The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 microgram i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p < 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p < 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; <4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where >80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p < 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p < 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p < 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p < 0.01). Follicular inhibin content after 2 h incubation in vitro was lower (p < 0.01) in small follicles but was unaffected by the estrogenicity of large follicles. Large estrogenic follicles incubated in vitro released more (p < 0.01) inhibin than large nonestrogenic follicles, which in turn released more inhibin (p < 0.01) than small follicles. In contrast, inhibin production by granulosa cells, on a per cell basis, was greatly elevated (p < 0.001) from cells isolated from large estrogenic follicles, but not from large nonestrogenic or small follicles. This indicates the existence of a population of large nonestrogenic follicles that releases appreciable quantities of inhibin, but does not necessarily reflect the capacity of those follicles' granulosa cells to synthesize inhibin. The ovarian secretion rate of inhibin, however, was more highly correlated with follicular inhibin content after 2 h incubation in vitro (r = 0.6; p < 0.001) than either follicular (r = 0.5; p < 0.01) or granulosa cell (r = 0.4; p < 0.01) inhibin production in vitro. We conclude that inhibin, like estradiol, is secreted by large estrogenic follicles but that inhibin, unlike estradiol, is also secreted in appreciable quantities by large nonestrogenic follicles and small follicles. This range of follicular sources and the insensitivity of their g