Vol.12, No. 1 53 Leptin Influences Ovarian Steroidogenesis/Spicer et al. Endocrine, vol. 12, no. 1, 53–59, February 2000 0969–711X/00/12/1:53–59/$11.75 © 2000 by Humana Press Inc. All rights of any nature whatsoever reserved. 53 To test the hypothesis that leptin signals metabolic information to the reproductive system in cattle by directly affecting IGF-I-induced ovarian cell function, granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in serum-free medium with added hormones. Recombinant human leptin at 30 and 300 ng/mL had no effect on basal thecal cell steroido- genesis or thecal cell numbers. However, 300 but not 30 ng/mL of leptin attenuated (p < 0.05) luteinizing hormone–induced androstenedione production by 24% in the absence of IGF-I and by 16% in the presence of IGF-I. Leptin had no effect on IGF-I-induced estradiol production in the presence of follicle-stimulating hormone (FSH), but at 100 ng/mL, leptin inhibited ( p < 0.05) FSH plus IGF-I-induced progesterone pro- duction and granulosa cell proliferation by 29 and 31%, respectively. Leptin did not compete for 125 I-IGF-I bind- ing to granulosa or thecal cells, whereas unlabeled IGF-I did. In conclusion, leptin has weak inhibitory effects on gonadotropin- and/or IGF-I-induced steroidogenesis of thecal and granulosa cells. Key Words: Leptin; granulosa cells; thecal cells; steroidogenesis; cattle. genetically obese (ob/ob) mice that lack endogenous leptin are injected with leptin, they experience decreased food intake, a loss in body weight, increased ovarian weight and number of follicles, and correction of a sterility defect (5–8,14,15). In mature cows, reproductive performance decreases as body fat content decreases (16). The latter observations indicate that leptin may have positive influences on the reproductive system in mice and cattle. However, recent in vitro evidence indicates that leptin may also have negative effects on ovarian cell function (17–19). Insulin-like growth factor-I (IGF-I) has long been known to have direct effects on ovarian cell function in several species including humans, rats, and cattle (20,21) . A decrease in systemic IGF-I concentrations is associated with the cessation of estrous cycles as cows are underfed to become nutritionally anestrus (22). Conversely, increases in systemic IGF-I concentrations are associated with the resumption of estrous cycles as nutritionally anestrous cows and prepubertal heifers are refed to gain weight (22,23). Thus, IGF-I may be one hormone, in addition to leptin, that communicates the metabolic status of an animal to the reproductive system in cattle. We hypothesized that leptin may directly influence IGF-I-stimulated ovarian follicular function in cattle and conducted experiments to determine the effect of leptin on IGF-I-induced proliferation and steroidogenesis of bovine granulosa and thecal cells in vitro. Results Experiment 1 Experiment 1 was conducted to evaluate the dose-response effect of leptin on basal and luteinizing hormone (LH)– induced thecal cell proliferation and/or steroidogenesis. The- cal cells from large ( 8 mm) follicles were cultured for 2 d in 10% fetal calf serum (FCS), and then cultured in serum-free medium for an additional 2 d with or without 100 ng/mL of LH in the presence of 0, 30, or 300 ng/mL of leptin. The doses of LH and leptin were selected based on previous studies (17,18,24,25). In the absence of leptin, LH increased (p < 0.05) thecal cell progesterone (Fig. 1A) and androstenedione (Fig. 1B) production by 2.5- and 3.5-fold, respectively. By contrast, leptin had no effect (p > 0.10) on basal or LH-induced progesterone (Fig. 1A) or on basal androstenedione (Fig. 1B) Ovarian Action of Leptin Effects on Insulin-Like Growth Factor-I–Stimulated Function of Granulosa and Thecal Cells Leon J. Spicer, Connie S. Chamberlain, and Cristina C. Francisco Department of Animal Science, Oklahoma State University, Stillwater, OK Received September 24, 1999; Revised November 27, 1999; Accepted December 2, 1999. Author to whom all correspondence and reprint requests should be addressed: Dr. Leon J. Spicer, Department of Animal Science, Oklahoma State University, Stillwater, OK 74078. E-mail: igf1Leo@okstate.edu. Introduction Leptin, the recently identified adipose obese (ob) gene product, is a plasma protein hormone that parallels the amount of fat reserves in rodents and humans (1–4) and is thought to regulate satiety (5–8). Whether systemic leptin concentrations correlate with body fat in cattle remains to be determined. The initial 167 amino acid sequence of leptin is highly conserved (i.e., 83–97% homology) as is the 145 amino acid sequence (the secreted form) of leptin (84–97% homology) across the four species (mouse, rat, human, monkey) in which it has been characterized (9–13). If