The use of roscovitine for developing an in vitro model to study the effects of elevated temperature on immature bovine oocytes

The overall objective of this thesis was to determine effects of elevated temperature on immature, but fully grown oocytes. Initial efforts were devoted to developing an in vitro model, using roscovitine, for the purpose of maintaining oocytes at the germinal vesicle stage after removal from antral follicles without compromising subsequent development. Doing so was critical for establishing a model for studying the effects of elevated temperature on fully grown, developmentally competent oocytes that had not resumed meiosis. To determine an effective dose of roscovitine for maintaining oocytes at the GV stage after removal from follicles, cumulus oocyte complexes were cultured with 0, 12.5, 25, or 50 µM roscovitine (a cell cycle inhibitor of p34^cdc2/cyclin B kinase) for 24 h. Culture of oocytes with 50 µM roscovitine was effective for maintaining an intact GV in oocytes and prevented expansion of cumulus (P<0.005). Moreover, oocytes cultured with 50 µM roscovitine resumed meiosis after release from inhibitor and progressed to MII. A second study was conducted to determine the duration of exposure to roscovitine that successfully maintained oocytes at the GV stage, yet still allowed oocytes to progress to MII after release from the inhibitor. Cumulus oocyte complexes were cultured with 50 µM roscovitine for 21, 42, or 66 h. Culture of oocytes for 21,42, or 66 h with roscovitine maintained oocytes at GV stage (P<0.14). However, exposure of oocytes to roscovitine for 66 h compromised ability to progress to MII after resuming meiosis (P<0.04). A third study was conducted to determine if roscovitine had a negative effect on oocytes to develop to blastocyst. Cumulus oocyte complexes were cultured in 0 or 50 µM roscovitine. After 24 or 48 h with roscovitine, oocytes were washed, matured, and then fertilized. Culture of oocytes with roscovitine did not compromise ability of oocytes to cleave and develop to blastocyst. Development of an in vitro model allowed for the study of direct effects of elevated temperature on immature bovine oocytes. Cumulus oocyte complexes were maintained at GV stage using roscovitine for 24 h. During that time, oocytes were exposed to 38.5°C or 41°C. Duration of heat shock was either 8, 12, or 24 h. Exposure to 41°C for 12 or 24 h decreased in the ability of immature oocytes to cleave after maturation and fertilization. However, development to blastocyst was similar regardless of initial treatment. Determining effects of elevated temperature on immature bovine oocytes will be important for devising strategies for reducing deleterious effects of elevated temperature on reducing reproductive performance in livestock species.