The Role And Mechanism Of IGFBP-4 In Ovarian Aging

Along with medicine progress and improvement of human life span,population aging and problems caused by aging has become a social concerned issue.As one of the earliest aging organs,ovarian aging is related to the occurrence of many diseases.As women age,follicular atresia leads to the loss of oocytes.Follicular atresia is not only an important reason for the reduction of ovarian reserve,but also the mechanism of ovarian aging.IGFBP-4 is a kind of insulin-like growth factor binding protein with the smallest molecular weight.In rat ovary,IGFBP-4 is specifically expressed in granulosa cells of atretic follicles.In addition,in human ovaries,IGFBP-4 is highly expressed in the follicular fluid of androgen dominated follicles with growth arrest or atresia.In contrast,IGFBP-4 is not detected in the follicular fluid of estrogen dominated growing follicles.Those correlation findings of IGFBP-4 and atresia suggest that IGFBP-4 may play a role in follicular development,dominant follicular selection and follicular atresia.However,there is a lack of causal relationship between IGFBP-4 and follicular atresia,and a lack of genetic evidence in animal models.In order to verify the role of IGFBP-4 in follicular atresia and ovarian aging,we used mice and human granulosa cell as the main research model to explore the expression of IGFBP-4 in vivo,functional regulation in vitro and physiological function in vivo.Firstly,we constructed single-cell acquisition strategy and analyzed the sequencing data of ovarian single-cell transcriptome in cynomolgus monkey whose genetic and physiological characteristics is similar to humans.We found that compared with the young group,the expression of IGFBP-4 gene increased significantly in the old group,suggesting that IGFBP-4 is related to ovarian aging;In order to verify the expression of IGFBP-4 in ovary,we constructed Igfbp-4-HA labeled mice.With HA antibody staining,cell morphological analysis and TUNEL staining,we found that IGFBP-4 was specifically expressed in granulosa cells of atretic follicles,but not in healthy growing follicles.In addition,in the same atretic follicle,the number of IGFBP-4 positive granulosa cells was positively correlated with the number of apoptotic granulosa cells;To further explore the function of IGFBP-4 in granulosa cells,we knocked down IGFBP-4 genes in granulosa cell line by sh RNA and lentivirus,and found the promotion of the granulosa cell proliferation;Subsequently,we established a method to purify IGFBP-4 protein.IGFBP-4 protein was added to the culture medium of human granulosa cells.The results showed that IGFBP-4could inhibit the proliferation of granulosa cells;To verify the physiological function of IGFBP-4 in vivo,we constructed the Amhr2-Cre;Igfbp-4 ^fl/fl mouse model of targeted knockout of IGFBP-4 in ovarian granulosa cells.Compared with the control group,in the ovary of the adult（8W）knockout mice,the number of atretic follicles decreased,ovulation and corpus luteum increased,suggesting that the conditional knockout of IGFBP-4 in granulosa cells improved the ovarian function of adult mice;Compared with the control group,the atresia follicles of knockout middling aging mice（12M）decreased,and the number of ovulation and corpus luteum increased,suggesting that the conditional knockout of IGFBP-4 in granulosa cells improved the ovarian function of middling aging mice and delayed ovarian aging;To verify the effect of IGFBP-4 on the reproductive ability of mice,we constructed mating cages for knockout mice and control mice respectively,and recorded the time and number of offspring.Compared with the control group,the total number of offspring in the knockout group increased by 78.6%,and the reproductive lifespan was significantly prolonged（11.5M vs 15M）,which was equivalent to 8 years in human age.The reproductive ability and ovarian function of mice were significantly improved.The conditional knockout of IGFBP-4 in granulosa cells delayed the ovarian aging.In conclusion,IGFBP-4 plays an important role in ovarian aging.This effect may be achieved by IGFBP-4 regulating the proliferation and function of granulosa cell,and then influencing follicular atresia.IGFBP-4 may become a new target to explore women’s reproductive lifespan,slow down ovarian aging and prolong women’s reproductive life.