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ABSTRACT

The aim of the present study was to investigate the sites and time of follistatin and inhibin α and β_A 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 β_A 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 β_A 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 β_A 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 β_A 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 β_A mRNAs during follicular development and atresia respectively. The marked expression of inhibin β_A in the cumulus cells implies a role of the oocyte in the differentiation of these cells.

ABSTRACT

The aim of this study was to investigate the sites of follistatin and α and β_A inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term=day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the α and β_A inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to α and β_A inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined. No FecB-specific differences in follistatin expression were noted with respect to stage of preantral follicular development and there were no obvious differences in the intensity of expression.

These results show that follistatin mRNA is expressed specifically in the granulosa cells and intraovarian rete. Expression of follistatin in rete cells was coincident with the increasing numbers of growing follicles within the fetal ovary, indicating that rete cell function may have a role in the ontogeny of early follicular growth. Our results suggest that follistatin and α and β_A inhibin may not be important for the initiation of follicle growth in the sheep ovary, since these genes are not expressed during the transformation of a primordial follicle to a primary structure. However, the evidence for follistatin mRNA expression in the ovine fetal ovary implies that this hormone is likely to play a role during the early stages of follicle growth.