Follicular atresia has been examined previously by various biochemical and histological methods. The aim of this study was to compare, for the first time, detection of granulosa cell apoptosis by biochemical DNA analysis and microscopic examination of fresh granulosa cell morphology with the established method of detecting atresia by histology in equine follicles. DNA extracted from granulosa cells was examined by staining with ethidium bromide and end-labelling with [(32)P]dideoxy-ATP, which labels the free 3'-end of DNA fragments. In 25 of 26 follicles (96%) there was agreement between end-labelling and staining of DNA with ethidium bromide (P < 0.001). Granulosa cell apoptosis was distinguished more easily in the end-labelled samples than by staining with ethidium bromide. Histological atresia and apoptosis as detected by biochemical DNA analysis were significantly correlated (P < 0.02) with 20 of 22 follicles (91%) receiving corresponding classifications with the two methods. No follicles with granulosa cell apoptosis as detected by biochemical DNA analysis were histologically viable, but some of the histologically early atretic follicles did not display DNA laddering. Stereomicroscopic evaluation of morphology of the fresh granulosa cells was significantly correlated (P < 0.001) with the histological findings, with 29 of 33 follicles (88%) receiving corresponding classifications. There was a potential error in determining follicle health by biochemical DNA analysis only, as both histologically early and late atretic follicles in some cases did not show DNA laddering. Thus, if relying solely on biochemical detection of apoptosis, severely atretic follicles could wrongly be classified as healthy follicles.
HG Pedersen, ED Watson and EE Telfer
FH Thomas, R Leask, V Srsen, SC Riley, N Spears and EE Telfer
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 vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (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 control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.