (Schleicher and Schuell). For subsequent normalization of data, replicate membranes were prepared from the same RNA samples for separate hybridization to the FSH receptor probe and the 28s rRNA probe. For both Northern and slot blot analyses, transferred RNA was covalently cross-linked to the nitrocellulose membranes using a UV-crosslinker (Stratagene, La Jolla, CA). Northern and slot blots were prehybridized for 2-4 h at 66 C in the presence of 50% formamide under standard conditions, followed by hybridization with the radiolabeled probe of interest [cRNA probe for FSH receptor (18,26)) or cDNA probe for 28s rRNA (27)] at the same temperature for 16-18 h. Membranes were washed in 2 x SSC-0.1% sodium dodecyl sulfate for 10 min at room temperature, followed by two or three consecutive 15-min (FSH receptor) or 30-min (28s rRNA) washes in 0.1 x SSC-0.1% sodium dodecyl sulfate at 66 C. Membranes were then exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) for l-7 days at -70 C. Data analysis The intensities of slot blot hybridization signals were quantitated using a scanning transmittance densitometer and a digitizing computer program (Hoeffer Scientific, San Francisco, CA). Data for treatment effects on FSH receptor mRNA levels were then normalized against levels of 28s rRNA in each sample and expressed as relative densitometric units. For all experiments, a representative Northern blot (of three replicates) is presented, whereas slot blot data depict the mean + SEM of results from three replicate experiments. Statistical analysis was performed based on analysis of variance, followed by Scheffe’s test. Results Time course of FSH action on its receptor mRNA levels Northern blot analysis indicated the existence of two predominant FSH receptor mRNA transcripts of approximately 7.0 and 2.5 kb in total RNA prepared from freshly isolated granulosa cells of immature estrogentreated rats (Fig. 1, left panel). After increasing lengths of culture (12, 24, 36, 48, and 60 h) in the absence of hormone treatment, a time-related decrease in the level of FSH receptor mRNA was observed compared to levels found in cells at the initiation of culture (designated time zero; Fig. 1, right panel). However, inclusion of ovine FSH (30 rig/ml) in the culture medium prevented the decrease in FSH receptor mRNA levels during culture
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FIG. 1. Effect of FSH treatment on FSH receptor mRNA transcripts in cultured granulosa cells collected from immature estrogen-treated rats. Total RNA (5 rg) was prepared from cells incubated for increasing lengths of time (O-60 h) in the absence [control (C)] or presence of 30 rig/ml FSH. Levels of FSH receptor mRNA were analyzed by Northern (left panel) and slot blot (right panel) analysis using a cRNA probe under highly stringent conditions. The Northern blot, which depicts levels of FSH receptor mRNA transcripts in freshly isolated granulosa cells [time zero (To); no culture], is representative of results from three replicate experiments. Data from the slot blot analysis, which represent the mean + SEM of three separate cultures, were normalized for 2% rRNA levels in each sample and expressed relative to FSH receptor mRNA levels at time zero (1.0). FIG. 2. Dose-dependent stimulation of FSH receptor mRNA levels by FSH. Left panel, Total RNA was prepared from granulosa cells cultured for 48 h in the absence [control CC)] or presence of 30 rig/ml FSH and analyzed by Northern blot for FSH receptor mRNA. Right panel, Total RNA was extracted from cells incubated for 48 h without and with increasing doses of FSH and analyzed by slot blot analysis for FSH receptor mRNA and 285 rRNA levels. Data for FSH receptor mRNA levels were normalized us. levels of 28s rRNA and expressed relative to the control value (1.0). Slot blot data represent the mean + SEM of results from four separate cultures, whereas the Northern blot is representative of three replicate experiments.
(12-60 h), maintaining levels comparable to those found in cells at time zero (P > 0.10; Fig. 1, right panel). Dose-dependent effects of FSH and forskolin on FSH receptor mRNA levels Culture of granulosa cells for 48 h in the presence of increasing concentrations (l-100 rig/ml) of FSH resulted in a dosedependent increase in FSH receptor mRNA levels, with a maximal 5.9 f 0.7-fold increase over controls observed in response to 30 rig/ml FSH (P < 0.05 vs. control; Fig. 2, right panel). The ED, for FSH action was estimated to be 4.5 ng/ ml. Northern blot analysis indicated an increased level of both the 7.0- and 2.5-kb transcripts in FSH-treated cells compared to controls (Fig. 2, left panel). The stimulatory effects of FSH were mimicked by the adenyl cyclase activator forskolin (O.l30 FM), which increased FSH receptor mRNA levels in a dosedependent manner (EDW, 0.2 PM) to a maximum 3.5 + 0.4-fold
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increase at a concentration of 10 PM (P C 0.05 vs. control; Fig. 3, right panel). The increased FSH receptor mRNA levels detected in forskolin-treated cells resulted from an increase in the levels of both receptor transcripts, as determined by Northern blot analysis (Fig. 3, left panel). Effects of EGF, bFGF, IGF-I, and GnRH on basal and FSH-maintained FSH receptor mRNA levels Treatment of granulosa cells for 48 h with EGF (0.310 rig/ml), bFGF (l-30 rig/ml), or IGF-I (l-30 rig/ml) did not significantly alter basal levels of FSH receptor mRNA (data not shown). However, treatment with EGF or bFGF dose-dependently attenuated the stimulatory actions of FSH (30 rig/ml) on FSH receptor mRNA levels (IDso of EGF, 0.6 rig/ml; ID60 of bFGF, 7 rig/ml; Fig. 4, right panel). A complete suppression of FSH action was
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FIG. 3. Effects of increasing concentrations of the adenyl cyclase activator forskolin (FOR) on FSH receptor mRNA levels in granulosa cells after a 48-h culture. Total RNA (5 rg) was extracted and analyzed by Northern (left panel; with or without 10 pM FOR) and slot blot (rig/rtpanel) analyses. Slot blot data for FSH receptor mRNA levels, which are the mean + SEM of three separate cultures, were normalized against 28s rRNA levels and expressed relative to the control value (C; 1.0). The Northern blot is representative of three replicate experiments. C
0.1
1
10
Forskolin
FIG. 4. Effects of different growth factors on FSH receptor mRNA levels. Left panel, Northern blot analysis of FSH receptor mRNA in 5 fig total RNA prepared from granulosa cells incubated for 48 h with medium alone [control (C)] or with 30 rig/ml FSH in the absence and presence of EGF (10 rig/ml), bFGF (FGF; 30 rig/ml), or IGF-I (IGF; 30 ng/ ml). The Northern blot is representative of three replicate experiments. Right panel, Slot blot analysis of FSH receptor mRNA levels in granulosa cells cultured for 48 h in medium alone [control (C)] or with 30 rig/ml FSH in the absence and presence of EGF (0.3-10 rig/ml), bFGF (l-30 rig/ml), or IGF-I (l-30 ng/ ml). These data, which represent the mean + SEM of results from three replicate cultures, were normalized us. levels of 28s rRNA in each sample and expressed relative to FSH receptor mRNA levels in FSH-stimulated cells (1.0).
observed in response to the highest dose of EGF or bFGF used (P < 0.05 vs. FSH alone), as determined by both Northern (Fig. 4, left panel) and slot blot (Fig. 4, right panel) analysis. In contrast, IGF-I did not alter the levels of FSH receptor mRNA maintained by 30 rig/ml FSH during a 48-h incubation (P > 0.10) (Fig. 4, both panels). Treatment of granulosa cells for 48 h with 100 nM GnRH suppressed basal levels of FSH receptor mRNA compared to control values (Fig. 5, left panel). Similarly, GnRH caused a dose-dependent attenuation of the stimulatory actions of FSH (30 rig/ml) on FSH receptor mRNA levels (IDbO, 8 nM), with a maximal inhibitory effect observed in response to 100 nM GnRH (Fig. 5, right panel). Northern blot analysis indicated that cotreatment of granulosa cells with 100 IXM GnRH completely suppressed the stimulatory effects of FSH on its own receptor mRNA levels (Fig. 5, leftpanel). The inhib-
30
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itory actions of GnRH (100 nM) on FSH-maintained FSH receptor mRNA levels were prevented by inclusion of an equimolar concentration of the GnRH antagonist [Ac-n-Phe’,D-pC1-Phe2,D_Trp3’6]GnRH in the culture medium (Fig. 6). The GnRH antagonist alone, however, did not alter basal or FSH-stimulated FSH receptor mRNA levels (Fig. 6). Discussion Granulosa cells isolated from immature rats contain two FSH receptor mRNA transcripts, the levels of which decreased in a time-related manner during culture, consistent with the reported loss in FSH-binding capacity (28). Although both transcripts are larger than the reported size of the FSH receptor-coding region (9), it remains to be established whether both transcripts are translated to mature functional proteins. Our data would
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FIG. 5. Inhibitory effects of GnRH on basal and FSH-maintained FSH receptor mRNA levels in cultured granulosa cells cultured for 48 h. Left panel, Northern blot analysis of FSH receptor mRNA levels in 5 pg total RNA prepared from granulosa cells incubated for 48 h without or with 30 rig/ml FSH in the absence and presence of 100 nM GnRH. Similar results were obtained in three separate experiments. Right panel, Dose-dependent suppression of FSH (30 ng/ml)maintained FSH receptor mRNA levels by GnRH, as determined by slot blot analysis of RNA samples. Data for FSH receptor mRNA levels (mean + SEM of three replicate cultures) were normalized against levels of 28s rRNA in each sample and expressed relative to FSH receptor mRNA levels present in FSH-stimulated cells (1.0).
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FSH + 6. Blockage of the inhibitory actions of GnRH on FSH receptor mRNA levels by a GnRH antagonist. Levels of FSH receptor mRNA in 5 rg total RNA, prepared from granulosa cells cultured for 48 h in medium alone [control (C)] or with 30 rig/ml FSH in the absence or presence of GnRH (G; 100 nM) and/or a GnRH antagonist (A, 100 nM), were determined. Data were normalized us. levels of 28s rRNA in each sample and expressed relative to the control value (1.0; mean + SEM of three replicate cultures).
FIG.
indicate that changes in FSH receptor number are associated with similar changes in the levels of both receptor mRNAs. Furthermore, treatment of granulosa cells with FSH maintained the levels of its receptor mRNA over a 60-h culture period, indicating that FSH may maintain its own receptor number by influencing the amount of its receptor mRNA. The stimulatory effects of FSH on its receptor mRNA levels in vitro are consistent with in uiuo studies demonstrating the ability of FSH to increase the levels of its own receptor mRNA (17) and binding capacity (1) within the ovary. However, in cultured granulosa cells, we did not observe a down-regulation of FSH receptor mRNA levels after FSH treatment, which has
A 1 \ 2 0-O C
FSH
1
10 FSH
+
100 GnRH
1000 WI
been reported to occur in viva after the injection of an ovulatory dose of FSH (17). The differential response may be due to differences in the levels of FSH used in the two studies and\or differences in the developmental state of the granulosa cells. The involvement of CAMP in mediating the actions of FSH on its receptor mRNA levels is evidenced by the finding that direct activation of adenyl cyclase with forskolin mimics the stimulatory effects of FSH. These data are consistent with previous reports linking the adenyl cyclase-CAMP system to the actions of FSH on other aspects of granulosa cell differentiation, including the induction of aroma&e (29, 30) and LH receptor expression (31). In contrast to the stimulatory actions of FSH and forskolin, culture of granulosa cells with EGF or bFGF did not affect basal FSH receptor mRNA levels, but attenuated the response of granulosa cells to FSH in a dose-dependent manner. Previous studies have demonstrated the presence of EGF-like factors within the ovary (32) and have shown that EGF and bFGF suppress the ability of FSH to induce LH receptor mRNA (27) and protein (33-35) levels in rat granulosa cells. Although EGF by itself does not affect FSH receptor number (34, 36, 37), a recent study has provided evidence that the induction of FSH-binding sites in rat granulosa cells by activin is suppressed by cotreatment of cells with EGF (36). However, EGF and FGF have been reported to increase FSH-binding capacity in porcine granulosa cells (38), suggesting an inherent species-specific difference in the actions of these growth factors. Our findings indicate that the ability of EGF to suppress hormoneinduced FSH receptor expression in rat granulosa cells is correlated with an inhibitory action of EGF at the mRNA level. Therefore, it is likely that the regulation of
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FSH RECEPTOR mRNA IN GRANULOSA FSH receptor expression on differentiating granulosa cells by various endocrine/paracrine factors (FSH, EGF, and bFGF) is due to the actions of these ligands on FSH receptor gene transcription and/or mRNA stability. Consistent with the reported ability of GnRH to block CAMP-induced FSH receptor expression in rat granulosa cells (28), our findings indicate that GnRH completely suppresses the induction of FSH receptor mRNA levels by FSH. The inhibitory actions of GnRH are comparable to previous reports of its suppressive effects on FSHstimulated granulosa cell differentiation (39) as well as inhibition of LH receptor mRNA levels induced by FSH (27). The actions of GnRH are presumably mediated via specific receptors found on the granulosa cells (40), as evidenced by the ability of a GnRH antagonist to completely reverse the inhibitory effects of GnRH on FSHinduced FSH receptor mRNA levels. The present study has provided new data concerning the identification and hormonal regulation of FSH receptor mRNA transcripts in cultured granulosa cells. Results described herein suggest that FSH may induce a positive feedback mechanism, involving increased transcription of its own receptor gene or increased stability of its receptor mRNA, leading to augmented expression of FSH receptors on the cell surface and increased responsiveness of the cells to subsequent FSH action. However, the actions of FSH on its receptor mRNA levels appear to be tightly regulated by the antagonistic actions of EGF, bFGF, and GnRH. The use of cultured rat granulosa cells provides a model system for future studies on the control of FSH receptor gene expression in the ovary. Acknowledgments The authors gratefully acknowledge the National Hormone and Pituitary Distribution Program of the NIH (Baltimore, MD) for ovine FSH, Dr. Andreas Sommer (Synergen, Boulder, CO) for basic FGF, and Dr. N. C. Ling (Whittier Institute, La Jolla, CA) for GnRH and the GnRH antagonist. We would also like to thank Dr. I. L. Gonzalez (Hahnemann University, Philadelphia, PA) for the plasmid containing the human 28s ribosomal RNA gene, and Ms. K. I. Kowalski for excellent technical assistance. References 1. Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley Jr AR, Reichert Jr LE 1976 Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle-stimulating hormone and luteinizine hormone. Endocrinology 991562-1570 2. Richards JS 1980 Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell development. Physiol Rev 60:51-89 3. Hsueh AJW, Adashi EY, Jones PBC, Welsh Jr TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76-127 4. Hsueh AJW, Bicsak TA, Jia X-C, Dahl KD, Fauser BCJM, Galway
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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AB, Czekala N, Pavlou SN, Papkoff H, Keene J, Boime I 1989 Granulosa cells as hormone targets: role of biologically active follicle-stimulating hormone in reproduction. Recent Prog Horm Rh?s 45:209-277 Ireland JJ, Richards JS 1978 Acute effects of estradiol and folliclestimulating hormone on specific binding of human [iz61]iodo-follicle-stimulating hormone to rat ovarian granulosa cells in uiuo and in vitro. Endocrinology 102:876-883 Nimrod A, Lamprecht SA 1980 Hormone-induced desensitization of cultured rat granulosa cells to FSH. Biochem Biophys Res Commun 92:905-911 Knecht M, Nanta T, Katz MS, Catt KJ 1983 Regulation of adenylate cyclase activity by follicle-stimulating hormone and a gonadotropin-releasing hormone agonist in cultured rat granulosa cells. Endocrinology 112:1247-1255 Louvet J-P, Vaitukaitis JL 1976 Induction of follicle-stimulating hormone (FSH) receptors in rat ovaries by estrogen priming. Endocrinology 99758-764 Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle-stimulating hormone: structure and functional exnression of cloned cDNA. Mol Endocrinol4:52530 McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: and unusual member of the G protein-coupled receptor famiiy. Science 245:494-499 Loosfelt H. Misrahi M. Ataer M. Salesse R. Thi MTVH. Jolivet A. Guiochon-Mantel A, Sar 5, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH/hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 Minegish T, Nakamura K, Takakura Y, Miyamoto K, Hasegawa Y, Ibuki Y, Igarashi M 1990 Cloning and sequencing of human LH/hCG receptor cDNA. Biochem Biophys Res Commun 172:1049-1054 Jia X-C, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJW 1991 Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine and ovine species. Mol Endocrinol5:759-768 Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, Van Sande J, Dumont JE, Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 2461620-1622 Nagayama K, Kaufman KD, Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184-1190 Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJW 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711-2719 LaPolt PS, Tilly JL, Aihara T, Nishimori K, Hsueh AJW 1992 Gonadotropin-induced up- and down-regulation of ovarian FSH receptor gene expression in immature rats: effects of PMSG, hCG and recombinant FSH. Endocrinology 130:1289-1295 LaPolt PS, Oikawa M, Jia X-C, Dargan C, Hsueh AJW 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 1263277-3279 Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro svnthesis of bioactive RNA and RNA hybridization probes from plasmids containing a bacteriophage _ SP6 promoter.-Nucleic Acids Res 12:7035-7056Erickson JM, Rushford CL, Dorney DJ, Wilson GN, Schmickel RD 1981 Structure and variation of human ribosomal RNA: molecular analysis of cloned fragments. Gene l&1-9 Gonzalez IL, Gorski JL, Campen TJ, Dorney DJ, Erickson JM, Sylvester JE, Schmickel RD 1985 Variation among human 28s ribosomal RNA genes. Proc Nat.1 Acad Sci USA 82:7666-7670 Feinbere AP. Voaelstein B 1983 A techniaue for radiolabeline DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Chan Y-L, Olvera J, Wool IG 1983 The structure of rat 28s ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene. Nucleic Acids Res 11:7819-7831
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24. Hadjiolov AA, Georgiev 01, Nosikov VV, Yavachev LP 1984 Primary and secondary structure of rat 28s ribosomal RNA. Nucleic Acids Res 12:3677-3693 25. Maniatis T, Fritsch EF, Sambrook J 1982 Isolation of mRNA from mammalian cells. In: Maniatis T, Fritsch EF, Sambrook J (eds) Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratorv, Cold Snrine: Harbor, DD 191-192 26. Ohlsson M,.Hsueh AJW; Ny T 1988 Hormonal regulation of tissuetype plasminogen activator ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin-releasing hormone. Mol Endocrinol2:854-861 27. Pique&e GN, LaPolt PS, Oikawa M, Hsueh AJW 1991 Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormane in cultured rat granulosa cells. Endocrinology 128244912546 28. Knecht M. Ranta T, Catt KJ 1983 Granulosa cell differentiation in vitro: induction and maintenance of follicle-stimulating hormone receptors by adenosine 3’,5’-monophosphate. Endocrinology 1X%949-956 29. Knecht M, Amsterdam A, Catt KJ 1981 The regulatory role of CAMP in hormone-induced granulosa cell differentiation. J Biol Chem 256X%28-10633 30. Wang C, Hsueh AJW, Erickson GF 1982 The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells. Mol Cell Endocrinol 25:73-83 31. Nimrod A 1981 The induction of ovarian LH receptors by FSH is mediated by cyclic AMP. FEBS Lett 131:31-33 32. Hsu C-J, Holmes SD, Hammond JM 1987 Ovarian epidermal
33. 34.
35. 36.
37. 38. 39. 40.
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growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement. Biochem Bionhvs _ _ Res Commun 147:242-247 Mondschein JS, Schomberg DM 1981 Growth factors modulate gonadotropin receptor induction in aranulosa cell cultures. Science 211:1179-i180 Knecht M, Catt KJ 1983 Epidermal growth factor and gonadotropin-releasing hormone inhibit CAMP-denendent luteinizine hormone receptor formation in ovarian granulosa cells. J Cefi Biol 21:209-217 Oury F, Darbon JM 1988 Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat manulosa cells. Biochem Biophys Res Corn&n 156634-643 Hasegawa Y, Miyamoto K, Abe Y, Nakamura T, Sugino H, Eto Y, Shibai H, Igarashi M 1988 Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell. Biochem Bionhvs Res Commun 156668-674 Knecht M, Catt KJ i983 Modulation of CAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 2582789-2794 May JV, Buck PA, Schomberg DW 1987 Epidermal growth factor enhances [‘2SI]iodo-follicle-stimulating hormone binding by cultured porcine granulosa cells. Endocrinology 120:2413-2420 Hsueh AJW, Jones PBC 1981 Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 2437-461 Jones PBC, Conn PM, Marian J, Hsueh AJW 1980 Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells. Life Sci 272125-2132
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Vol. 130, No. 3 Printed
Society
Hormonal Regulation of Follicle-Stimulating Receptor Messenger Ribonucleic Acid Levels Rat Granulosa Cells* JONATHAN
L. TILLYt,
PHILIP
S. LAPOLTS,
AND
AARON
in U.S.A
Hormone in Cultured
J. W. HSUEH
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
epidermal growth factor (EGF; 0.3-10 rig/ml), basic fibroblast growth factor (bFGF; l-30 rig/ml), or insulin-like growth factorI (IGF-I; l-30 rig/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 rig/ml) on its own receptor mRNA levels. Similarly, GnRH (IO1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (IDW, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-tr-Phe’,p-pC1-Phez,&Trp~6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitorv actions of EGF. bFGF. and GnRH. Thus. the use of cultured
ABSTRACT. The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2&y culture period. However, inclusion of FSH (30 rig/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (l-100 rig/ml) increased FSH receptor mRNA levels in a dose-dependent manner (EDm, 4.5 rig/ml), with a maximal 5.9 + 0.7-fold increase observed in response to 30 rig/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 PM), suggesting the involvement of CAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with
rat” granulosa
cells’
provides
a model
systdm
to analyze
the
hormonal regulation of FSH receptor gene expression in the ovary. (Endocrinology 130: 1296-1302, 1992)
T
HE REQUIREMENT for FSH in the differentiation and maturation of ovarian follicles has been well established (for review, see Refs. l-4). The actions of FSH are mediated through specific receptors, located exclusively on the granulosa cell plasma membrane, which are functionally coupled via membrane-associated G-proteins to the adenyl cyclase-CAMP-generating system. Previous studies have shown that FSH increases the levels of its own receptor (1, 5), although prolonged exposure to FSH may also down-regulate the FSH receptor and the responsiveness of granulosa cells to subsequent FSH challenge (6, 7). Estradiol alone does not
appear to alter the distribution, number, or affinity of FSH-binding sites within the rat ovary (1, 8); however, estrogens do synergize with FSH in vivo to increase the number of FSH receptors per granulosa cell through a process involving both induction and maintenance (1,5). Recent cloning of the rat FSH receptor cDNA has provided data indicating that this protein belongs to the G-protein-coupled receptor family, with the characteristic seven-transmembrane-spanning region (9). Additionally, evaluation of cDNA-derived amino acid sequences indicates a high homology of the FSH receptor to LH/ CG (10-13) and TSH (14, 15) receptors. However, little information is available concerning the hormonal regulation of FSH receptor gene expression in the ovary. In light of the limited and conflicting data available regarding the hormonal regulation of FSH-binding sites within the ovary, the ability to study changes in FSH receptor mRNA levels would provide a clearer insight into the regulation of FSH receptor expression. Using a specific cRNA probe for the FSH receptor nucleotide sequence,
Received September 9,199l. Address all correspondence and requests for reprints to: Dr. Aaron J.-W. Hsueh, Division of Reproductive Bioloav, Department of Gynecology and Obstetrics, Stanford University School of Medicine,-300 Pasteur Drive. Stanford. California 94305-5317. * This work was supported by NIH Grant HD-23273. 7 Postdoctoral Fellow, supported by a grant from the Lalor Foundation. $ Postdoctoral Fellow, supported by National Research Service Award HD-07252. 1296
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FSH RECEPTOR mRNA IN GRANULOSA we analyzed FSH receptor mRNA transcripts within cultured rat granulosa cells to elucidate the hormonal regulation of ovarian FSH receptor gene activation. Materials
and Methods
Hormones Ovine FSH (NIH FSH S17; 20 x NIH FSH Sl U/mg) was a gift from the National Hormone and Pituitary Distribution Program (Baltimore, MD). Receptor grade murine EGF and human insulin-like growth factor-I (IGF-I) were obtained from Collaborative Research, Inc. (Waltham, MA), whereas recombinant basic fibroblast growth factor (bFGF) was generously provided by Dr. Andreas Sommer (Synergen, Boulder, CO). GnRH and the GnRH antagonist, [Ac-D-Phe’,D-pCl-Phe*,DTrp3B6]GnRH, were provided by Dr. N. C. Ling (Whittier Institute, La Jolla, CA). Forskolin was obtained from Calbiochem (La Jolla, CA). Granulosa cell culture Granulosa cells were obtained from immature 26-day-old Sprague-Dawley rats (Johnson Laboratories, Bridgeview, IL) implanted with diethylstilbestrol capsules 4 days earlier, as previously described (16). After estimation of cell number and viability with trypan blue exclusion, 7 x lo5 viable cells were pipetted into 12 x 75-mm polypropylene tubes and cultured in 0.5 ml McCoy’s 5a medium (Gibco, Santa Clara, CA) at 37 C in a humidified 95% air-5% CO, atmosphere. For culture, the McCoy’s 5a medium was supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin sulfate, and 100 nM androstenedione in the absence and presence of different hormones. Preparation of nucleic acid probes A cRNA probe for bases 621-1031 of the rat FSH receptor nucleotide sequence (9) was synthesized using a 410-basepair (bp) cDNA template obtained by the reverse transcriptionpolymerase chain reaction (17), essentially as previously described (17-19). This stretch of FSH receptor cDNA has low (48%) sequence homology with the rat LH receptor (10) and, under the stringent hybridization conditions used in the present study, the FSH receptor probe does not cross-hybridize with LH receptor mRNA (17). For normalization of FSH receptor mRNA data, a 1.6-kilobase (kb) cDNA probe for human 28s rRNA (BumHI-EcoRI fragment pAaz) (20, 21) was prepared using the random priming method (22). Sequence analysis of the 1.6-kb human ribosomal (r) RNA gene fragment used in the present studies predicts 89% nucleotide homology to a corresponding conserved sequence in the rat 28s rRNA gene (23, 24). RNA isolation and analysis After culture, supernatant was removed from culture tubes with a glass Pasteur pipette, and the remaining cell pellet was immediately snap-frozen in a dry ice-ethanol bath and stored at -70 C. Total RNA was then extracted from the cultured cells using the Nonidet P-40 method (25) and quantitated by measuring the absorbance of samples at 260 nm. For Northern
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blot analysis, 5 pg total RNA from the various treatment groups were fractionated on denaturing agarose gels and subsequently transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) by overnight capillary electrophoresis with 20 x sodium chloride-sodium citrate (SSC). For slot blot analysis, RNA samples were denatured in the presence of 4 M formaldehyde for 15 min at 60 C and quickly chilled on ice. Samples were adjusted to 6 x SSC and applied directly to nitroceilulose membranes using a slot blot apparatus (Schleicher and Schuell). For subsequent normalization of data, replicate membranes were prepared from the same RNA samples for separate hybridization to the FSH receptor probe and the 28s rRNA probe. For both Northern and slot blot analyses, transferred RNA was covalently cross-linked to the nitrocellulose membranes using a UV-crosslinker (Stratagene, La Jolla, CA). Northern and slot blots were prehybridized for 2-4 h at 66 C in the presence of 50% formamide under standard conditions, followed by hybridization with the radiolabeled probe of interest [cRNA probe for FSH receptor (18,26)) or cDNA probe for 28s rRNA (27)] at the same temperature for 16-18 h. Membranes were washed in 2 x SSC-0.1% sodium dodecyl sulfate for 10 min at room temperature, followed by two or three consecutive 15-min (FSH receptor) or 30-min (28s rRNA) washes in 0.1 x SSC-0.1% sodium dodecyl sulfate at 66 C. Membranes were then exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) for l-7 days at -70 C. Data analysis The intensities of slot blot hybridization signals were quantitated using a scanning transmittance densitometer and a digitizing computer program (Hoeffer Scientific, San Francisco, CA). Data for treatment effects on FSH receptor mRNA levels were then normalized against levels of 28s rRNA in each sample and expressed as relative densitometric units. For all experiments, a representative Northern blot (of three replicates) is presented, whereas slot blot data depict the mean + SEM of results from three replicate experiments. Statistical analysis was performed based on analysis of variance, followed by Scheffe’s test. Results Time course of FSH action on its receptor mRNA levels Northern blot analysis indicated the existence of two predominant FSH receptor mRNA transcripts of approximately 7.0 and 2.5 kb in total RNA prepared from freshly isolated granulosa cells of immature estrogentreated rats (Fig. 1, left panel). After increasing lengths of culture (12, 24, 36, 48, and 60 h) in the absence of hormone treatment, a time-related decrease in the level of FSH receptor mRNA was observed compared to levels found in cells at the initiation of culture (designated time zero; Fig. 1, right panel). However, inclusion of ovine FSH (30 rig/ml) in the culture medium prevented the decrease in FSH receptor mRNA levels during culture
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FIG. 1. Effect of FSH treatment on FSH receptor mRNA transcripts in cultured granulosa cells collected from immature estrogen-treated rats. Total RNA (5 rg) was prepared from cells incubated for increasing lengths of time (O-60 h) in the absence [control (C)] or presence of 30 rig/ml FSH. Levels of FSH receptor mRNA were analyzed by Northern (left panel) and slot blot (right panel) analysis using a cRNA probe under highly stringent conditions. The Northern blot, which depicts levels of FSH receptor mRNA transcripts in freshly isolated granulosa cells [time zero (To); no culture], is representative of results from three replicate experiments. Data from the slot blot analysis, which represent the mean + SEM of three separate cultures, were normalized for 2% rRNA levels in each sample and expressed relative to FSH receptor mRNA levels at time zero (1.0). FIG. 2. Dose-dependent stimulation of FSH receptor mRNA levels by FSH. Left panel, Total RNA was prepared from granulosa cells cultured for 48 h in the absence [control CC)] or presence of 30 rig/ml FSH and analyzed by Northern blot for FSH receptor mRNA. Right panel, Total RNA was extracted from cells incubated for 48 h without and with increasing doses of FSH and analyzed by slot blot analysis for FSH receptor mRNA and 285 rRNA levels. Data for FSH receptor mRNA levels were normalized us. levels of 28s rRNA and expressed relative to the control value (1.0). Slot blot data represent the mean + SEM of results from four separate cultures, whereas the Northern blot is representative of three replicate experiments.
(12-60 h), maintaining levels comparable to those found in cells at time zero (P > 0.10; Fig. 1, right panel). Dose-dependent effects of FSH and forskolin on FSH receptor mRNA levels Culture of granulosa cells for 48 h in the presence of increasing concentrations (l-100 rig/ml) of FSH resulted in a dosedependent increase in FSH receptor mRNA levels, with a maximal 5.9 f 0.7-fold increase over controls observed in response to 30 rig/ml FSH (P < 0.05 vs. control; Fig. 2, right panel). The ED, for FSH action was estimated to be 4.5 ng/ ml. Northern blot analysis indicated an increased level of both the 7.0- and 2.5-kb transcripts in FSH-treated cells compared to controls (Fig. 2, left panel). The stimulatory effects of FSH were mimicked by the adenyl cyclase activator forskolin (O.l30 FM), which increased FSH receptor mRNA levels in a dosedependent manner (EDW, 0.2 PM) to a maximum 3.5 + 0.4-fold
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increase at a concentration of 10 PM (P C 0.05 vs. control; Fig. 3, right panel). The increased FSH receptor mRNA levels detected in forskolin-treated cells resulted from an increase in the levels of both receptor transcripts, as determined by Northern blot analysis (Fig. 3, left panel). Effects of EGF, bFGF, IGF-I, and GnRH on basal and FSH-maintained FSH receptor mRNA levels Treatment of granulosa cells for 48 h with EGF (0.310 rig/ml), bFGF (l-30 rig/ml), or IGF-I (l-30 rig/ml) did not significantly alter basal levels of FSH receptor mRNA (data not shown). However, treatment with EGF or bFGF dose-dependently attenuated the stimulatory actions of FSH (30 rig/ml) on FSH receptor mRNA levels (IDso of EGF, 0.6 rig/ml; ID60 of bFGF, 7 rig/ml; Fig. 4, right panel). A complete suppression of FSH action was
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FIG. 3. Effects of increasing concentrations of the adenyl cyclase activator forskolin (FOR) on FSH receptor mRNA levels in granulosa cells after a 48-h culture. Total RNA (5 rg) was extracted and analyzed by Northern (left panel; with or without 10 pM FOR) and slot blot (rig/rtpanel) analyses. Slot blot data for FSH receptor mRNA levels, which are the mean + SEM of three separate cultures, were normalized against 28s rRNA levels and expressed relative to the control value (C; 1.0). The Northern blot is representative of three replicate experiments. C
0.1
1
10
Forskolin
FIG. 4. Effects of different growth factors on FSH receptor mRNA levels. Left panel, Northern blot analysis of FSH receptor mRNA in 5 fig total RNA prepared from granulosa cells incubated for 48 h with medium alone [control (C)] or with 30 rig/ml FSH in the absence and presence of EGF (10 rig/ml), bFGF (FGF; 30 rig/ml), or IGF-I (IGF; 30 ng/ ml). The Northern blot is representative of three replicate experiments. Right panel, Slot blot analysis of FSH receptor mRNA levels in granulosa cells cultured for 48 h in medium alone [control (C)] or with 30 rig/ml FSH in the absence and presence of EGF (0.3-10 rig/ml), bFGF (l-30 rig/ml), or IGF-I (l-30 ng/ ml). These data, which represent the mean + SEM of results from three replicate cultures, were normalized us. levels of 28s rRNA in each sample and expressed relative to FSH receptor mRNA levels in FSH-stimulated cells (1.0).
observed in response to the highest dose of EGF or bFGF used (P < 0.05 vs. FSH alone), as determined by both Northern (Fig. 4, left panel) and slot blot (Fig. 4, right panel) analysis. In contrast, IGF-I did not alter the levels of FSH receptor mRNA maintained by 30 rig/ml FSH during a 48-h incubation (P > 0.10) (Fig. 4, both panels). Treatment of granulosa cells for 48 h with 100 nM GnRH suppressed basal levels of FSH receptor mRNA compared to control values (Fig. 5, left panel). Similarly, GnRH caused a dose-dependent attenuation of the stimulatory actions of FSH (30 rig/ml) on FSH receptor mRNA levels (IDbO, 8 nM), with a maximal inhibitory effect observed in response to 100 nM GnRH (Fig. 5, right panel). Northern blot analysis indicated that cotreatment of granulosa cells with 100 IXM GnRH completely suppressed the stimulatory effects of FSH on its own receptor mRNA levels (Fig. 5, leftpanel). The inhib-
30
()IM)
l&r
W-1
c
FSH
0.8
t
FSli
l
a
Qrowth
T
10
Factor
30
~&II)
itory actions of GnRH (100 nM) on FSH-maintained FSH receptor mRNA levels were prevented by inclusion of an equimolar concentration of the GnRH antagonist [Ac-n-Phe’,D-pC1-Phe2,D_Trp3’6]GnRH in the culture medium (Fig. 6). The GnRH antagonist alone, however, did not alter basal or FSH-stimulated FSH receptor mRNA levels (Fig. 6). Discussion Granulosa cells isolated from immature rats contain two FSH receptor mRNA transcripts, the levels of which decreased in a time-related manner during culture, consistent with the reported loss in FSH-binding capacity (28). Although both transcripts are larger than the reported size of the FSH receptor-coding region (9), it remains to be established whether both transcripts are translated to mature functional proteins. Our data would
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FSH RECEPTOR mRNA IN GRANULOSA
FIG. 5. Inhibitory effects of GnRH on basal and FSH-maintained FSH receptor mRNA levels in cultured granulosa cells cultured for 48 h. Left panel, Northern blot analysis of FSH receptor mRNA levels in 5 pg total RNA prepared from granulosa cells incubated for 48 h without or with 30 rig/ml FSH in the absence and presence of 100 nM GnRH. Similar results were obtained in three separate experiments. Right panel, Dose-dependent suppression of FSH (30 ng/ml)maintained FSH receptor mRNA levels by GnRH, as determined by slot blot analysis of RNA samples. Data for FSH receptor mRNA levels (mean + SEM of three replicate cultures) were normalized against levels of 28s rRNA in each sample and expressed relative to FSH receptor mRNA levels present in FSH-stimulated cells (1.0).
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CFSH G&H-
-+--+
7 2. Q
FSH + 6. Blockage of the inhibitory actions of GnRH on FSH receptor mRNA levels by a GnRH antagonist. Levels of FSH receptor mRNA in 5 rg total RNA, prepared from granulosa cells cultured for 48 h in medium alone [control (C)] or with 30 rig/ml FSH in the absence or presence of GnRH (G; 100 nM) and/or a GnRH antagonist (A, 100 nM), were determined. Data were normalized us. levels of 28s rRNA in each sample and expressed relative to the control value (1.0; mean + SEM of three replicate cultures).
FIG.
indicate that changes in FSH receptor number are associated with similar changes in the levels of both receptor mRNAs. Furthermore, treatment of granulosa cells with FSH maintained the levels of its receptor mRNA over a 60-h culture period, indicating that FSH may maintain its own receptor number by influencing the amount of its receptor mRNA. The stimulatory effects of FSH on its receptor mRNA levels in vitro are consistent with in uiuo studies demonstrating the ability of FSH to increase the levels of its own receptor mRNA (17) and binding capacity (1) within the ovary. However, in cultured granulosa cells, we did not observe a down-regulation of FSH receptor mRNA levels after FSH treatment, which has
A 1 \ 2 0-O C
FSH
1
10 FSH
+
100 GnRH
1000 WI
been reported to occur in viva after the injection of an ovulatory dose of FSH (17). The differential response may be due to differences in the levels of FSH used in the two studies and\or differences in the developmental state of the granulosa cells. The involvement of CAMP in mediating the actions of FSH on its receptor mRNA levels is evidenced by the finding that direct activation of adenyl cyclase with forskolin mimics the stimulatory effects of FSH. These data are consistent with previous reports linking the adenyl cyclase-CAMP system to the actions of FSH on other aspects of granulosa cell differentiation, including the induction of aroma&e (29, 30) and LH receptor expression (31). In contrast to the stimulatory actions of FSH and forskolin, culture of granulosa cells with EGF or bFGF did not affect basal FSH receptor mRNA levels, but attenuated the response of granulosa cells to FSH in a dose-dependent manner. Previous studies have demonstrated the presence of EGF-like factors within the ovary (32) and have shown that EGF and bFGF suppress the ability of FSH to induce LH receptor mRNA (27) and protein (33-35) levels in rat granulosa cells. Although EGF by itself does not affect FSH receptor number (34, 36, 37), a recent study has provided evidence that the induction of FSH-binding sites in rat granulosa cells by activin is suppressed by cotreatment of cells with EGF (36). However, EGF and FGF have been reported to increase FSH-binding capacity in porcine granulosa cells (38), suggesting an inherent species-specific difference in the actions of these growth factors. Our findings indicate that the ability of EGF to suppress hormoneinduced FSH receptor expression in rat granulosa cells is correlated with an inhibitory action of EGF at the mRNA level. Therefore, it is likely that the regulation of
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FSH RECEPTOR mRNA IN GRANULOSA FSH receptor expression on differentiating granulosa cells by various endocrine/paracrine factors (FSH, EGF, and bFGF) is due to the actions of these ligands on FSH receptor gene transcription and/or mRNA stability. Consistent with the reported ability of GnRH to block CAMP-induced FSH receptor expression in rat granulosa cells (28), our findings indicate that GnRH completely suppresses the induction of FSH receptor mRNA levels by FSH. The inhibitory actions of GnRH are comparable to previous reports of its suppressive effects on FSHstimulated granulosa cell differentiation (39) as well as inhibition of LH receptor mRNA levels induced by FSH (27). The actions of GnRH are presumably mediated via specific receptors found on the granulosa cells (40), as evidenced by the ability of a GnRH antagonist to completely reverse the inhibitory effects of GnRH on FSHinduced FSH receptor mRNA levels. The present study has provided new data concerning the identification and hormonal regulation of FSH receptor mRNA transcripts in cultured granulosa cells. Results described herein suggest that FSH may induce a positive feedback mechanism, involving increased transcription of its own receptor gene or increased stability of its receptor mRNA, leading to augmented expression of FSH receptors on the cell surface and increased responsiveness of the cells to subsequent FSH action. However, the actions of FSH on its receptor mRNA levels appear to be tightly regulated by the antagonistic actions of EGF, bFGF, and GnRH. The use of cultured rat granulosa cells provides a model system for future studies on the control of FSH receptor gene expression in the ovary. Acknowledgments The authors gratefully acknowledge the National Hormone and Pituitary Distribution Program of the NIH (Baltimore, MD) for ovine FSH, Dr. Andreas Sommer (Synergen, Boulder, CO) for basic FGF, and Dr. N. C. Ling (Whittier Institute, La Jolla, CA) for GnRH and the GnRH antagonist. We would also like to thank Dr. I. L. Gonzalez (Hahnemann University, Philadelphia, PA) for the plasmid containing the human 28s ribosomal RNA gene, and Ms. K. I. Kowalski for excellent technical assistance. References 1. Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley Jr AR, Reichert Jr LE 1976 Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle-stimulating hormone and luteinizine hormone. Endocrinology 991562-1570 2. Richards JS 1980 Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell development. Physiol Rev 60:51-89 3. Hsueh AJW, Adashi EY, Jones PBC, Welsh Jr TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76-127 4. Hsueh AJW, Bicsak TA, Jia X-C, Dahl KD, Fauser BCJM, Galway
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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AB, Czekala N, Pavlou SN, Papkoff H, Keene J, Boime I 1989 Granulosa cells as hormone targets: role of biologically active follicle-stimulating hormone in reproduction. Recent Prog Horm Rh?s 45:209-277 Ireland JJ, Richards JS 1978 Acute effects of estradiol and folliclestimulating hormone on specific binding of human [iz61]iodo-follicle-stimulating hormone to rat ovarian granulosa cells in uiuo and in vitro. Endocrinology 102:876-883 Nimrod A, Lamprecht SA 1980 Hormone-induced desensitization of cultured rat granulosa cells to FSH. Biochem Biophys Res Commun 92:905-911 Knecht M, Nanta T, Katz MS, Catt KJ 1983 Regulation of adenylate cyclase activity by follicle-stimulating hormone and a gonadotropin-releasing hormone agonist in cultured rat granulosa cells. Endocrinology 112:1247-1255 Louvet J-P, Vaitukaitis JL 1976 Induction of follicle-stimulating hormone (FSH) receptors in rat ovaries by estrogen priming. Endocrinology 99758-764 Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle-stimulating hormone: structure and functional exnression of cloned cDNA. Mol Endocrinol4:52530 McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: and unusual member of the G protein-coupled receptor famiiy. Science 245:494-499 Loosfelt H. Misrahi M. Ataer M. Salesse R. Thi MTVH. Jolivet A. Guiochon-Mantel A, Sar 5, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH/hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 Minegish T, Nakamura K, Takakura Y, Miyamoto K, Hasegawa Y, Ibuki Y, Igarashi M 1990 Cloning and sequencing of human LH/hCG receptor cDNA. Biochem Biophys Res Commun 172:1049-1054 Jia X-C, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJW 1991 Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine and ovine species. Mol Endocrinol5:759-768 Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, Van Sande J, Dumont JE, Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 2461620-1622 Nagayama K, Kaufman KD, Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184-1190 Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJW 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711-2719 LaPolt PS, Tilly JL, Aihara T, Nishimori K, Hsueh AJW 1992 Gonadotropin-induced up- and down-regulation of ovarian FSH receptor gene expression in immature rats: effects of PMSG, hCG and recombinant FSH. Endocrinology 130:1289-1295 LaPolt PS, Oikawa M, Jia X-C, Dargan C, Hsueh AJW 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 1263277-3279 Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro svnthesis of bioactive RNA and RNA hybridization probes from plasmids containing a bacteriophage _ SP6 promoter.-Nucleic Acids Res 12:7035-7056Erickson JM, Rushford CL, Dorney DJ, Wilson GN, Schmickel RD 1981 Structure and variation of human ribosomal RNA: molecular analysis of cloned fragments. Gene l&1-9 Gonzalez IL, Gorski JL, Campen TJ, Dorney DJ, Erickson JM, Sylvester JE, Schmickel RD 1985 Variation among human 28s ribosomal RNA genes. Proc Nat.1 Acad Sci USA 82:7666-7670 Feinbere AP. Voaelstein B 1983 A techniaue for radiolabeline DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Chan Y-L, Olvera J, Wool IG 1983 The structure of rat 28s ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene. Nucleic Acids Res 11:7819-7831
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FSH RECEPTOR
mRNA IN GRANULOSA
24. Hadjiolov AA, Georgiev 01, Nosikov VV, Yavachev LP 1984 Primary and secondary structure of rat 28s ribosomal RNA. Nucleic Acids Res 12:3677-3693 25. Maniatis T, Fritsch EF, Sambrook J 1982 Isolation of mRNA from mammalian cells. In: Maniatis T, Fritsch EF, Sambrook J (eds) Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratorv, Cold Snrine: Harbor, DD 191-192 26. Ohlsson M,.Hsueh AJW; Ny T 1988 Hormonal regulation of tissuetype plasminogen activator ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin-releasing hormone. Mol Endocrinol2:854-861 27. Pique&e GN, LaPolt PS, Oikawa M, Hsueh AJW 1991 Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormane in cultured rat granulosa cells. Endocrinology 128244912546 28. Knecht M. Ranta T, Catt KJ 1983 Granulosa cell differentiation in vitro: induction and maintenance of follicle-stimulating hormone receptors by adenosine 3’,5’-monophosphate. Endocrinology 1X%949-956 29. Knecht M, Amsterdam A, Catt KJ 1981 The regulatory role of CAMP in hormone-induced granulosa cell differentiation. J Biol Chem 256X%28-10633 30. Wang C, Hsueh AJW, Erickson GF 1982 The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells. Mol Cell Endocrinol 25:73-83 31. Nimrod A 1981 The induction of ovarian LH receptors by FSH is mediated by cyclic AMP. FEBS Lett 131:31-33 32. Hsu C-J, Holmes SD, Hammond JM 1987 Ovarian epidermal
33. 34.
35. 36.
37. 38. 39. 40.
CELLS
Endo l 1992 Voll30. No 3
growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement. Biochem Bionhvs _ _ Res Commun 147:242-247 Mondschein JS, Schomberg DM 1981 Growth factors modulate gonadotropin receptor induction in aranulosa cell cultures. Science 211:1179-i180 Knecht M, Catt KJ 1983 Epidermal growth factor and gonadotropin-releasing hormone inhibit CAMP-denendent luteinizine hormone receptor formation in ovarian granulosa cells. J Cefi Biol 21:209-217 Oury F, Darbon JM 1988 Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat manulosa cells. Biochem Biophys Res Corn&n 156634-643 Hasegawa Y, Miyamoto K, Abe Y, Nakamura T, Sugino H, Eto Y, Shibai H, Igarashi M 1988 Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell. Biochem Bionhvs Res Commun 156668-674 Knecht M, Catt KJ i983 Modulation of CAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 2582789-2794 May JV, Buck PA, Schomberg DW 1987 Epidermal growth factor enhances [‘2SI]iodo-follicle-stimulating hormone binding by cultured porcine granulosa cells. Endocrinology 120:2413-2420 Hsueh AJW, Jones PBC 1981 Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 2437-461 Jones PBC, Conn PM, Marian J, Hsueh AJW 1980 Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells. Life Sci 272125-2132
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