357 CALCIUM RELEASE INDUCED BY THIMEROSAL AND INOSITOL 1,4,5-TRIPHOSPHATE IN HEAT-SHOCKED PORCINE OOCYTES

Abstract

Elevated ambient temperature has been known to be deleterious to the developmental competence of mammalian oocytes and embryos, although the mechanism is still unclear. The objective of this study was to determine the effect of heat shock (HS) on the alteration of intracellular calcium concentrations ([Ca²⁺]_i) of matured pig oocytes by two different calcium releasing agents. Porcine cumulus–oocyte complexes were aspirated from the follicles (3–6 mm) and subjected to standard in vitro maturation procedure for 42 h. Matured oocytes were then randomly allocated to different heat treatments at 41.5°C for 0 (Control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group was cultured for 4 h without heat shock (C4h). Oocytes were incubated with 2 µM fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 in Ca²⁺-free PBS (40 min) following heat shock, and then washed with Ca²⁺-free PBS (30 min) for detection of [Ca²⁺]_i. Fluorescent images were captured with alternative excitation wavelengths at 340/380 nm by a rotating chopper disk equipped with an Axon imaging system. Data from both experiments were analyzed by ANOVA using the General Linear Model (GLM) of the SAS (SAS Institute, Inc., Cary, NC, USA). In Experiment 1, matured oocytes were activated by 200 mM thimerosal (10 min) following heat treatment. The maximal [Ca²⁺]_i in the HS2h group was the highest among all treatment groups. The lowest maximal peak of [Ca²⁺]_i was observed in the HS4h group, but it was still higher than that in the C4h group (P < 0.05). The total amount of Ca²⁺ release represented by the total area of the peaks in C4h was lower than in any other groups except HS4h (P < 0.05). In Experiment 2, each matured oocyte was injected with approximately 10 pL of inositol 1,4,5-triphosphate (IP₃, 0.5 mM); the Ca²⁺ transient was recorded as described in the previous experiment. The maximal value of [Ca²⁺]_i in the C4h group was still the lowest among the heat-shocked and C0h groups (P < 0.05). The total Ca²⁺ release in the HS2h group was the highest among all treatment groups, but only significantly higher than the HS1h and C4h groups (P < 0.05). A similar pattern of Ca²⁺ release in HS-oocytes was induced by thimerosal and IP₃ stimulations. These results indicate that Ca²⁺ releasing capacity of matured pig oocytes is enhanced by a shorter duration of heat shock, but declines after prolonged exposure of heat shock and/or in vitro culture. The differential Ca²⁺ releasing capacity of heat-shocked oocytes prior to fertilization revealed physiological changes of pig oocytes after heat shock. This finding provides further insight for the low fertilization and developmental competence that occurs in farm species during hot seasons.