Effects Of Cysteamine, Cystine And Cumulus Cells On The Intracellular Glutathione Level And Developmental Cpacity Of Goat Cumulus-denuded Oocytes

Removal of cumulus cells before in vitro maturation (IVM) was detrimental to oocyte maturation, and co-culture with cumulus oocyte complexes (COCs) or cumulus cells restored partially the developmental potential of cumulus-denuded oocytes (DOs). However, the removal of cumulus cells from oocytes or zygotes at various stages of development is inevitable for some embryo manipulation techniques. Therefore, an efficient IVM system for DOs will provide a technical approach to such procedures as germinal vesicle (GV) transfer, somatic cell haploidization and oocyte cryopreservation at the GV stage. In addition, the mechanisms by which cumulus cells improve oocyte maturation are poorly understood. To improve in vitro maturation of DOs, we compared the effects of different forms of cumulus cells, including dispersed cumulus cells (DCCs), monolayer of cumulus cells (MCCs) and cumulus oocyte complexes (COCs) on the in vitro maturation and embryo development of goat DOs. And we also examined the inhibition of FSH, LH and 17β-E2 which added to the medium on the luteinization of cumulus cells during its cultured to monolayer. Moreover, we especially observed the interactive effects of cysteamine, cystine and cumulus cells on the GSH level and developmental capacity of goat IVM oocytes. The results are summarized as follows:1. The rate of goat COCs maturation and blastocyst development is higher than DOs(p<0.05), any forms of cumulus cells can improved nuclear maturation as well as morulation of DO(sp<0.05), but has no beneficial effects on blastulation of DOs. When co-cultured with MCCs, more DOs can developed to morula after chemical activation than co-cultured with DCCs and COCs(p<0.05).2. Cumulus cells cultured for 5 days in TCM-199 without hormones produced much more profesterone than cumulus cells cultured with FSH, LH and 17β-E2(p<0.05), which was not different significantly from that contained in the control TCM-199 cultured for the same period without cells.This indicted that the hormones in the cultural medium had inhibited luteinization of cumulus cells.3. When goat COCs were cultured in TCM-199 supplemented with different concentrations of cysteamine, the content of intracellular GSH increased with cysteamine. However, the highest rate of blastocyst formation was achieved just at 100-μM cysteamine, with blastocyst rates decreasing at higher concentrations. A similar pattern of changes was observed in the average cell number per blastocyst among cysteamine concentrations.4. When goat COCs were cultured in TCM-199 supplemented with different concentrations of cystine, the content of intracellular GSH increased with cystine. However, the highest rate of blastocyst formation was achieved only at 200-μM cystine, with blastocyst rates decreasing at higher concentrations. A similar pattern of changes was observed in the average cell number per blastocyst among cystine concentrations.5. When both cysteamine (100-μM) and cystine(200-μM) were added to the IVM medium, the rate of blastocyst formation was further increased(P<0.05). However, the level of GSH was similar with COCs matured in TCM-199 contained either 100-μM cysteamine or 200-μM cystine(P>0.05). Increasing the concentrations of cysteamine and cystine to 200-μM and 500-μM respectively did not have any fuether effects on both GSH synthesis and blastulation of goat COCs(P>0.05).6. The addition of 100-μM cysteamine to TCM-199 improved GSH synthesis and blastulation as well as nuclear maturation of DOs. The addition of 200-μM cystine, however, did not have any beneficial effect on the DOs. When cysteamine and cystine were added together, GSH content and rate of blastocysts further increased (P<0.05) relative to those obtained when cysteamine was added alone. These results indicated that goat DOs can not takeup cystine itself, unless assisted by cysteamine, but can use cysteamine directly.7. In the presence of neither cysteamine nor cystine, co-culture with cumulus monolayer increased nuclear maturation and GSH synthesis (P<0.05), but not blastulation of DOs. Co-cultured with cumulus cell monolayer, the addition of either cysteamine or cystine improved both GSH synthesis and blastulation of DOs. When cysteamine and cystine were added together, both the GSH level and the rate of blastocysts of the co-cultured DOs increased further to the same level as that of COCs cultured with no thiol supplementation.