Investigation into the Effects of Oxidative Stress on Reproductive Development.

Abstract

Nuclear transfer (NT), or cloning, which is the transfer of a donor nucleus to a recipient enucleated oocyte, has been successfully achieved to produce viable offspring in many species. The process is very inefficient, as reprogramming of the donor nucleus is required, and losses are high throughout development. Placentation abnormalities are a common feature amongst cloned animals. Incomplete nuclear reprogramming and erroneous epigenetic imprinting may contribute to aberrant protein transcription and DNA mutations, affecting mitochondrial metabolism and inducing cellular stress. In vitro produced embryos under high oxygen culture conditions may also suffer oxidative stress, with the resulting reactive oxygen species causing mitochondrial DNA mutations and cellular stress similar to clones.

In this study, expression of oxidative stress protein markers (Hsp60, SOD2, Hsp70) in NT cotyledons were compared to artificial insemination (AI) at different time points of gestation (days 50, 100, and 150). As a continuum of the oxidative stress investigation in cloned cotyledons, in vitro produced embryos were cultured under 20% oxygen compared to the control 7% oxygen laboratory standard culture, with oxidative stress protein markers examined between the groups at blastocyst stage (day 7) and day 15. Embryo morphology was also observed to determine apparent physiological differences between the treatment and control embryos. No previous studies to date have investigated the developmental effects of oxidative stress in day 15 bovine embryos.

The significant differences in oxidative stress proteins observed at several time points in the NT and AI groups were not repeatable, possibly due to sample freeze/thaw degradation. Morphological differences observed between embryos cultured in 20% oxygen and control groups were visually apparent, although not quantified. At day 15 manganese superoxide dismutase expression was significantly lower in the 20% group compared to control. The 20% oxygen group did not show higher heat shock protein 60 expression than control, however the same results have been observed in another study at blastocyst stage. The results of this study suggest that the effect of oxidative stress on embryonic development is evident yet inconclusive in bovine NT cotyledons, however does not appear apparent in day 15 embryos following culture in 20% oxygen.