| [Background]Cryopreservation and autotransplantation of ovarian tissue are currently the most ideal way to preserve the fertility of surviving cancer patients,which makes it possible to extend the endocrine and reproductive functions of the ovaries.Hundreds of ovarian transplant operations have been performed worldwide,and more than 160 live births have been obtained through this technology,and about 60%of patients recover their endocrine function within three months after transplantation.Therefore,the initial stage of ovarian transplantation faces the establishment of new blood vessels and ischemia-reperfusion injury.The ischemia that lasts for several days is most likely to deepen and become irreversible.During this period of time,approximate 60-70%of the primordial follicles are lost,which obviously affects the recovery of the endocrine function of the ovarian tissue after transplantation and shortens the life of the graft.Therefore,it is particularly important to protect the follicles in the transplanted ovarian tissue,especially the primordial follicles.There are two reasons for follicle loss:one is that most of the primordial follicles are directly atresia and undergo a degenerative process and die,which belongs to apoptosis or another kind of programmed cell death;the other is that they are over-activated,continue to grow and enter the growing follicle pool,and then atresia occurs.Therefore,this study used the mouse ovarian cryopreservation-autotransplantation model to explore the reasons for the loss of primordial follicles and develop corresponding interventions from the following two parts.The results of this experiment will provide a new strategy to improve the effect of cryopreservation-autotransplantation of human ovarian tissue.Part Ⅰ:Study on the role of pyroptosis in the process of cryopreservation and autotransplantation of mouse ovarian tissue[Object]Studies have shown that pyroptosis plays an important role in the ischemia-reperfusion injury of the heart,liver,kidney,brain and other organs.So far,it is not clear whether ovarian tissue cryopreservation-autotransplantation will trigger pyroptosis.Therefore,this study used the mouse ovarian cryopreservation-autotransplantation model to explore the role and the molecular regulation mechanism of pyroptosis during ovarian tissue cryopreservation-transplantation and the effect of pyroptosis inhibitors on the function of mouse ovarian grafts,providing support for the improvement of cryopreservation-transplantation technology of human ovarian.tissue.[Method]1.Experimental grouping:6-week-old ICR mice,divided into four groups,10 mice in each group:(1)Control group:neither cryopreservation nor autotransplantation;(2)Fresh transplantation group:On the day of ovarian removal,autologous ransplantation was directly performed without freezing;(3)Frozen transplantation group:the ovarian tissue was cryopreserved on the day of ovarian removal,and autologous transplantation was performed after freezing for 2 weeks;(4)Frozen pyroptosis suppression group:on the day of ovarian removal,the ovarian tissues were cryopreserved for 2 weeks and then transplanted.Ac YVAD-CMK(Caspase-1 inhibitor)was injected intraperitoneally at a dose of 2 mg/kg for 3 consecutive days on the day after transplantation,1 day after surgery,and 2 days after surgery.2.Three days after transplantation,the ovarian grafts were taken out.Western blot was used to detect the expression of Caspase-1 and NLRP3 protein in the ovarian tissues of the four groups of mice;the ELISA method was used to detect the levels of IL-1β and IL-18 in the ovarian tissues of the four groups of mice;Western blot and immunohistochemistry was used to detect the expression of Caspase-1 and Caspase-11 protein in the ovarian tissue of the natural control group and the frozen transplantation group.3.14 days after transplantation,the serum estradiol concentration of the four groups of mice was detected by ELISA;the ovarian grafts were taken out,and the orphology and density of follicles were observed using HE staining.[Result]1.Western blot results:the expression levels of Caspase-1 and NLRP3 protein in ovarian grafts in the fresh transplantation group,frozen transplantation group and pyroptosis inhibition group were significantly higher than those in the natural control group(P<0.05);The expression levels of Caspase-1 and NLRP3 protein in the ovarian transplantation group were significantly lower than those in the frozen transplantation group(P<0.05);compared with the fresh transplantation group,the expression levels of Caspase-1 and NLRP3 protein in the frozen transplantation group were significantly increased(P<0.05);the expression levels of Caspase-1 and Caspase-11 protein in the ovarian grafts of the frozen transplantation group were significantly higher than those of the natural control group(P<0.05).2.The levels of IL-1β and IL-18 in tissue homogenates:the levels of IL-1β and IL-18 in the fresh transplantation group,frozen transplantation group and pyroptosis inhibition group were significantly higher than those of the natural control group(P<0.05);compared with fresh transplantation group,the levels of IL-1β and IL-18 in the frozen transplantation group were significantly increased(P<0.05),while the levels of IL-1β and IL-18 in the pyroptosis inhibition group were significantly lower than those in the frozen transplantation group(P<0.05).3.The estradiol concentration at 14 days after transplantation:the estradiol concentration of the fresh transplantation group,the frozen transplantation group and the pyroptosis suppression group was significantly lower than that of the natural control group(P<0.05);compared with the fresh transplantation group,The level of estradiol was significantly reduced in the frozen transplantation group(P<0.05);compared with the frozen transplantation group,the level of estradiol in the pyroptosis suppression group was significantly increased(P<0.05).4.Follicle density of ovarian graft(1)Comparison of total follicle density:The total follicle density of ovarian grafts in the fresh transplant group,frozen transplantation group and pyroptosis inhibition group was significantly lower than that of the natural control group(P<0.05);the density of follicles in pyroptosis inhibition group was significantly higher than that in the frozen transplantation group(P<0.05)(2)Comparison of primordial follicle density:The primordial follicle density of ovarian grafts in the fresh transplantation group,frozen transplantation group and pyroptosis inhibition group was significantly lower than that of the natural control group(P<0.05);The density of primordial follicles of ovarian grafts in the pyroptosis inhibition group was significantly higher than that in the frozen transplantation group(P<0.05)(3)Percentage of growing follicles in each group:Compared with natural controFgroup,the proportion of growing follicles in fresh transplantation group and frozen transplantation group increased(P<0.05);compared with the frozen transplantation group,the proportion of growing follicles in the pyroptosis inhibition group decreased(P<0.05).[Conclusion]1.Pyroptosis occurred in the process of cryopreservation and autotransplantation of ovarian tissue.2.Under the experimental conditions,the application of pyroptosis inhibitors can protect the number of primordial follicles of mouse ovarian tissue after cryopreservation-autologous transplantation,without significant influence on the continued development of follicles and damage of ovarian endocrine function,which is expected to extend the life of the graft.3.The process of cryopreservation and autotransplantation of ovarian tissue causes an increase in the number of growing folliclesPartⅡ:Study on the role of inhibition of mTOR signaling pathway during cryopreservation and autotransplantation of mouse ovarian tissue[Object]In view of the first part of the experimental results,cryopreservation-transplantation of ovarian tissue stimulated an increase in the proportion of growing follicles,and it is considered that the primordial follicles were over-activated during this process.Studies have shown that mTOR plays a key role in the initiation of granulosa cell activation.Excessive activation of the mTOR signal in granulosa cells will accelerate the differentiation of granulosa cells,leading to the early activation of resting primordial oocytes.Therefore,the mTOR signal may be related to the shortened life span of the ovarian graft.In this study,the mouse ovarian cryopreservation-autotransplantation model was used to explore the role of mTOR signaling pathway in ovarian tissue cryopreservation-autotransplantation,in order to further clarify the molecular regulation mechanism of ovarian tissue cryopreservation-autotransplantation as well as the effect of mTOR specific inhibitor rapamycin on the function of mouse ovarian tissue during cryopreservation and autotransplantation,which will provide potential new targets for optimizing and improving this important fertility preservation technology.[Method]1.Experimental grouping:6-week-old ICR mice,divided into four groups,10 mice in each group:(1)Control group:neither cryopreservation nor autotransplantation;(2)Fresh transplantation group:On the day of ovarian removal,autologous transplantation was directly performed without freezing;(3)Frozen transplantation group:the ovarian tissue was cryopreserved on the day of ovarian removal,and autologous transplantation was performed after freezing for 2 weeks;(4)mTOR suppression group:on the day of ovarian removal,the ovarian tissues were cryopreserved for 2 weeks and then transplanted.rapamycin was injected intraperitoneally at a dose of 2 mg/kg for 3 consecutive days on the day after transplantation,1 day after surgery,and 2 days after surgery.2.Seven days after transplantation,the plasma estradiol and AMH levels of the four groups of mice were detected by ELISA;the ovarian grafts were taken out,and the follicle morphology and density were observed by HE staining;Western blot and immunohistochemistry were used to detect the expression of P-S6K protein.3.At 28 days after transplantation,the plasma estradiol and AMH levels of the four groups of mice were detected by ELISA method;the ovarian grafts were taken out,and the follicle morphology and density were observed by HE staining.[Result]1.The density of follicles in ovarian grafts(1)Comparison of total follicle density:At 7 and 28 days after autotransplantation of mouse ovarian tissue,the total follicle density of ovarian grafts in the fresh transplantation group,frozen transplantation group and mTOR inhibition group was significantly lower than that of the natural control group(P<0.05);There was no significant difference in the total follicle density of ovarian grafts in the mTOR inhibition group compared with the frozen transplantation group.(2)Comparison of primordial follicle density:At 7 and 28 days after autotransplantation of mouse ovarian tissue,the primordial follicle density of ovarian grafts in the fresh transplantation group,frozen transplantation group and mTOR inhibition group was significantly lower than that of the natural control group(P<0.05);the primordial follicle density of ovarian transplants in the mTOR inhibition group was significantly higher than that in the frozen transplantation group(P<0.05).(3)The proportion of growing follicles in each group;At 7 and 28 days after autotransplantation of mouse ovarian tissue,the proportion of growing follicles increased in the fresh transplantation group and frozen transplantation group compared with the natural control group(P<0.05);Compared with the frozen transplantation group,the proportion of growing follicles was lower in mTOR inhibition group(P<0.05).2.Western blot and immunohistochemical results:The expression level of P-S6K protein in the fresh transplantation group,frozen transplantation group,and mTOR inhibition group was significantly higher than that of the natural control group(P<0.05);the protein expression in mTOR inhibition group was significantly lower than that in freezing transplantation group(P<0.05).3.Estradiol and AMH levels:At 7 days and 28 days after autotransplantation of mouse ovarian tissue,the estradiol and AMH levels of the fresh transplantation group,frozen transplantation group and mTOR inhibition group were significantly lower than those of the natural control group(P<0.05);At 7 days after transplantation,the levels of estradiol and AMH in the mTOR inhibition group were significantly lower than those in the frozen transplantation group(P<0.05).At 28 days after transplantation,the mTOR inhibition group had lower estradiol levels compared with the frozen transplantation group(P<0.05),but there was no significant difference in AMH levels.[Conclusion]1.Cryopreservation-autotransplantation of ovarian tissue causes excessive activation of primordial follicles.2.mTOR and its signaling pathways are involved in the activation of primordial follicles;3.Under the conditions of this experiment,the application of mTOR inhibitor rapamycin can protect the number of primordial follicles from cryopreservation-autotransplantation of mouse ovarian tissue,and has no significant effect on the continued development of follicles and does not damage the endocrine function of the ovary,so it has the potential to extend the life of the graft.4.The concentration of estradiol is correlated with the density of antral follicles,and the level of AMH is correlated with the density of primary and secondary follicles.The combination of the two can monitor the development of follicles after autologous transplantation. |
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