Regeneration of atretic sheep ovarian follicles in vitro

Abstract

Large (4-6 mm diameter) and small (2-3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size. The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles, respectively. This could not be accounted for by cell division which remained low throughout the culture period. Non-atretic follicles showed high mitotic activity during the first 24 h in culture. This was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo. Total steroid secretion (estradiol-17.beta., testosterone plus progesterone) into the culture medium (pmol/mg tissue per 24 h) was the same for atretic and non-atretic follicles of comparable size. There was a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was estrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of estrogen. Addition of FSH [follitropin] to the culture medium did not restore aromatizing capacity to the atretic follicles.