FOLLICULAR HETEROGENEITY AND OOCYTE MATURATION INVITRO IN PIGS

Experiments were conducted to confirm that co-culture of follicular shells and immature pig oocytes improved the rate of male pronuclear (MPN) formation and to test the hypothesis that the maturational state of the follicle used in co-culture would significantly affect dhe quality of oocytes matured in vitro. In a preliminary experiment, co-culture of oocyte complexes with follicular shells did not affect nuclear maturation, slightly inhibited penetrability (p = 0.04) but greatly enhanced (p = 0.0004) MPN rate. In the main experiment, oocyte complexes (10-15 per dish), obtained from follicles 36 h after eCG injection of prepubertal gilts, were co-cultured with single 36-h small (3.5-5.0 mm), 36-h large (6-9 mm), 72-h small (4-7 mm), or 72-h large (7.5-11.0 mm) follicular shells prior to insemination. MPN formation in penetrated oocytes was significantly affected by follicular size (small: 60.14% vs. large: 72.08%, p = 0.014) but not by time of recovery (36 h: 72.32% vs. 72 h: 59.90%, p = 0.39). Overall, MPN formation rate was significantly correlated with follicular diameter (r = 0.45, p = 0.005), follicular fluid progesterone (r = 0.37, p = 0.02), and estradiol (r = 0.40, p = 0.01), and to the ratios of follicular fluid progesterone:testosterone (r = 0.39, p = 0.018) and follicular fluid estradiol:testosterone (r = 0.43, p = 0.007). There were no simple correlations between rate of MPN formation and steroid concentrations and their ratios in culture media collected at the completion of culture. However, multiple stepwise regression showed that, in addition to follicular diameter, MPN rate may be affected by the ratio of progesterone: testosterone (positively correlated) and the progesterone content (negatively correlated) of the culture medium. It is concluded that the maturational status of the follicle has a critical influence on oocyte cytoplasmic maturation and that this effect may mainly be mediated by nonsteroidal factors.