Inhibitory Effect of Gonadotrophin-releasing Hormone (GnRH) on Rat Granulosa Cell Deoxyribonucleic Acid Synthesis

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1 MOLECULAR REPRODUCTION AND DEVELOPMENT 47: (1997) Inhibitory Effect of Gonadotrophin-releasing Hormone (GnRH) on Rat Granulosa Cell Deoxyribonucleic Acid Synthesis PATRICIA E. SARAGÜETA, GUILLERMO M. LANUZA, AND J. LINO BARAÑAO* Instituto de Biología y Medicina Experimental-CONICET and Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina ABSTRACT Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-b (TGF-b), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of 3 H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFb (2.5 ng/ml), at concentrations as low as M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED M) and native GnRH (ED M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide M could prevent the inhibition elicited by M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: , r 1997 Wiley-Liss, Inc. Key Words: gonadotropin releasing hormone; granulosa cell; rat; DNA synthesis; LHRH INTRODUCTION Many reports have indicated that GnRH directly affects ovarian function in mammals (Hsueh and Jones, 1981; Shinohara et al., 1985; Yoshimura et al., 1990). The administration of GnRH to hypophysectomized rats has been shown to result in a variety of both inhibitory and stimulatory responses, affecting follicular and luteal function and ovulation (Hsueh and Jones, 1981; Shinohara et al., 1985). The inhibitory actions of GnRH are mainly evidenced as a blockade of gonadotropin-stimulated granulosa cell differentiation (Knecht et al., 1982; Tureck et al., 1982; Darbon et al., 1984). These direct effects appear to be mediated by receptors essentially indistinguishable from those present in the pituitary (Reeves et al., 1980) and to involve activation of the phosphoinositide-derived pathways (Leung and Wang, 1989). r 1997 WILEY-LISS, INC. In addition to its inhibitory action, GnRH also has been shown to stimulate many granulosa cell functions such as the secretion of prostaglandin E and, to a lesser extent, of prostaglandin F in vitro (Sharpe, 1982), estrogen, and progesterone production (Dorrington et al., 1983), protein synthesis (Hsueh and Jones, 1982), plaminogen activator activity (Wang, 1983), and fibronectin secretion (Dorrington and Skinner, 1986). The demonstration of GnRH-like substances (Birnbaumer et al., 1985; Ireland et al., 1988), and the gene transcript of GnRH (Goubau et al., 1992) in the ovary have led to the hypothesis that the GnRH receptor may be involved in an autocrine or paracrine intraovarian system regulating granulosa cell function. In a previous study (Dain et al., 1993), we have shown that human granulosa-lutein cells produce a factor(s) capable of inhibiting gonadotropin-stimulated DNA synthesis in rat granulosa cells, suggesting the existence of negative loops controlling granulosa cell growth. The present study aims at determining the possible involvement of GnRH in the regulation of the granulosa cells proliferation. MATERIALS AND METHODS Hormones and Chemicals Ovine FSH (ofsh-17) and GnRH, amide form, were obtained from the National Hormone and Pituitary program, [methyl- 3 H]thymidine (10 Ci/mmol) from American Radiolabeled Chemicals (St. Louis, MO); Diethylstilbestrol (DES), Leuprolide, Buserelin, and Antide were from Sigma (St. Louis, MO). Transforming growth factor-b1 (TGF-b1) from porcine platelets was obtained from R&D Systems (Minneapolis, MN). Granulosa Cell Preparation and Culture Ovaries were obtained from day-old female Sprague-Dawley rats after 3 days of DES treatment (sc Silastic implants containing 5 mg DES). Granulosa cells were prepared and cultured as previously de- Contract grant sponsor: Rockefeller Foundation and the Buenos Aires University. *Correspondence to: J. Lino Barañao, Instituto de Medicina y Biología Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. Received 22 October 1996; Accepted 21 November 1996

2 EFFECT OF GnRH ON RAT GRANULOSA CELL 171 scribed (Bley et al., 1991). Briefly, the ovaries were punctured with a 30-gauge needle and incubated in Dulbecco s Modified Eagle s medium (4.5g glucose/liter)- Ham s F12 (DMEM:F12; 1:1, Gibco, Grand island, NY), EDTA (6.8 mm), and HEPES (10 mm; 15 min at 37 C), and then washed twice and incubated in DMEM-F12 (1:1), sucrose (0.5 M), and HEPES (10 mm; 5 min at 37 C). After incubation, the medium was diluted with 2 vol of DMEM-F12 and HEPES (10 mm), and ovaries were allowed to sediment. Granulosa cells were obtained by pressing ovaries within two pieces of nylon mesh (Nytex 50, Geneva, Switzerland). The cell suspension was layered over a 40% Percoll solution in saline and centrifuged at g for 20 min. The granulosa cell layer was aspirated from the top of Percoll solution and resuspended in DMEM-F12 (1:1) and bicarbonate solution (2.2 g/l; ph:7.4). Unless otherwise indicated, cells were seeded on plastic 96-well plates (Nunc) precoated with rat tail collagen. The initial plating density was viable cells/cm 2. Cells were maintained at 37 C with 5% CO 2. After 2 hr, media were changed to remove nonattached cells and were replaced by fresh media containing different factors to test. DNA Synthesis Assay DNA synthesis was determined by 3 H-thymidine incorporation according to a method previously validated in these culture conditions (Bley et al., 1991, 1992; Dain et al., 1993). Briefly, granulosa cells were cultured in 96-well plates in presence of different hormones and peptides. Tritiated thymidine (4 µci/ml) was added to the cultures 24 hr after plating. Cells were harvested 24 hr later in glass hollow fibers with a multiwell cell harvester (Nunc, Copenhagen, Denmark), radioactivity was measured in a scintillation counter. Statistical Analysis Results are expressed as the mean 6 SEM of triplicate cultures. Statistical comparisons of the results were made using one-way analysis of variance and Scheffe s test for multiple comparisons (Li, 1964). Calculation of the ED 50 s were done using a computer program based on a four-parameter logistic equation (DeLean et al., 1978). Experiments were carried out at least three times with similar results. Fig. 1. Effect of GnRHa on hormonally stimulated DNA synthesis in rat granulosa cell cultures. Cells were cultured with increasing concentrations of GnRHa (Leuprolide), in the presence of FSH (20 ng/ml) or a combination of estradiol (E 2, 100 ng/ml) and FSH (20 ng/ml). 3 H-thymidine incorporation was performed for 24 hr starting 24 hr after plating. Values are means 6 SEM of triplicate cultures. RESULTS Effect of GnRH on Hormonally Stimulated DNA Synthesis Previous studies have demonstrated that treatment with a combination of FSH and estradiol elicits a stimulatory effect on immature rat granulosa cell DNA synthesis (Bley et al., 1991; Dain et al., 1993). As can be seen in Figure 1, GnRHa alone had no effect on basal DNA synthesis. However, when rat granulosa cells were cultured with FSH (20 ng/ml) and E 2 (100 ng/ml), addition of GnRHa produced a dose-dependent inhibition of thymidine incorporation, with an ED 50 of M. Effects of GnRHa on Interaction Among FSH, Estradiol, and Transforming Growth Factor-b We next determined whether the GnRHa could also affect the synergism between FSH and TGF-b (Barañao et al., 1993). Figure 2 shows that the combination of FSH and TGF-b produced a 10-fold increase in thymidine incorporation. This effect was almost completely blocked by GnRHa at concentrations as low a M. The slight stimulation elicited by TGF-b alone was not significantly affected by the agonist (Control: ; GnRHa 0.5 ng/ml: ; TGF-b: ; TGF-b 1GnRHa: cpm/well). Estradiol supplementation further enhanced the stimulatory effect of FSH plus TGF-b (Barañao et al., 1993). This effect was also inhibited by GnRHa in the same range of concentrations (Fig. 2). Another GnRH agonist, Buserelin (Fig. 3) and the native GnRH (Fig. 4) were also able to abolish the interaction between FSH and TGF-b, but the effective concentrations required to elicit a comparable inhibition were markedly higher that those of Leuprolide (ED 50 s: and M, for Buserelin and native GnRH, respectively). Effect of a GnRH Antagonist The GnRH antagonist, Antide, was able to revert the inhibitory action of the GnRHa on DNA synthesis stimulated by a combination of FSH and TGF-b. As shown in Figure 5, the competitive effects of antide

3 172 P.E. SARAGÜETA ET AL. Fig. 2. Effects of GnRHa on the interaction among FSH, estradiol, and TGF-b. Cells were cultured with increasing concentrations of GnRHa (Leuprolide), with the following additions: TGF-b1(3 ng/ml), FSH (20 ng/ml) or a combination with estradiol (100 ng/ml). 3 H- thymidine incorporation was performed for 24 hr starting 24 hr after plating. Values are means 6 SEM of triplicate cultures. Fig. 4. Effect of native GnRH Cells were cultured with increasing concentrations of GnRHa (Leuprolide) and native GnRH, in presence of a combination of TGF-b1 (3 ng/ml) and FSH (20 ng/ml). 3 H-thymidine incorporation was performed for 24 hr starting 24 hr after plating. Values are means 6 SEM of triplicate cultures. Fig. 3. Effect of Buserelin on the interaction among FSH, estradiol, and TGF-b. Cells were cultured with increasing concentrations of Buserelin, in presence of a combination of TGF-b1 (3 ng/ml), FSH (20 ng/ml), and estradiol (100 ng/ml). 3 H-thymidine incorporation was performed for 24 hrs starting 24 hr after plating. Values are means 6 SEM of triplicate cultures. were dose dependent. The effect of M of Leuprolide was blocked by M of Antide. DISCUSSION GnRH and its analogues are known to bind to specific cell surface receptors in granulosa cells and suppress Fig. 5. Effect of a GnRH antagonist. Cells were cultured with increasing concentrations of a antagonist of GnRH (Antide) in presence of M GnRHa (Leuprolide), TGF-b1 (3 ng/ml), and FSH (20 ng/ml). 3 H-thymidine incorporation was performed for 24 hr starting 24 hr after plating. Values are means 6 SEM of triplicate cultures.

4 EFFECT OF GnRH ON RAT GRANULOSA CELL 173 gonadotropin-induced changes associated with follicular development, such as increases in aromatase activity, LH receptors, and biosynthesis of progestagens (Hsueh and Erickson, 1979; Hsueh et al., 1980; Jones et al., 1980; Hsueh and Jones, 1981). The concentrations of GnRH agonist required to inhibit DNA synthesis in the present study were similar to those required to inhibit FSH-stimulated camp and progesterone secretion (Ranta et al., 1982) and would be consistent with the affinity of the GnRH receptors in rat granulosa cells (Kd M). These findings, together with the reversion of the inhibition of DNA synthesis with the specific antagonist, strongly suggest that the inhibitory effects of GnRHa on DNA synthesis are mediated by homologous receptors. In recent years, GnRH analogs have proved to be a potential therapeutic tool for the control of proliferation of breast, ovary, and prostate-derived tumor cells. The antiproliferative effects of GnRH analogs seem to be due not only to the suppression of gonadal steroids through the inhibition of gonadotropin action, but also to direct effects on tumor cell growth (Emons and Schally, 1994). There is convincing in vitro evidence (Sharoni et al., 1989; Emons and Schally, 1994) that indicates that GnRH analogues can directly inhibit the proliferation of breast cancer and epithelial ovarian cells. Therefore, GnRH agonists might have direct antiproliferative effects on granulosa cells. However, the seemingly minor effect of GnRH on TGF-b-stimulated thymidine incorporation suggests that the effect of GnRH in FSH/TGF-b stimulated granulosa cells would be mainly due to an antigonadotrophic effect. The agonists used in this study were 4 5 orders more potent than native GnRH. The increased potency was consistent with the known effect of the replacement of peptidase sensitive amino acids in the molecule (Sharpe, 1982). Regarding the possible mechanism involved in the inhibitory effect of GnRH, the suppression of FSHstimulated steroidogenesis has been shown to be tightly coupled to an increase in phophoinositide metabolism (Davis and Clark, 1983), suggesting that protein kinase C (PKC) could be mediating the antigonadotrophic action (Imai et al., 1993). Further studies will be required to determine whether activation of PKC pathway is also involved in the inhibition of gonadotropinstimulated DNA synthesis. It is to be noted that the inhibitory effects of GnRH on DNA synthesis were not associated with a decreased cell viability. Furthermore, preliminary data indicate a marked increase in the overall protein secretion in GnRH treated cultures. This effect would be consistent with the previous demonstration of a stimulatory effect of GnRH on the production of specific proteins such as fibronectin (Dorrington and Skinner, 1986), plaminogen activator (Wang, 1983), and insulin-like growth factor binding protein-4 (Erickson et al., 1994), and would suggest that the inhibition of cell DNA synthesis is associated with the maintenance of a secretory phenotype, characteristic of immature granulosa cells (Lerner et al., 1995). However, recent reports have conclusively demonstrated that in vivo treatment with GnRH of immature rats directly induces apoptotic cell death in granulosa cells (Billig et al., 1994). The possible causal relationship between the inhibition of DNA synthesis demonstrated here and the induction of apoptotic cell death cannot be clearly established since, to our knowledge, the induction of this later phenomenon by GnRH has not been demonstrated in granulosa cell cultures. It is noteworthy that Erickson et al. (1994) have shown that GnRH treatment of hypohysectomized estrogen-primed rat caused a rapid and dramatic decrease in the mitotic index of granulosa cells of atretic as well as of healthy antral follicles, followed by a stimulation of granulosa cell piknosis. This would suggest that the inhibition of DNA synthesis is an early event that precedes the onset of apoptosis and would be consistent with the hypothesis that apoptosis and mitosis may be mechanistically related or even coupled (Steller, 1995). It is also possible that GnRHa triggers apoptosis by means of an independent mechanism. This would explain the massive induction of DNA fragmentation in granulosa cells after GnRHa treatment (Billig et al., 1994), even though only a small population is engaged in DNA synthesis (Erickson et al., 1994). There is evidence for a physiological role of a GnRHlike material in the ovarian function (Birnbaumer et al., 1985). Moreover, the demonstration of primary GnRH gene transcripts in rat ovaries (Goubau et al., 1992) has led to the hypothesis that either this or a related peptide produced from the same precursor mrna may play a role as an intraovarian regulator. However, the lack of effect observed with the GnRH antagonist when added alone suggests that, in our culture conditions, the endogenous GnRH may not be produced in significant concentrations by granulosa cells. Data presented herein indicate that in addition to its previously demonstrated inhibitory effect on cell differentiation, GnRH also may exert a marked inhibition on hormonally stimulated granulosa cell proliferation. ACKNOWLEDGMENTS The authors thank Ms. Silvia Lugo for her skillfull technical assistance, and the National Hormone and Pituitary Distribution Program, NIH, for providing FSH and GnRH. This work was supported by grants from the Rockefeller Foundation and the Buenos Aires University (to J.L.B.). 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