Date of Award
Doctor of Philosophy
James D. Godkin
Judy Grizzle, Mary Ann Handel, Patricia Tithof, J. Lannett Edwards
Previous studies demonstrated that retinol administration to ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro. In vivo studies with sheep and in vitro experiments with bovine embryos, were designed to evaluate the effects of retinol and to understand its mechanism(s) of action.
The primary objective of the first experiment was to analyze ovine oocyte metabolism, and to assess the effects of retinol on this process. Sheep oocytes were matured in vitro over a 24 hour period in the presence of different radiolabeled substrates. Results revealed that oxidative metabolism measured by glutamine showed no significant differences over all time periods. Pyruvate oxidation was highest early in maturation and then decreased. Glycolysis was highest at the middle time period. Differences in metabolism between oocytes from retinol-treated ewes and those from control ewes were not detected.
Next, varying concentrations of retinol were added either during in vitro maturation (IVM) or in vitro culture (IVC) of bovine oocyte and embryos, respectively. Our results demonstrated that 5mM retinol supplementation during IVM tended to improve embryonic development measured by the rate of blastocyst development. This concentration proved even more beneficial if the control blastocyst rate was below 20%. Furthermore, 10mM retinol appeared detrimental during IVC but not during IVM.
In the third experiment, we evaluated the effects of 5mM retinol and 100mM cysteamine on bovine oocyte glutathione content. In addition, we investigated the combinatorial effects of retinol and cysteamine on in vitro bovine embryonic development. We did not observe an increase in glutathione levels in bovine oocytes treated with retinol. However, in the presence of cysteamine bovine oocytes exhibited an increase in glutathione content. Retinol and cysteamine treatment during IVM and IVC increased bovine blastocyst development, which may indicate that retinol is increasing the utilization or uptake of cysteine from the medium.
Next, we evaluated the role of exogenous retinol supplementation to superovulated ewes on the glutathione content of mature oocytes collected from the oviducts. Our results did not reveal differences in glutathione content of oocytes from retinol-treated ewes versus those from control ewes. Antioxidant transcripts encoding for Mn-SOD, Cu-Zn SOD, GS, and GSTp, were detected in ovine oocytes matured in vivo. However, retinol did not appear to impact the expression levels of these transcripts.
Livingston, Tracy Elizabeth, "The Effects of Retinol during In Vivo and In Vitro Oocyte Maturation and Embryonic Development.. " PhD diss., University of Tennessee, 2003.