Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

The contribution of specific follicle populations to dimeric inhibin produc tion and inhibin subunit mRNA expression by the rat ovary has been investig ated in two model systems, granulosa. tells isolated from 25-day-old diethy l-stilboestrol (DES)-treated rats and pr,st-natal rat, ovaries, dispersed i n culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR, Media from FSH-stimulated granulosa cell cultures fractionated b y gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of a subunit activity which were attributed to alpha sub unit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were lo w. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produ ced by granulosa cells. All three inhibin subunit mRNAs were expressed by g ranulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the i solation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated. cul tures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin p roduction, Consistent with these results, follicles within these ovaries ex pressed all three inhibin subunit mRNAs, with maximal expression observed i n the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline ill the mRNA levels of each of the subunit s but was most evident for the beta subunits. There was a profound influenc e of secondary preantral follicles on dimeric inhibin-A production, with FS H stimulation increasing inhibin-A relative to alpha subunit levels in cult ures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicl es exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce in hibin-B in response to FSH stimulation. Transforming growth factor-beta (TG F-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not r esponsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit, Preant ral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was d own-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are i ndividually regulated are likely to br similar during the post-natal period , when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.