DIFFERENTIAL RESPONSES OF BOVINE OOCYTES AND PREIMPLANTATION EMBRYOS TO HEAT-SHOCK

The authors sought to determine whether developmental differences in t he magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. in this regard, responses of oocytes, two-cell embryos, four- to eight-c ell embryos, and compacted morulae to heat shock were compared. An add itional goal was to define further the role of cumulus cells and gluta thione in thermoprotection of oocytes, In experiment 1, heat shock (41 degrees C for 12 hr) decreased the number of embryos developing to th e blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell ( 25% vs. 10%) embryos but did not affect morulae (37% vs. 42%), In expe riment 2, exposure of two-cell embryos to 41 degrees C for 12 hr reduc ed the number of four- to eight-cell embryos present 24 hr after the e nd of heat shock (88% vs. 62%). In experiment 3, heat shock reduced th e number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occu rred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocy tes were cultured with an inhibitor of glutathione synthesis, DL-buthi onine-[S, R]-sulfoximine (BSO), for 24 hr and exposed to 41 degrees C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39 degrees C), 18% (41 degrees C), 17% (39 degrees C+BSO), and 11% ( 41 degrees C+BSO). Far experiment 5, oocytes were either denuded or le ft with cumulus intact and were then radiolabeled with [S-35]methionin e and [S-35]cysteine at 39 degrees C or 41 degrees C for 12 hr. Exposu re of oocytes to 41 degrees C for 12 hr reduced overall synthesis of S -35-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 v s. 23,246). Analysis by two-dimensional SDS PAGE and fluorography reve aled that heat shock protein 68 (HSP68) and two other putative heat sh ock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 b ut decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the tw o-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also i ndicate a role for glutathione in oocyte function. (C) 1997 Wiley-Liss , Inc.