Different hormonal requirements of pig oocyte-cumulus complexes during maturation in vitro

Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml^-1), hCG (10 iu ml^-1) and oestradiol (1 μg ml^-2) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 ± 72.6 ng ml^-1) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 ± 13.3 ng ml^-1) during the first 20 h of culture (42.8 ± 19.4 ng ml^-1) and also during the second 20 h period of culture without hormonal supplements (0.925 ± 0.544 ng ml^-1). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 ± 0.2 ng ml^-1 at 0 h to 6.9 ± 1.5 ng ml^-1 after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.