Regulation of Steroid Production in Bovine Fetal Ovaries.

Abstract

The formation of the primordial follicle pool, a process essential to female reproductive capacity, begins around day 90 of bovine gestation (gestational length= 281 days). Previous results indicate that bovine fetal ovaries produce steroids and that ovarian estradiol (E2) and progesterone (P4) decline around the time of follicle formation. Furthermore, we showed that in vitro E2 and P4 can inhibit follicle formation and prevent primordial follicles from acquiring the capacity to activate (initiate growth). Although it is known that the fetal ovary is capable of producing steroids and that E2 and P4 can affect the formation and development of follicles, little is known about the mechanisms that regulate fetal steroid production in vivo. To begin to investigate the regulation of fetal steroid production, pairs of fetal ovaries (n=4, 81-97 days) were collected at an abattoir and cut into pieces (1 mm3). Ovarian pieces were cultured in 24-well culture plates (2 pieces/well) and treatments were applied to duplicate wells within each experiment (fetus). Medium was collected and replaced every 2 days for 10 days and measured for E2 and P4 by radioimmunoassay. To test the hypothesis that gonadotropins can stimulate steroid production, ovarian pieces were cultured in the presence or absence of LH (luteinizing hormone) and/or FSH (follicle-stimulating hormone) at 100 ng/ml. Average cumulative levels of E2 and P4 in control cultures over 10 days of culture were 0.40 and 0.72 ng/well, respectively. LH, FSH, and LH+FSH stimulated E2 production, whereas only LH+FSH stimulated P4 accumulation above control levels (P < 0.05). The combination of LH+FSH enhanced E2 levels more than FSH alone (P < 0.05). To test the hypothesis that availability of androgen substrate limits fetal ovarian E2 production, ovarian pieces were cultured with testosterone (T, 0.5 μM) in the presence or absence of LH and/or FSH (n=4 fetus). Estradiol secretion was stimulated by T (18-fold), LH+T (24-fold) and FSH+T (35-fold), compared with control medium and FSH+T increased E2 values more than T alone (P < 0.05). The high levels of E2 observed in the presence of exogenous T support our hypothesis that androgen is limiting to E2 synthesis in vitro. Unexpectedly, T decreased P4 production by 98% compared to control medium (P < 0.05). Testosterone was also inhibitory to P4 production in the presence of gonadotropins (P < 0.05). To determine if T inhibits P4 directly or indirectly through aromatization to E2, ovarian pieces (n=3 fetuses) were cultured in control medium or with T or E2 (0.5 μM). The average cumulative level of P4 in control medium was 1.18 ng/well. Treatment with T or E2 decreased P4 accumulation by 92 and 81%, respectively, relative to control medium (P < 0.05), suggesting that the effects of T on progesterone production are exerted, at least in part, by its aromatization to E2. Further studies are needed to elucidate the mechanism(s) of steroidal inhibition of P4 production and the physiological role of this inhibition. Together these results show that fetal bovine ovaries are responsive to both LH and FSH, that ovarian production of androgens may be limiting to E2 synthesis and that steroid hormones may play a role in regulating fetal steroid production. (This project was supported by National Research Initiative Competitive Grant no. 2008-35203-05989 from the USDA National Institute of Food and Agriculture).