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.
