LOW AND HIGH-CONCENTRATIONS OF GONADOTROPINS DIFFERENTIALLY REGULATE HORMONE PRODUCTION BY THECA INTERNA AND GRANULOSA-CELLS FROM BOVINE PREOVULATORY FOLLICLES

Mammalian preovulatory follicles produce primarily estradiol and androgens before the preovulatory gonadotropin surge. In cattle, the LH/FSH surge triggers a rapid decrease in estradiol and androgen production and a dramatic increase in progesterone and oxytocin biosynthesis. It is unclear how changes in gonadotropin concentrations in vivo regulate this follicular/luteal phase shift in hormone production. To address this question, theca interna and granulosa cells were isolated from preovulatory follicles of Holstein heifers similar to 24 h before the expected time of the LH/FSH surge and cultured in defined medium containing insulin (1 mu g/ml), with varying doses of LH or FSH (0, 1, 2, 4, 8, 16, 32, 64, or 128 ng/ml). Media were collected and replaced every 24 h for 3 days, and assayed for androstenedione and progesterone in theca interna cultures, and for estradiol, progesterone and oxytocin in granulosa cell cultures. After the first day of culture (24-72 h), only very low doses of LH (2 or 4 ng/ml) enhanced (p < 0.05) androstenedione secretion by theca interna cultures above control levels, whereas progesterone secretion was increased by a wide range of LH concentrations (p < 0.05), with maximal progesterone secretion at high doses of LH. Likewise, after the first day of culture (24-72 h), estradiol secretion by granulosa cells was stimulated only by low doses of FSH and was inhibited at higher concentrations relative to control cultures (p < 0.05), whereas the production of oxytocin and progesterone was enhanced maximally by high concentrations of FSH (p < 0.05). Although low doses of LH and FSH stimulated androstenedione and estradiol secretion, respectively, even in their presence the secretion of these two steroids declined significantly with time in culture. All doses of LH were inhibitory to estradiol secretion by granulosa cells and stimulatory to progesterone and oxytocin secretion (p < 0.01). To determine whether the responses of granulosa cells to gonadotropins could be replicated under different culture conditions, granulosa cells were cultured with 0, 2, 8, 128, or 256 ng/ml of FSH or LH in medium containing insulin (1 mu g/ml), 1% fetal calf serum (PCS), or insulin (1 mu g/ml) + 1% FCS. After the first 48 h of culture, estradiol secretion in control cultures was greater in cultures containing insulin + 1% FCS than in cultures containing insulin or 1% FCS alone (p < 0.01). In contrast, progesterone and oxytocin secretion in control cultures was nor affected by the culture conditions. The stimulatory effects of low doses of FSH on estradiol production were dramatically accentuated in medium containing 1% FCS alone (p < 0.01), whereas medium with insulin + 1% FCS accentuated the inhibitory effects. In contrast, the stimulatory effects of high doses of gonadotropins on progesterone and oxytocin secretion were observed in all three culture conditions. However, the magnitude of the responses to gonadotropins was influenced by the different culture conditions. The results reveal that low doses of LH and FSH stimulate follicular-phase steroid production in theca and granulosa cells, respectively whereas higher doses accentuate the natural tendency of both cell types to shift from androgen or estradiol secretion typical of the follicular phase to progesterone or progesterone and oxytocin secretion typical of the luteal phase.