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.
FH Thomas, R Leask, V Srsen, SC Riley, N Spears and EE Telfer
E. Soloy, V. Sršeň, A. Pavlok, P. Hyttel, P. D. Thomsen, S. D. Smith, R. Procházka, M. Kubelka, R. Høier, P. Booth, J. Motlík and T. Greve
The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.