Investigation Of Effects And Potential Mechanisms Of Rapamycin On Oocytes During In Vitro Maturation

Objective: Rapamycin may have positive effects on the in vitro maturation(IVM)process of oocytes,yet the optimal concentration,specific effects,possible mechanisms,and the prospect and safety of clinical application still need to be further explored.Therefore,the purpose of our study is to comprehensively evaluate the effects of rapamycin at optimal concentration on the quality and developmental competence of mouse oocytes during IVM,to explore possible mechanisms,and to further assess the effectiveness of rapamycin in improving the IVM outcomes of human oocytes.Methods: Immature oocytes from female ICR mice were collected for in vitro culture,and different concentrations of rapamycin(1 n M-10 μM)were added to the culture medium to select the optimal dose,by comparing the maturation rates and activation rates.Subsequently,rapamycin at optimal concentration was added to the culture medium of the treatment group,and the developmental competence after parthenogenetic activation,spindle assembly,chromosome arrangement,mitochondrial membrane potential(MMP)levels,and reactive oxygen species(ROS)levels of in vitro matured oocytes in the treatment and control groups were compared.To explore the possible mechanism,the DNA damage levels were further evaluated and alteration of effects of rapamycin brought by the mTOR activator was assessed.Moreover,the expression levels of mTORC1pathway-related genes,spindle assembly-related genes,antioxidant genes,and key genes for DNA damage repair in the oocytes of both groups were also compared.Additionally,the human oocytes failing to mature in vivo during clinical routine ovulation were collected and cultured with or without rapamycin.The maturation rates,normal fertilization rates,high-quality embryo rates,and blastocyst formation rates of oocytes in the treatment and control groups were compared.Results: The effects of rapamycin on the oocyte IVM process was related to its concentrations.Mouse oocytes treated with 10 n M rapamycin presented higher maturation rates and parthenogenetic activation rates after IVM,thus 10 n M was selected as the optimal concentration.In subsequent culture,the cleavage rates and blastocyst formation rates of activated embryos were also significantly increased in the 10 n M rapamycin group.Compared with the control group,the chromosome arrangement of mature oocytes in the10 n M rapamycin group was improved,and the ROS levels and DNA damage levels were significantly reduced,while no significant differences were observed in spindle morphology and MMP levels.The mTORC1 pathway was inhibited in oocytes in the rapamycin group,and simultaneously the m RNA levels of antioxidant genes NRF2,DNA damage repair genes ATM,ATR,DNAPK,and RAD51 were significantly increased,compared with the control group.Furthermore,during IVM of human oocytes,the addition of 10 n M rapamycin significantly increased the germinal vesicle breakdown(GVBD)rates,48 h maturation rates,and high-quality embryo rates after fertilization of oocytes and decreased the levels of DNA damage.Conclusion: Rapamycin at 10 nM significantly improved the quality and developmental competence of in vitro matured oocytes in both mouse and human.The mechanism might be to reduce DNA damage in oocytes and maintain the genetic stability,by reducing oxidative stress and promoting DNA damage repair,thereby improving IVM outcomes.