Regulation Of Post-ovulatory Aging Of Mouse Oocytes By Inhibiting Resumption Of The Second Meiosis And Oxidative Stress

Most mammalian oocytes would be blocked at MII stage after maturation in vivo or invitro until sperm penetrating. There are some morphological and physiological changeshappened in oocytes as fertilization is delayed, which is usually called oocyte aging. Agedoocytes have poor developmental potential accompanied with low fertilization rate andsimultaneously higher polyspermous fertilization rate. Organelles in aged oocytes such asspindel and cortical granules(CGs) are wrong assembled and localized. When these oocytesare activated by chemical treatment, cytoplasmic fragmentation ratio would increase withincreased activation rate. Many experimental designs in research involve culture of maturedoocytes prior to micromanipulation or insemination. Therefore，it is necessary to find asuitable way to inhibit the aging of oocytes. Post-ovulatory aging of mouse oocytes wasregulated by inhibiting resumption of the second meiosis and oxidative stress. To study therole of MG132, caffeine, cysteamine and cystine in mouse oocyte aging inhibition, wecompared different concentration, treatment duration, different temperature and procedure ofthese reagents on chemical activation(CA) ability and embryos developmental potential oftreated oocytes, and we also studied spindle assemblement,cortical granules(CGs) locationand protein Bcl-2level of mouse oocyte treated by these reagents at15℃. At the same time,we study the role of cumulus cell during OA inhibiting mouse oocyte aging.Experiments results as below:1. Low temperature can inhibit oocyte aging. The chemical activation ratio of controlincreases at6h at37℃, while it maintains low chemical activation up to24h at25℃and60hours at15℃.2. MG132has different inhibition of oocyte aging at different temperature. Oocyte matured invivo aging was effectively inhibited by2μM,2.5μM,3μM and3.5μM MG132treated for6h at37℃. When3μM and3.5μM MG132were used to inhibit oocyte aging, the oocytescould develop to blastocyst which was similar to the control group treated for6h withoutMG132at37℃.1μM,2μM and4μM MG132could effectively inhibit oocyte aging whenwe prolong the treatment duration to12h at37℃, however, the oocytes blastulationdecreased to about10%. Oocyte aging could be inhibited by1μM,2μM and4μM MG132treated for12h at25℃, but the oocytes blastulation was lower than control after the treated oocytes recovered in medium without MG132for6h at37℃.When we went on decreasingthe temperature to15℃, we found oocyte aging could be effectively inhibited by1μM,2μMand4μM MG132at15℃.Then, when oocytes treated by different concentration of MG132for different hours were recovered for6h at37℃, we found that the oocytes treated by1μMand2μM MG132for12h could develop to blastocyst which was similar to the controltreated for6h withour MG132at37℃. The blastocyst was lower than control when weprolong the treatment duration to24h at15℃.3. We detected the spindle morphous, cortical granules(CGs) distribution and protein Bcl-2level in oocytes treated by1μM MG132for12h or24h at15℃. Oocytes had no integratedspindles before recovery and regained normal morphology of tine pole spindles after recovery.Though about half of the oocytes preserved at15℃for12h were at the early cortical granulemigration, most of the oocytes restored normality of cortical granule distribution afterrecovery culture. Most of the oocytes preserved at15℃for24h suffered from at the earlyand late cortical granule migration, and some oocytes preserved at15℃for24h remained atearly stage of cortical granule migration after recovery culture. Whereas Bcl-2level was highboth before and after recovery when oocytes were preserved at15℃for12h, Bcl-2level waslower than control which were preserved at37℃for6h both before and after recovery whenoocytes were preserved at15℃for24h.4. Oocyte matured in vivo aging was efficiently inhibited by8mM,10mM and12mMcaffeine treatment for6h at37℃.When8mM caffeine was used to inhibit oocyte aging, theoocytes could develop to blastocyst which was similar to the control group.4mM,6mM and8mM caffeine could effectively inhibit oocyte aging when we prolong the treatment durationto12h at37℃, however, the oocytes blastulation was lower than control. Caffeine could notinhibit oocyte aging at25℃.2mM and4mM caffeine effectively inhibited oocyte aging at15℃, however, the oocytes blastulation was lower than control after the treated oocytesrecovered in medium without caffeine for6h at37℃.5.150μM Cysteamine and300μM cystine could not only effectively inhibit oocyte aging butalso maintain the development competence of oocytes treated for6h at37℃. AlthoughCysteamine and cystine could effectively inhibit oocyte aging, they could not maintainoocytes development competence when we prolong the treatment duration to12h at37℃.100μM Cysteamine and200μM cystine,150μM Cysteamine and300μM cystine couldinhibit oocyte aging at25℃, and after oocytes treated for12h at25℃were recoveried inmedium without Cysteamine and cystine for6h at37℃, the ratio of blastocyst was similar tocontrol group.100μM Cysteamine and200μM cystine could inhibit oocyte aging at15℃, and after oocytes treated for24h at15℃were recoveried in CZB without Cysteamine andcystine for6h at37℃, the oocytes could develop to blastocyst which was similar to thecontrol group. Thus, antioxidants could maintain development competence of oocytes, whichwas confirmed by Vitamin C addition.6. We detected the spindle morphous, cortical granules(CGs) distribution and protein Bcl-2level in oocytes treated by200μM Vc for12h and24h or by100μM Cysteamine and200μM cystine for24h and36h at15℃. Oocytes had no integrated spindles before recoveryand almostly formed normal morphology of tine pole spindles after recovery. Though abouthalf of the oocytes preserved in presence of200μM Vc at15℃for12h were at the earlycortical granule migration, and most of the oocytes restored normality of cortical granuledistribution after recovery culture. Most of the oocytes preserved in presence of200μM Vc at15℃for24h suffered from at the early and late cortical granule migration, and some oocytespreserved at15℃for24h remained at early stage of cortical granule migration afterrecovery culture. Whereas Bcl-2level was high both before and after recovery when oocyteswere preserved at15℃for12h, Bcl-2level was lower than control which were preserved at37°C for6h both before and after recovery when oocytes were preserved at15℃for24h.Though a lot of oocytes preserved in presence of100μM Cysteamine and200μM cystine at15℃for24h were at the early or late cortical granule migration, and most of the oocytesrestored normality of cortical granule distribution after recovery culture. Though most of theoocytes preserved in presence of100μM Cysteamine and200μM cystine at15℃for36hsuffered from late cortical granule migration, some oocytes preserved remained at early stageof cortical granule migration after recovery culture. Whereas Bcl-2level was high both beforeand after recovery when oocytes were preserved at15℃for12h in presence of200μM Vc orfor24h in presence of100μM Cysteamine and200μM cystine, Bcl-2level was lower thancontrol which were preserved at37℃for6h both before and after recovery when oocyteswere preserved at15℃for24h in presence of200μM Vc or for36h in presence of100μMCysteamine and200μM cystine.7. After oocytes were treated by2μM MG132,10.27mM pyruvate and100μM Cysteamineand200μM cystine for9h at37℃and recoveried in CZB for6h at37℃, we found thatthe oocytes could develop to blastocyst which was similar to the control group. The oocytescould maintain development competence after oocytes were treated by10.27mM pyruvate and100μM Cysteamine and200μM cystine for48h at15℃and recoveried in CZB for6hat37℃. 8. During oocyte aging was inhibited by different concentration of okadaic acid supplied inCZB without energy, cumulus cells inhibit oocyte aging.