| Removal of cumulus cells before in vitro maturation was detrimental to oocyte maturation. 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 denuded oocytes (DOs) will provide a technical approach to such procedures as germinal vesicle transfer, oocytes cryopreservation and so on. In addition, the mechanisms by which cumulus cells improve oocyte maturation are poorly understood. So it means very much to establish such efficient IVM system for DOs. The level of glutathione (GSH) is defined as one of the indications of cytoplasmic maturation. The addition of cystine or cysteamine to the maturation medium in order to enhancing GSH synthesis has been used by many researchers, but the mechanism by which cystine and cysteamine improve the GSH synthesis is still not clear. So in this rearch, to improve in vitro maturation of DOs, we observed the interactive effects of cysteamine, cystine and cumulus cells as well as oocytes on the GSH synthesis, and developmental capacity of mouse IVM oocytes, and thereby to establish an efficient IVM system for DOs. Our results demonstrated that:1) Mouse oocytes can’t utilize cystine or cysteamine supplied to the medium to synthesis GSH, however, cystine and cysteamine interacted to promote the GSH synthesis in mouse oocytes.2) Goat oocytes can utilize both cystine and cysteamine respectively.3) When mouse DOs were cultured alone or cocultured with mouse CCs, supplementation with either cysteamine or cytine had no effect, but supplementation with both increased DOs’intracellular GSH level significantly. When cocultured with goat CCs, however, supplementation with either cysteamine or an optimal concentration of cytine promoted mouse DOs’GSH synthesis.4) Although supplementation of either thiol significantly increased the intracellular GSH concentration of goat CC monolayer, the GSH level of mouse CCs increased only with supplementation of both thiols.5) When M16was incubated alone or with mouse CCs, cysteine was detectable only when both cysteamine and cystine were supplemented. When incubated with goat CCs, supplementation with either cystine alone or both cystine and cysteamine produced cysteine.6) Supplementation of cystine alone did not increase the developmental potential of matured COCs. The optimal combinations between cysteamine and cystine to improve blastulation were100or200μM cysteamine plus200μ M cystine.7) When cocultured with mouse CCs, supplementation with either cysteamine or cystine had no effect, but supplementation with both thiols increased blastulation of mature DOs to a level as high as that achieved in COCs matured with the optimum thiol supplementation.8) Oocytes matured under different conditions were inseminated in vitro. Rates for blastocyst formation did not differ between COCs and the cocultured DOs matured with supplementation of cysteamine and cystine.9) The embryos transfer experiment suggested that similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients.10) The mRNA of xCT, which is the regulatory subunit of system Xc-(cystine/glutamate exchange system), is expressed in both mouse CCs and goat CCs. This means that the reason why mouse CCs can’t utilize cytsine is not because of the absence of system Xc-11)The activation of cystine reducase in goat CCs is higher than in mouse CCs, which may explain the difference between mouse and goat CCs in utilization of cystine.It is concluded that1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either-one;2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation;3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs;4) The low activity of cystine reducase maybe the reason why mouse CCs can’t utilize cytine to synthesize GSH.With the fast development of our society, the pressure that people faced is becoming stronger and stronger. In recent years, females often serve several parts in the daily life. All of these bring heavy pressure to them. Women are more susceptible than men to stress-related mental illness and twice as likely to experience depression. Intensive research in recent years has substantiated that stress can harm the health of female. The effects of stress on reproduction in female have been demonstrated that stress can bring disorder to the reproductive endocrine system, and induce anaplasia in reproductive system of women. In the aspect of genetics, either acute or chronic stress can do some genetic damage to the organs of human body. As we know, aneuploidy of oocytes is a major obstacle in achieving reproductive success, and whether stress can do some harm to chromosomes of oocytes has been little concentrated on. So in this research, we investigated the effects of restraint stress(24h), the emotional stress modle we established before, on the abnormality of chromosomes of mouse oocytes. Besides, in order to reveal the reasons why restraint stress can induce chromosome abnormalities to mouse oocytes, we also examined the spinle assembly, chromosomes alignment and the number and structure of chromosomes in mouse MI ooytes, and detected the expression of MAD2, which is the main member of spindle assemble checkpoint(SAC). Finally, we investigated the GSH and GSH/GSSG in mouse oocytes, so that we can determine whether acute restraint stress can bring oxidative stress to mouse oocytes or not. Our research demonstrated that:1) Restraint stress applied to mouse can decreased the blastocyst developmental potential and the blastomere number of blastocyst. The proceeding of parthenogenetic embryos in stimulated mouse is slower than intact mouse.2) No matter the oocytes were cultured in TCM-199or in a-MEM medium, the increased levels of aneuploidy was obseaved in restraint treated mouse.3) Restraint treatment to mouse has no effects on the premature separation of sister chromatids.4) There are more aneuploidy embryos in stimulated mouse, while the level of mosaic embryos between the two groups has no difference. These resultes sugessted that the chromosomes abnormality of embryos is caused by the abnormality of oocytes chromosomes.5) The time that MI and A/TI lasted is much shorter in restraint treated mouse oocytes, and the first polar body is extruded earlier in stimulated mouse oocytes.6) Restraint stress can do some harm to the spindle assembly and chromosomes alignment in mouse MI oocytes, but has no effects on the number of homologs and premature homologous chromoses separation.7) The expression of MAD2is changed by restraint treatment. MAD2expression is higher in GV oocytes from stimulated mouse, while in pMI stage oocytes, its expression is lower in stimulated mouse.8) The level of GSH is decreased by restraint treatment, and the GSH/GSSG is lower in the oocytes of stimulated mouse, which means oxidative stress was introduced by acute restraint stress.On the whole, oxidative stress is most likely mechanism of restraint stress. Oxidative stress can induce the abnormality of MI spindle assembly and chromosomal distribution, as well as override the spindle checkpoint. All of these may provide a link between aneuploidy oocurrence and oxidative stress. |
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