Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in v itro. Preantral follicles were cultured for 12 days in serum-free medium co ntaining ascorbic acid (50 mug ml(-1)). Half of the medium was replaced eve ry 2 days, and conditioned medium was analysed for oestradiol and matrix me talloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was as sessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absen ce of serum, there was significant (P < 0.05) follicle growth and oestradio l secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle grow th (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in con trol follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. I n conclusion, this culture system was capable of supporting follicle differ entiation over the 12 day culture period. Furthermore, ascorbic acid mainta ins bovine follicle health and basement membrane remodelling in vitro.