Study On The Role And Mechanism Of HIF-1α In Follicular Overactivation In Ovarian Tissue Transplantation

Objective The purpose of this study is to study the relationship between ischemia and hypoxia inducible factor α(HIF-1 α)and overactivation of primordial follicles in ovarian transplantation,so as to explore the cause and mechanism of follicular depletion in the process of autologous ovarian frozen transplantation.The study will provide a theoretical basis for improving the survival rate of follicles after ovarian transplantation and further optimizing the fertility preservation of female patients with malignant tumors,and is of great significance to guide the clinical practice of fertility preservation.Method Part one: the animal models of different degrees of ischemia and hypoxia after ovarian transplantation were established by heterotopic transplantation of ovarian tissue to different parts of the blood supply environment(subcutaneous of neck,muscle of back,subcapsular of kidney).The dynamic changes of estrous cycle and anti-Mullerian hormone(AMH)were observed.And the changes of ovarian weight,total follicle density count,primordial follicle density count and follicular morphology on the day of sampling to understand the effects of different degrees of ischemia and hypoxia on primordial follicle activation.In the second part,the subrenal capsule was used as the site of autologous ovarian transplantation in rats,and different ovarian states(whole ovary transplantation and ovarian mass)were used to compare the effects of two different degrees of ischemia and hypoxia on the recovery of ovarian function and follicular activation.On the basis of whole ovary renal capsule transplantation,rats were treated with HIF-1 α activator DMOG and inhibitor 2ME to understand the effects of activation and inhibition of HIF-1 α on ovarian function and the changes of primordial follicle activation related factors.The third part: the ovarian granulosa cell line COV434 was used to study the changes of ovarian granulosa cell status and factors related to primordial follicle activation in different hypoxia time(12 hours,24 hours,36 hours,48 hours).Then,with hypoxia for 36 hours,2ME and DMOG were used to inhibit / activate HIF-1 α under hypoxia to explore the changes of primordial follicle activation related factors in granulosa cells induced by down-regulation /up-regulation of HIF-1-α.Finally,the HIF-1 α gene of lentivirus knock-down granulosa cell line was used to understand the effects of low expression of HIF-1α on granulosa cell cycle,apoptosis and primordial follicle activation related factors.Result In the first part,ectopic transplantation of ovarian tissue will cause different degrees of follicular depletion,the proportion of primordial follicles decreased,and the consumption of primordial follicles was the least in subcapsular transplantation.The time of recovery of estrus in subcapsular group was 5.97 ±0.81 days,and the duration of first estrus was 1.83 ±0.75 days,which was less than that in subcutaneous group of neck and intramuscular group of back.On the 3rd day after operation,AMH decreased to the lowest in the three groups of ovarian transplantation group,and then increased slowly;in the renal subcapsular transplantation group and cervical subcutaneous transplantation group,AMH reached the highest value on the 28 th day after operation,and then decreased.On the day of sampling,the weight of the recovered ovarian mass in the three groups was lower than that before transplantation,and the high total follicle density count in the subcapsular transplantation group was higher than that in the other two groups by HE staining,and the primitive follicle density count was lower than that in the sham operation group but higher than that in the other two groups,and the abnormal rate of follicle morphology was the lowest.In the second part,both whole ovary kidney subcapsule transplantation and ovarian block kidney subcapsule transplantation could restore estrus in estrus inhibition rats ahead of time,but the duration of first estrus in whole ovary transplantation group was 4.16 ±1.66 days,which was longer than that in ovary block transplantation group(2.33 ±0.51 days).In the dynamic monitoring of postoperative AMH,the AMH value of the whole ovary subcapsular transplantation group was similar to that of the ovarian block transplantation group,but the degree was more serious than that of the ovarian mass subcapsular transplantation group,and the peak value of AMH was 0.891 ±0.234ng/ml.In the comparison of ovarian weight,follicular density count and follicular morphology,the whole ovary renal capsule transplantation group was less than the ovarian mass renal capsule transplantation group.Although the use of HIF-1α agonist DMOG after whole ovary renal subcapsular transplantation could not prematurely restore the estrous cycle,the duration of estrus was 2.32 ±0.21 d,compared with 4.98 ±0.62 d in the control group,the difference was statistically significant.The rising trend of AMH in 2ME group was slower than that in DMOG group and control group.The density of total follicles and primordial follicles in DMOG group was higher than that in control group and 2ME group.The expression of HIF-1 α protein and gene in ovarian tissue increased in control group and DMOG group,compared with 2ME group,the difference was statistically significant,while the expression of primordial follicle activation related proteins BMP15,PCNA and GDF9 decreased.In the third part,different hypoxia time inhibited the growth of COV434 cells in different degrees,and aggravated with the increase of time,and the expression of HIF-1 α protein increased with the increase of hypoxia time,but its expression did not increase with the increase of hypoxia time.The mRNA and protein expression of VEGF and primordial follicle activating factor KitL and PCNA increased with the increase of hypoxia time,while the inhibitor of primordial follicle activation FOXL2 decreased with the increase of hypoxia time.2ME,an inhibitor of HIF-1α,decreased the expression of HIF-1 α in granulosa cells under hypoxia,resulting in a decrease in the expression of FOXL2 and an increase in the expression of KitL and PCNA.In contrast,HIF-1 α agonist DMOG could stabilize the expression of HIF-1 α under hypoxia,so the expression of FOXL2 increased and the expression of KitL and PCNA decreased.After the use of lentivirus to knock down HIF-1 α,the S phase cells in the cell cycle increased,the apoptosis rate decreased,the expression of KitL and PCNA increased,and FOXL2 decreased.Conclusion Ischemia and hypoxia after ovarian transplantation lead to over-activation of primordial follicles,which leads to massive depletion of follicles.The degree of primordial follicle activation increases with the aggravation of ischemia and hypoxia,and its mechanism may be that HIF-1 αmediates the change of primordial follicle activation inhibitor in granulosa cells and oocytes.The use of HIF-1 α agonist can improve primordial follicle activation,reduce follicle consumption and prolong ovarian function during ovarian transplantation.