Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro

Abstract

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.