Protective Effect Of Melatonin On Vitrified Cryopreservation Of Mouse Ovarian Tissue

Cryopreservation of ovarian tissue is an important method to protect the fertility of female cancer patients.The cryopreservation,recovery and transplantation of ovarian tissue not only retain the reproductive potential of young women,but also restore the endocrine function of the ovaries.Vitrification is a technology that can change tissue into vitrified solid state by using rapid cooling,which can keep the normal distribution of molecules and ions inside and outside cells.Vitrification has become an important technique for ovarian tissue freezing because of its advantages of high speed,high efficiency and low cost.Unfortunately,the technology of ovarian tissue cryopreservation is not mature,and there is no recognized,standardized and optimal formulation of vitrified solution.A number of studies have shown that oxidative stress is one of the key factors affecting the effect of vitrification.Reactive oxygen species(ROS)produced in oxidative stress can lead to apoptosis or dysfunction of ovarian cells,decrease the survival rate of oocytes,and reduce the development ability of ovarian tissue.Melatonin(MLT),a neuroendocrine hormone,has many physiological functions,such as enhancing immunity,anti-aging and anti-tumor.Especially as an effective antioxidant,MLT can resist oxidative stress reactions through various mechanisms.At present,it has been reported that melatonin has a protective effect on sperm cryoresuscitation,but there is no report on the application of melatonin in the vitrification of ovarian tissue at home and abroad.In this study,we found that melatonin can improve the morphology of follicle cells and protect the structural integrity of ovary in KM mice,which may be related to the increase of antioxidant capacity of ovarian tissue,the reduction of production of reactive oxygen species and the inhibition of apoptosis of follicle cells.This experiment may provide a new idea for the optimization of ovarian tissue freezing technology.Objective:1.To investigate whether melatonin has protective effect on the ovarian tissue of KM mice during vitrification.2.To find the optimal concentration of melatonin to protect the integrity of ovarian tissue structure.3.To explore the possible mechanism of melatonin's protective effect on ovarian tissue in the process of vitrification.Methods:1.Collection and grouping of ovarian tissue: 53 SPF adolescent female KM mice were subjected to bilateral ovariectomy and randomly divided into a fresh control group(n=6)and an experimental group(n=100)undergoing vitrification.The experimental group was divided into 5 groups according to the concentration of melatonin in the frozen solution and the resuscitation solution,which were 0m M MLT group,0.001 m M MLT group,0.01 m M MLT group,0.1m M MLT group and 1m M MLT group(n=20).2.After vitrification and resuscitation of ovarian tissue,HE staining microscopy was used to measure the number and diameter of follicles in each experimental group,3.The apoptosis rate of follicular cells in each experimental group was detected by TUNEL test after vitrification of ovarian tissue.4.RT-q PCR was used to detect the m RNA expression level of antioxidant related genes(Nrf2,HO-1,Hsp90 AA1)and apoptosis related genes(Bcl-2,Bax)in each experimental group.5.Western blot was used to detect the expression levels of antioxidant-related protein Hsp90 and apoptosis-related protein(Bcl-2,Bax)in each experimental group.6.The activity of antioxidant enzyme(GSH-Px,T-SOD,CAT),antioxidant enzyme(GSH),total antioxidant capacity(T-AOC),lipid peroxidation product MDA and LDH were detected by antioxidant kit.Results:1.Melatonin increased the normal rate of follicle morphology in the experimental groupAfter melatonin was added during vitrification of KM mice's ovarian tissue,the normal rate of Primordial follicle,primary follicle,secondary follicle and antral follicle in each experimental group was higher than that in the group without melatonin,and the normal rate of Primordial follicle(P< 0.01),primary follicle(P<0.001),secondary follicle(P<0.001)and antral follicle(P< 0.05)in the 0.1m M MLT group was the highest.2.Melatonin reduced the follicular diameter in the experimental groupAfter melatonin was added during vitrification of KM mice's ovarian tissue,the diameter of Primordial follicle,primary follicle and secondary follicle in each experimental group was lower than that in the group without melatonin,and the diameter of Primordial follicle,primary follicle and secondary follicle in 0.1m M MLT group was the lowest(P<0.001).The diameter of antral follicles in 0.01 m M,0.1m M and 1m M MLT group were all lower than that in the experimental group without melatonin,and the diameter of antral follicles in 0.1m M MLT and 1m M MLT group was the lowest(P<0.001).3.Melatonin reduced the apoptosis rate of follicles in the experimental groupAfter adding melatonin in the vitrification of ovarian tissue of KM mice's ovarian tissue,the apoptosis rate of follicles in each experimental group was lower than that in the group without melatonin,and the apoptosis rate of follicles in 0.1m M and 1m M MLT groups was the lowest(P<0.01).4.Melatonin up regulates the expression of Nrf2,HO-1,Hsp90AA1 and Bcl-2 m RNA in the ovary of KM miceAfter adding melatonin in the vitrification of ovarian tissue of KM mice's ovarian tissue,the relative expression of Nrf2 m RNA in 0.001 m M,0.01 m M and 0.1m M MLT group was higher than that in the group without melatonin.The relative expression of Nrf2 m RNA in 0.1m M MLT group was the highest(P<0.01),and there was no difference between the 1m M MLT group and the 0m M MLT group(P = 0.113).The relative expression level of HO-1m RNA in the experimental group with melatonin addition was higher than that in the group without melatonin addition,and the relative expression level of HO-1 m RNA in the 0.1m M and 1m M MLT groups was the highest(P<0.05).The relative m RNA expression level of Hsp90AA1 and Bcl-2 in the experimental group added with melatonin was higher than that in the group without melatonin,and the relative m RNA expression level of Hsp90AA1 and Bcl-2 in the 0.1m M MLT group was the highest(P<0.05).5.Melatonin down regulates Bax m RNA expressionAfter melatonin was added to the vitrified ovarian tissue of KM mice,the relative expression level of Bax m RNA in each experimental group was lower than that in the group without melatonin,and the relative expression level of Bax m RNA in the 0.1m M MLT group was the lowest(P< 0.01).6.Melatonin up regulates the protein expression of Hsp90 and Bcl-2 in ovarian tissueAfter adding melatonin in the vitrification of ovarian tissue of KM mice's ovarian tissue,the relative expression of Hsp90 and Bcl-2 protein in each experimental group was higher than that in the group without melatonin,and the relative expression of Hsp90(P<0.01)and Bcl-2 protein(P<0.05)in the 0.1m M MLT group was the highest,and the relative expression of Hsp90 protein in the 1m M MLT group was lower than that in the other experimental groups(P<0.001).7.Melatonin down regulates Bax protein expressionAfter melatonin was added to the vitrified ovarian tissue of KM mice,the relative expression level of Bax protein in each experimental group was lower than that in the group without melatonin and the relative expression level of Bax protein was lowest in the 0.1m M MLT group(P<0.001).8.Melatonin significantly increased the activities of GSH-Px,GSH,T-SOD,CAT and T-AOCAfter melatonin was added to the ovarian tissue of KM mice during vitrification,the activities of GSH-Px,GSH,CAT and T-AOC in each experimental group were higher than those in the group without melatonin,among which the activities of GSH-Px(P<0.01),GSH(P<0.001),CAT(P<0.01)and T-AOC(P<0.05)in the 0.1m M MLT group were the highest.The activity of T-SOD in the experimental group with melatonin was higher than that in the group without melatonin,and the activity of T-SOD was higher in the 0.01 m M,0.1m M and 1m M MLT group(P<0.01).9.Melatonin significantly reduces MDA activityAfter adding melatonin in the vitrification of ovarian tissue of KM mice,the MDA activity of each experimental group was lower than that of the group without melatonin,and the MDA activity of 0.1m M and 1m M MLT groups was the lowest(P<0.01).10.Melatonin had no significant effect on LDH activity of ovarian tissue in each experimental groupThere was no significant difference in LDH activity among the experimental groups(P = 0.145).Conclusion:1.During the vitrification and resuscitation of KM mouse ovarian tissue,the addition of melatonin can improve the morphology of follicular cells and protect the structural integrity of ovarian tissue.The optimal concentration is 0.1m M,and this concentration has less toxic and side effects.2.The protective effect of melatonin on ovarian tissue may be related to the increase of antioxidant capacity of ovarian tissue,the reduction of production of reactive oxygen species and the inhibition of follicular cell apoptosis.