Studies On The Effect And Mechanism Of Oocyte Control Of Cumulus Cell Expression Of Genes Related To GSH Metabolism

Glutathione,termed c-L-glutamyl-L-cysteinyl-glycine tripeptide,is a non-proteinaceous thiol compound widely distributed within the body including somatic cells and gametes.Although under physiological conditions,both thiol-reduced(GSH)and disulfide-oxidized(GSSG)glutathione exists,more than 90% of glutathione are found to be present in the form of GSH.Catalyzed by glutathione peroxidase,GSH converts to GSSG by reacting with hydrogen peroxide and other peroxides,thereby antagonizing the oxidative stress exposed to cells,which is essential for the maintenance of the normal functions and survival of the cells.GSSG converts back to GSH under the action of glutathione reductases.Glutathione synthesis and metabolism also play important roles during the process of follicle and oocyte growth,development and maturation.Glutathione synthesis and metabolism are one of the key facets of oocyte cytoplasmic maturation,which are prerequisites for normal fertilization and subsequent embryo development.The concentration of GSH in oocytes reflects the maturity degree of cytoplasm,and is considered to be one of the most effective mark for the cytoplasm maturation during oocyte in vitro maturation procedure.The levels of GSH in oocytes are affected by the presence of follicular granulosa cells,especially the cumulus cells surrounding the oocytes,suggesting that the bi-directional communication between these two types of cells are crucial for the maintenance of the normal levels of GSH in both oocytes and cumulus cells,as well as those within the whole microenviroment of the follicle.However,the mechanism controlling glutathione synthesis and metabolism in the follicular microenvironment,especially the effect and mechanism of oocytecontrol of cumulus cell glutathione synthesis and metabolism is not clear.In this study,we first established a culture system for studying the interaction between oocytes and cumulus cells using the ICR strain of mice that are commonly used in biomedical research in China.Then using this culture system,we studied the effect and mechanism of the oocyte and its secreted papracrine factors,GDF9 and BMP15,on the expression of genes related to glutathione synthesis and metabolism in culmulus cells,as well as the potential physiological means underlying this control.By analysis of our previously collected data of oocyte and cumulus cell transcriptomes,we found that genes encoding key enzymes involved in glutathione metabolic processes were expressed at higher levels in cumulus cells than in oocytes.By quantitative real time RT-PCR analysis,we revealed that these genes are expressed differently among mural granulosa cells,cumulus cells and oocytes.By culture of oocytectomized cumulus complexes(OOX),and treatments with recombinant GDF9 and/or BMP15,and inhibitors of SMAD signaling pathways,we found that the differences of expression of these genes were regulated by specific oocyte secreted paracrine factors GDF9,and were mediated by the SMAD2-dependent pathway.By Tunel staining and Western blot analysis,we found that one of the biological effects of oocyte control of cumulus cell expression of genes related to glutathione metabolism is to promote cumulus cells to antagonize apoptosis.In sum,oocytes control the expression of genes related to glutathione metabolism in granulosa cells through a SMAD2-dependent paracrine mechanism,which could contribute to the regulation of glutathione metabolism in follicle,and thereby influence the redox state within the follicular micoenviroment,and eventually promote the healthy development and maturation of the follicles and oocytes.