88 Increasing cytoplasmic glutathione in bovine oocytes with modified in vitro maturation systems

In vitro-matured (IVM) bovine oocytes have lower developmental competence than in vivo-matured oocytes. One of the suspected reasons for this is a reduced accumulation of the antioxidant glutathione (GSH) during IVM. Lower GSH levels are thought to explain lower fertilisation rates following intracytoplasmic sperm injection (ICSI) for bovine IVM oocytes due to the role of GSH in sperm decondensation and male pronuclei formation. For this experiment, maturation rates, ICSI fertilisation rates, and IVF cleavage and blastocyst rates were examined, as well as the levels of glutathione accumulation between groups. Abattoir-derived bovine oocytes were matured in TCM 199 maturation medium with FSH, LH, bovine fibroblast growth factor, and 10% fetal bovine serum supplemented with either 0.6, 1.2, or 2.4 mM cysteine (GSH precursor) or 2 or 4 mM of the cell permeable substrate GSH ethyl ester (GSE). Controls for both series was the base maturation medium. Glutathione accumulation was measured using cellTracker blue staining, fluorescence microscopy, and quantification using ImageJ (National Institutes of Health). ICSI was performed on oocytes matured with either 0.6 mM cysteine (n = 116) or 4 mM GSE-IVM (n = 103), and IVF was performed on oocytes matured with 4 mM GSE-IVM (n = 113). Both IVF and ICSI results were analysed using a Yate’s corrected chi-squared, and ImageJ output (arbitrary fluorescence units) of glutathione levels was analysed via Kruskal–Wallis one-way ANOVA. Supplementing maturation medium with cysteine at any level did not significantly increase glutathione levels within the oocytes (n = 183). However, the addition of 4 mM GSE did significantly increase glutathione levels when compared to 2 mM GSE or controls (n = 108) (P < 0.05). There were no differences seen in maturation rates with 0.6 mM cysteine (85.2% vs. 86.7%) or 4 mM GSE IVM compared to controls (87.7% vs. 93%), respectively. Fertilisation rates post-ICSI, assessed via pronuclei formation, were similar for both groups as well, with cysteine (35.3% vs. 40.4%) and GSE (35.8% vs. 39.3%) groups showing no significant differences compared to controls (P < 0.05). Following IVF, there were no significant differences between cleavage (85% vs. 87.6%) or blastocyst (53.1% vs. 48.7%) rates when compared to control IVF (P < 0.05). The addition of 4 mM GSE to maturation medium did increase levels of glutathione within the oocytes, but did not lead to changes in fertilisation rates with ICSI or development after IVF. Measured GSH levels were highly variable throughout all treatment groups and each included a portion of individual oocytes which had a relatively high GSH level. This variation may explain why the difference in mean GSH levels for 4 mM GSE supplementation was not reflected in increased fertilisation after ICSI or increased development following IVF.

Support was provided by USDA-NIFA 2020-67016-31134.