The effect of heat shock on the developmental competence and activation of bovine oocytes

Low reproductive performance has long been an unsolved problem for animal producers in tropical and subtropical areas but is believed to be related to fertilization failure and early embryonic mortality. The mechanism of heat-induced oocyte or embryo damage is unclear. Two series of studies were conducted to investigate the direct impact of heat shock on mature bovine oocytes. In the first series of studies (Chapters 2 through 5), thermal tolerance of bovine oocytes and embryos was tested. In vitro-matured oocytes were allocated to different hyperthermic treatments (40.5 to 43°C) in an atmosphere of 5% CO2 in air. Fertilization, cleavage, blastocyst development and cell lineage analysis were used as criteria to assess viability of oocytes and embryos after heat shock. Results showed that viability of mature oocytes was not affected by heat shock for 2 hr at 40.5 to 42°C, but oocytes lost viability after 45 min at 43°C. No heat shock response was induced from both Day 3 and Day 4 embryos. Effect of heat shock on the membrane surface ultrastructures was further examined using scanning electronic microscopy (SEM) and immunocytochemical staining. Matured bovine oocytes were not efficiently activated by heat shock as reported for mouse and rabbit oocytes. However, the ultrastructure of both the zona pellucida and vitelline membrane of heat-shocked (42°C for 30 min) oocytes manifested surface alteration from a microvilli-dominant pattern to cytoplasmic protrusion-dominant pattern. Furthermore, the activation rate of the heat-shocked oocytes increased during 8 to 16 hr of incubation following heat shock treatment indicating that physiological conditions may have been changed after heat shock. These results suggest that fertilization and pregnancy rates of farm animals during periods of high ambient temperature may be attributed in part to the alteration of oocyte cytoskeleton and surface ultrastructure and the incomplete activation of the oocyte caused by heat stress.; In the second series of studies (Chapters 6 through 7), different activation agents, including electric pulse, ethanol, calcium ionophore, cycloheximide (CHX) with cytochalasin D (CD), 6-dimethylaminopurine (6-DMAP), and their combined effect were tested. Single activation agents cannot fully activate mature oocytes reflected by low pronuclear formation and metaphase III arrest. Cytoskeletal reorganization, including microtubules and microfilaments occurred when oocytes were activated by different parthenogenetic agents and the combined treatments. An immediate disappearance of meiotic spindle and enhancement of cell cycle progression occurred after 6-DMAP treatment indicating phosphorylation is required for maintaining meiotic spindle and normal cell function. A dramatic relocation of microfilaments was found in the oocyte treated with 6-DMAP and CHX/CD. After removal of the chemicals, oocytes reversed microfilament structure corresponding to their developmental stages. These studies provided a solid basis of thermotolerance and a possible mechanism of thermal injury of bovine oocyte, as well as novel phenomena of cytoskeletal reorganization after oocyte activation. Further molecular approach of the signaling pathways in response to thermal stress and activation is required.