The role of inhibins and activins in follicle development in the sheep

The growth and development of ovarian follicles is controlled primarily by FSH and LH secreted by the anterior pituitary. In the ewe, the secretion of FSH and LH by the pituitary is regulated by a number of ovarian factors, including oestradiol, progesterone, and the glycoprotein dimers inhibin and activin. Two forms of inhibin have been identified to date, inhibin A and inhibin B, with both consisting of a common a-subunit and differing p-subunits. Although well researched in a number of species, the levels of inhibins in the circulation and the expression of their subunits in the gonads have yet to be examined as thoroughly in the sheep, a species in which the ovulatory quota is rigorously controlled.

In order to identify the source of inhibin A in the ewe, ovaries were removed at each of three points during the oestrous cycle (mid-luteal, early follicular and late follicular), all visible antral follicles dissected out and hormone release measured during incubation and in their follicular fluids. Large oestrogenic follicles were found to be the main source of inhibin A in the ewe with large non-oestrogenic follicles also releasing significant amounts of the hormone. The levels of inhibin A released by both small antral follicles and large oestrogenic follicles during the early follicular phase were significantly reduced compared to the luteal phase, possibly reflecting the fall in FSH concentration at this time.

Conventional immunocytochemistry and dual label confocal microscopy were used to investigate the expression of inhibin a, PA and PB subunit proteins in different compartments of the ovarian follicle. All three subunits were found in the granulosa cells of antral follicles with little variation in the relative amounts of the proteins in follicles during the oestrous cycle. In addition, there was significant immunostaining for both P-subunits (but not a subunit) in theca cells, with mRNA expression of all three subunits confined to the granulosa cells. These findings are compatible with the hypothesis that pA and PB activins diffuse from the granulosa layer through the theca and possibly the adjacent stroma, where they may exert a paracrine influence.

Significant amounts of inhibin B have not been detected in the blood or follicular fluid of sheep using a specific two-site ELISA assay. The nature of inhibin/activin proteins present in ovarian cells and follicular fluid was therefore investigated using Western blotting. A small amount of high molecular weight protein (consistent with inhibin B precursor) was detected in protein extracts of ovarian cells. Conversely, there were no inhibin B proteins present in follicular fluid, which contained large quantities of inhibin A forms. Therefore, although all three inhibin subunit mRNAs and proteins are expressed in the granulosa cells of antral follicles, it seems unlikely that inhibin B is secreted outside the cell.

In order to explore further the forms of inhibin produced by the sheep, subunit expression in the male was investigated. In most species studied (e.g. human, rat), the male secretes exclusively inhibin B. In this work, both α and ß subunit proteins were found in the seminiferous tubules of rat testes. In contrast, all three subunit proteins were present in the testis of the ram, which like the ewe, apparently only secretes inhibin A. Together, these findings are consistent with the hypothesis that ßA subunit is preferentially bound to a subunit when both P subunits are expressed in the species studied, and that inhibin B is only secreted in the absence of ßA protein.

To further examine the hypothesis that inhibin and activin may influence ovarian function through paracrine and autocrine actions, differences in the expression of inhibin subunits in ewes carrying the fecundity gene, which leads to precocious follicular maturation, were examined. It was noted that expression of both a and PA subunits was significantly lower in medium sized antral follicles in carriers of the mutation. Furthermore, the level of diffusion of PB subunit from medium and large antral follicles was significantly higher in animals carrying the fecundity gene. The level of PB-subunit associated proteins diffusing from the granulosa cells is increased in antral follicles, possibly reflecting an increase in activin diffusion which may affect the growth of smaller follicles.

In conclusion, this work has 1) identified the sources of inhibin A in the ovine ovary; 2) provided evidence for the possible diffusion of activins throughout ovarian tissue; 3) supported the hypothesis that the ewe does not produce inhibin B; 4) reported a possible increase in activin diffusion from specific follicle populations in ewes with increased ovulation rates; and finally 5) shown that in some species there may be preferential binding of certain inhibin subunits during dimerisation.