In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes

Abstract

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 °C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture ( P < 0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (±S.E.M.) embryonic development in HS2h (cleavage: 42 ± 29%; blastocyst: 11 ± 10%) and HS4h (cleavage: 36 ± 28%; blastocyst: 11 ± 8%) were decreased when compared to those in C0h (cleavage: 63 ± 12%; blastocyst: 24 ± 14%) and C4h (cleavage: 66 ± 8%; blastocyst: 21 ± 11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups ( P < 0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.