Effect Of Addition Of MnTBAP To Cryoprotectant On Vitrified Cryopreservation Of Mouse Ovarian Tissues

OBJECTIVE: Based on the antioxidant activity of Mn TBAP,the experiment aimed to investigate the protective effect and its protective mechanism of Mn TBAP on ovarian tissue vitrification,and to provide a reference for optimizing ovarian tissue vitrified conditions.METHODS: The ovarian tissues of mice were randomly divided into fresh control group and experimental group with different concentrations(0nmol/L,2.5 nmol/L,5 nmol/L,10 nmol/L,20 nmol/L)of Mn TBAP for vitrification.The percentage of morphologically normal follicles was analyzed by hematoxylin-eosin staining,and the follicle viability was analyzed by neutral red staining.After screening the optimal concentration group of Mn TBAP,the SOD,CAT activity and MDA content were analyzed by the kits.The follicular apoptosis rate of ovarian tissues was analyzed by TUNEL.The relative m RNA expression of Bcl-2,Bax,Caspase-3,FOXO3,CX43 and CX37 and Nrf2-associated pathway factors were detected by q PCR.The degree of fibrosis was analyzed by sirius red staining.The expression of FOXO3 a and Cleaved-Caspase-3 was analyzed by immunohistochemistry.RESULTS: Compared with the fresh control group,the percentage of morphologically normal follicles and follicle viability were significantly lower in the experimental group with 0 nmol/L Mn TBAP(P < 0.05).Compared with the experimental group with 0 nmol/L Mn TBAP,the percentage of morphologically normal follicles and follicle viability were significantly higher in the experimental groups with 2.5 nmol/L,5 nmol/L,10 nmol/L,and 20nmol/L Mn TBAP(P < 0.05).The experimental group with 5 nmol/L Mn TBAP was closer to the fresh control group in follicle morphology and follicle viability.So the concentration of 5 nmol/L Mn TBAP was selected as the optimal concentration.Compared with the experimental group with 0 nmol/L Mn TBAP,the rate of follicular apoptosis was significantly reduced(P < 0.05).The relative m RNA expression of Bax and Caspase-3 was down-regulated and the relative m RNA expression of Bcl-2 was up-regulated in the experimental group with 5 nmol/L(P < 0.05).The protein expression levels of Cleaved-Caspase-3 was down-regulated(P < 0.05).The sirius red staining showed that the degree of fibrosis was reduced in the experimental group with5 nmol/L Mn TBAP(P < 0.05).The relative m RNA expression of CX43 was up-regulated(P < 0.05).The antioxidant enzymes(SOD,CAT)activity was increased and the redox product MDA content was reduced in the experimental group with 5 nmol/L Mn TBAP(P < 0.05).The q PCR showed that the relative m RNA expression of Nrf2,HO-1 and NQO1 were up-regulated(P < 0.05).In terms of primordial follicle activation,the activation rate of primordial follicles was significantly reduced in the experimental group with 5 nmol/L Mn TBAP.The relative m RNA expression of FOXO3 was upregulated(P < 0.05).CONCLUSION: Mn TBAP can regulate redox endostasis through the endogenous Nrf2/HO-1 antioxidant pathway,thereby improving ovarian tissue morphology and follicular viability after the vitrification of ovarian tissues.Mn TBAP can reduce the apoptosis,the degree of fibrosis and the activation of primordial follicles after vitrification of ovarian tissues,and the expression of gap connection factor CX43 is upregulated.