Expression of mRNA for follistatin and inhibin/activin subunits during follicular growth and atresia

Abstract

The aim of the present study was to investigate the sites and time of follistatin and inhibin alpha and beta 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 alpha or inhibin beta 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 alpha riboprobe hybridized to the granulosa cells of most preantral and all non-atretic antral follicles. In the preantral follicles, the strongest inhibin alpha expression was observed in the cells that were in close proximity to the oocyte. The inhibin beta 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 beta 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 beta A mRNA was observed only in the cells of the cumulus, whereas inhibin alpha and follistatin mRNAs were still present in the granulosa cells. As atresia progressed mRNA for inhibin alpha, and later also follistatin, disappeared. Our results suggest that there is a sequential appearance and disappearance of follistatin, inhibin alpha and inhibin beta A mRNAs during follicular development and atresia respectively. The marked expression of inhibin beta A in the cumulus cells implies a role of the oocyte in the differentiation of these cells.

Ovarian Expression of Inhibin-Subunits, 3.BETA.-Hydroxysteroid Dehydrogenase, and Cytochrome P450 Aromatase during the Estrous Cycle and Pregnancy of Shiba Goats (Capra hircus)

“…Immunostaining intensity was weak to moderate in pre-antral stages and strongest at the antral stage, confirming that the mature large follicles are the major source of inhibin in goats and that the pre-antral follicles contribute minimally to inhibin production. Further confirmation of this is provided by the presence of mRNA encoding inpresence of mRNA encoding inpresence of mRNA encoding inhibin α and β A in the granulosa cells of most pre-antral and all non-atretic antral follicles, and its absence in primordial follicles or primary follicles with less than two layers of granulosa cells [1]. Furthermore, the amounts of mRNAs for inhibin α and β A subunits increased coincident with follicle size, whereas inhibin β A mRNAs remained unchanged [10].…”

Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

“…In follicles where such a section could not be found, diameters were measured in the largest estimated cross-section. Antral follicles were examined for the presence of pycnotic and/or apoptotic cells and classified as non-atretic, early atretic (stage I), advanced (stage II) and late atretic (stage III) according to Braw-Tal (1994). Briefly, follicles designated as non-atretic were those with occasional mitotic figs and none or up to five pycnotic/apoptotic nuclei among the granulosa cells.…”

Regulation of ovarian function by the TGF-beta superfamily and follistatin

“…Within the ovary, the granulosa cells in antral follicles and luteinized granulosa cells are the main sites that express follistatin mRNA and protein, whereas other structures, such as the theca cells, stroma and oocytes, appear to be devoid of follistatin mRNA (Shimasaki et al, 1989;Roberts et al, 1993;Braw-Tal, 1994;Tisdall et al, 1994), although follistatin mRNA has been observed in human oocytes (Sidis et al, 1998). However, follistatin protein was detected in the theca cell layers of human small antral follicles in the absence of follistatin mRNA expression, but then became undetectable with the development of a dominant follicle (Roberts et al, 1993).…”

“…Moreover, the follistatin protein appears to be present in only the selected dominant follicles (Nakatani et al, 1991). At the same time, primordial through to primary follicles do not express follistatin mRNA (Kogawa et al, 1991;Braw-Tal, 1994;Tisdall et al, 1994), implying that follistatin may not be involved in the initial stage of recruitment of follicles. Species differences occur, as the expression of follistatin is maintained in the corpus luteum in primates, whereas in other species, expression falls markedly after ovulation in the developing corpus luteum (Kogawa et al, 1991;Roberts et al, 1993;Wada et al, 1996).…”