121 HEAT SHOCK TO PIG OOCYTES DOES NOT INDUCE APOPTOSIS BUT REDUCES EMBRYO DEVELOPMENT

Abstract

Oocytes are susceptible to heat shock (HS) during the maturation process. It has been demonstrated that HS induces apoptosis and/or the expression of HS protein 70 (hsp 70) in in vitro-produced oocytes and embryos. The objectives of this study were to analyze the effects of HS on the development and apoptosis of pig oocytes and embryos. Porcine ovaries were collected from a local slaughterhouse and the cumulus-oocyte complexes (COCs) were aspirated from follicles 3–6 mm in diameter and subjected to standard in vitro maturation procedures at 39°C for 42 h. The in vitro matured oocytes were then randomly allocated to different HS treatments at 41.5°C for 0 (control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group of oocytes was cultured for 4 h without HS (C4h). Data were analyzed by chi-square test. In Experiment 1, anti-hsp 70 (SPA-810AP, Stressgen, San Diego, CA, USA) and Western blotting were used to examine the expression of hsp 70. Results indicated that no significant difference of hsp 70 expression in metaphase II porcine oocytes occurred between controls and HS groups (P > 0.05, 7 replicates). In Experiment 2, apoptosis of metaphase II oocytes after HS was identified by annexin V-FITC (Sigma, St. Louis, MO, USA) staining and TUNEL (Roche, Indianapolis, IN, USA). No significant apoptotic signal was detected in the HS groups compared to the controls. The intensity of annexin V staining was not affected by HS, but it increased with the time of culture (P < 0.05, n = 24–37). In Experiment 3, the apoptotic rate and developmental competence of the HS-oocytes were evaluated by TUNEL assay (n = 123–137, 4 replicates). Parthenogenetic activation (n = 123–137) was performed by an electric pulse (2.2 kV cm⁻¹) combined with 6-dimethyaminopurine treatment (6-DMAP, 2.5 μM, 4 h, Sigma). The cleavage rates in HS2h (43 ± 29%) and HS4h (35 ± 28%) decreased (P < 0.05) compared to those in C0h (62 ± 12%) and C4h (66 ± 8%). In addition, the blastocyst formation rates and total cell numbers reduced (P < 0.05) after 2 h (11 ± 10%, 20 ± 16) and 4 h (11 ± 8%, 19 ± 8) of HS treatments compared to those in C0h (23 ± 14%, 32 ± 22) and C4h (21 ± 11%, 27 ± 17), all respectively. The numbers of blastocysts with TUNEL-positive signals were not significantly different between the HS and control groups, but the signals increased (P < 0.05) before the 8-cell stage in HS groups (22–24%) compared to the C0h and C4h controls (16 and 11%), respectively. These results indicate that reduction in developmental competence of in vitro-matured pig oocytes after heat shock is not closely correlated to the expression of hsp 70 in the oocytes and to the apoptotic cell numbers in the blastocyst. Whether detection of apoptosis by TUNEL or annexin V-FITC in oocytes is a good indicator requires further investigation.