248 INVOLVEMENT OF THE SPHINGOLIPID, CERAMIDE, IN HEAT SHOCK-INDUCED APOPTOSIS IN BOVINE OOCYTE

Abstract

Programmed cell death through the sphingomyelin pathway has been suggested to underlie the mechanism by which heat shock disturbs oocyte developmental competence. Serial experiments were performed to characterize the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation could be regulated. In all experiments, bovine cumulus–oocyte complexes (COC) were aspirated from ovaries collected at the abattoir. Cumulus–oocyte complexes were matured (22 h, 38.5°C, 5% CO₂), in vitro fertilized (18 h, 38.5°C, 5% CO₂), and cultured for 8 days (KSOM; 38.5°C, 5% CO₂, 5% O₂). In the first experiment, COC were matured at 38.5°C (n = 673) or 41°C (heat shock; n = 718). Exposure of COC to heat shock during maturation reduced cleavage (92.3 v. 84.2%; P < 0.05) and blastocyst formation rates (24.1 v. 14.6%; P < 0.05) relative to the control groups. In the second experiment, COC (n = 563) were exposed to either long-term (22 h) or short-term (0.5 to 3.5 h) heat shock during maturation. An Annexin V-FITC assay (Bender) was used to identify the turnover of phosphatidylserine, one of the features of early-stage apoptosis. In addition, a subgroup of matured oocytes (n = 384) were denuded and fixed in 2% paraformaldehyde, followed by immunofluorescent staining using anti-ceramide (Alexis) to detect ceramide levels and counterstaining with Hoechst (Sigma). Immunofluorescent staining showed no difference in endogenous ceramide levels between groups. Similarly, annexin expression did not differ between groups at the end of the maturation but was higher (P < 0.05) in oocytes exposed to short-term heat shock than in those subjected to long-term heat shock, suggesting that early features of apoptosis should be examined at a specific time of heat exposure. Another experiment was performed to examine the effect of ceramide on oocyte developmental competence. Cumulus–oocyte complexes (n = 1185) were matured with or without 10, 30, and 50 μm C2-ceramide (Sigma). An Annexin V-FITC assay was used with a subgroup of oocytes (n = 137) that were matured with or without 50 μm C2-ceramide for 2 h. To examine the major pathway of ceramide generation, heat-shocked COC were matured with or without 5 μm fumonisin B1 (FB1; Sigma), a specific inhibitor of dihydroceramide synthase (i.e. de novo formation) and ceramide synthase (i.e. salvage pathway), or with 5 μm desipramine hydrochloride (DH; Sigma), a specific inhibitor of the acid sphingomyelinase (i.e. hydrolysis). Results revealed that maturation in 50 μm C2-ceramide increased (P < 0.05) annexin expression in association with reducing cleavage rate and blastocyst formation (P < 0.05). Although not significant, maturation with FB1 moderated the heat-shock effect on oocyte developmental competence. Similarly, 5 μm DH increased the blastocyst formation rate for heat-shocked oocytes from a level of 17% to the control level (22.5%). In summary, ceramide appears to play an important role in heat-shock-induced apoptosis because blocking ceramide formation alleviated its deleterious effect.

Research was supported in part by USDA Grant 2007-35203 and BARD Grant 3986-07