The aim of the present study was to investigate the sites and time of follistatin and inhibin α and βA subunit gene expression during ovine follicular development and atresia. Prepubertal ovaries of 2-, 8- and 14-week-old ewe lambs (n=9) were used. Regardless of age, the ovaries contained many follicles at different stages of development up to 2 mm in diameter, but large antral follicles were not found.
Ovarian sections were hybridized with 35S-labelled antisense RNA probes transcribed from follistatin, inhibin α or inhibin βA cDNA. Ovaries from mature gonadotrophin-stimulated ewes were used as controls. All three probes hybridized exclusively to granulosa cells, and not to other follicular or stromal cells. None of the probes hybridized to primordial follicles or primary follicles with less than two layers of granulosa cells. Follistatin mRNA was expressed strongly in the granulosa cells of all preantral follicles with two or more layers of cells, and in all non-atretic antral follicles. In addition, follistatin mRNA was found in some cells of the ovarian rete tubules. The inhibin α riboprobe hybridized to the granulosa cells of most preantral and all non-atretic antral follicles. In the preantral follicles, the strongest inhibin α expression was observed in the cells that were in close proximity to the oocyte. The inhibin βA riboprobe hybridized exclusively to the granulosa cells of antral follicles. The labelling was observed either in the cumulus oophorus or in the cumulus oophorus and periantral granulosa cells of the non-atretic antral follicles. In adult ovaries, which were used as controls, the inhibin βA riboprobe hybridized strongly to all granulosa cells of non-atretic large antral follicles. During follicular atresia, expression of all three mRNAs progressively decreased. In early atresia, inhibin βA mRNA was observed only in the cells of the cumulus, whereas inhibin α and follistatin mRNAs were still present in the granulosa cells. As atresia progressed mRNA for inhibin α, and later also follistatin, disappeared.
Our results suggest that there is a sequential appearance and disappearance of follistatin, inhibin α and inhibin βA mRNAs during follicular development and atresia respectively. The marked expression of inhibin βA in the cumulus cells implies a role of the oocyte in the differentiation of these cells.