Inhibitory Effect of Gonadotrophin-releasing Hormone (GnRH) on Rat Granulosa Cell Deoxyribonucleic Acid Synthesis PATRICIA E. SARAGU ¨ ETA, GUILLERMO M. LANUZA, AND J. LINO BARAN ˜ AO* Instituto de Biologı´a 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 5 3 10 211 M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED 50 5 1.58 6 0.22 3 10 210 M) and native GnRH (ED 50 5 1.4 6 0.3 3 10 26 M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10 28 M could prevent the inhibition elicited by 10 27 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: 170–174,1997. r 1997 Wiley-Liss, Inc. Key Words: gonadotropin releasing hormone; granu- losa 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 follicu- lar 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 gonadotro- pin-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 essen- tially indistinguishable from those present in the pitu- itary (Reeves et al., 1980) and to involve activation of the phosphoinositide-derived pathways (Leung and Wang, 1989). 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 fibro- nectin secretion (Dorrington and Skinner, 1986). The demonstration of GnRH-like substances (Birn- baumer 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 exis- tence 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 24–25-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 Baran ˜ ao, Instituto de Medicina y Biologı ´a Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. Received 22 October 1996; Accepted 21 November 1996 MOLECULAR REPRODUCTION AND DEVELOPMENT 47:170–174 (1997) r 1997 WILEY-LISS, INC.