Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

Abstract

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 ± 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 ± 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 ± 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 ± 74.1 nmol/egg in unfertilised eggs, 146.0 ± 50.0 nmol/egg in DSH eggs and 39.7 ± 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

NMR spectroscopy of single sub-nL ova with inductive ultra-compact single-chip probes

“…Spectrophotometry of intracellular extracts, on the other hand, has shown that up to 30 pmol/oocyte of glutathione (GSH) are contained in oocytes of goat34 and pig3536 and can change in reaction to environment and developmental stage37. Variations of a few pmol/oocyte of GSH in time scales of the order of several hours have been reported in hamster38 and rat39 oocytes. In these studies, the intracellular GSH content and its evolution is directly measured, but the ensemble measurements hide possible heterogeneities among single entities.…”

“…The direct application of NMR on single embryos using miniaturized high sensitivity probes is potentially advantageous for this aim. We suggest that systematic and extensive NMR studies on single cultured ova may provide new data that could shed light on cryptic processes involved in embryonic development3233343536373839 and provide new methodologies to estimate embryonic health3740.…”