Impacts Of Age On Mitochondrial Function Of Ovarian Granulosa Cells

The ovary is the center of female fertility regulation and one of the organs most sensitive to senescence.Older age is related to a decrease in the number and quality of follicles in the ovary,but the mechanism is not completely clear,which may be related to the acceleration of follicular atresia and the shrinkage of the original follicular pool.Decreased Ovarian Reserve(DOR)refers to the decrease in the number of recruitable follicles remaining in the ovary and the quality of the oocytes,which leads to insufficient or absent secretion of sex hormones and a decline in female fertility,which can further develop into ovarian failure.Clinically,DOR is characterized by a decrease in anti-Müllerian hormone(AMH)and sinus follicle number(AFC),and an increase in basic follicle stimulating hormone(bFSH).With age,the occurrence and development of DOR obviously accelerated.The follicle is the basic functional unit of the ovary,consisting of oocytes and surrounding granulosa cells.Some reports suggest that the developmental competence of oocyte is highly dependent on the bidirectional dialogue between the granulosa cells and the oocyte.Mitochondria are the main places for cells to carry out aerobic respiration.Ovarian granulosa cells are rich in mitochondria,which can metabolize glucose in the cytoplasm to pyruvate.Pyruvate is transported into oocytes to produce ATP,which maintains oocyte development.With age,the mitochondrial morphology in granulosa cells changes,the mitochondrial membrane potential decreases,the level of ATP synthesis significantly decreases,and the apoptosis of granulosa cells increases,which may affect the function of oocytes or cause changes in ovarian reserve.In this study,human follicle granulosa cells were collected to clarify the correlation between granulosa cells and oocytes by examining the changes of mitochondria-related functions and tracking the outcome of assisted pregnancy.Secondly,44?48 weeks old mice were selected as the experimental model to explore the effects of the changes of mitochondrial dynein and autophagy function in ovarian granulosa cells on apoptosis of granulosa cells and oocytes,and providing new measures for improving the quality of oocytes in elderly pregnant women.The first part is the human follicle granulosa cell experiment,which mainly includes:1.Detection of people in the age-appropriate group(<38 years old),the advanced age group(38-41 years old)and the ultra-old age group(?42 years old)by qRT-PCT Changes in mRNA levels of mitochondrial-related factors in follicular granulosa cells,the results show that the expression of P62,PINK and PARKIN were not significantly different between the age-appropriate group and the 38?41 year old age group;but there were significant differences between the age-appropriate group and the ultra-old age group(P<0.05).2.Comparative analysis of fertilization,embryogenesis and clinical pregnancy of functional oocytes of different ages,the statistical results show that the oocyte collection rate and fertilization rate of the three groups are significantly different(P<0.05),but there is no significant difference in the number of embryogenesis,excellent embryos,biochemical pregnancy and clinical pregnancy.The second part is the fertility experiment of aged mice,mainly including:1.Pair age-appropriate(6?8 weeks old)and older age(44?48 weeks old)female mice with age-appropriate male mice,record the number of litters to compare the fertility of mice,statistics show that the number of litters in older mice group decreased significantly.2.HE staining was used to observe ovarian histological changes and to count the number of follicles at all levels in two female mice group,the ovarian morphological structure of age-appropriate mice is complete and the follicles at all levels are well developed,however,the ovarian tissue fibrosis is increased in the older mice group,the follicles at all levels are dysplasia and the number is obviously reduced,the number of locked follicles is obviously increased.3.Observe the mitochondrial membrane potential and apoptosis of ovarian granulosa cells by immunofluorescence staining technique:compared with age-appropriate mice group,the ovarian granulosa cells of the older age group mostly showed green fluorescence and negligible red fluorescence,indicating that the mitochondrial membrane potential of the granulosa cells of the older age mice group decreased;The proportions of FITC~+and PI~+in granulosa cells of older mice group significantly increased,indicating increased apoptosis..4.We detected changes in mRNA and protein levels of mitochondrial motility-related proteins Drp1 and Opa1,autophagy labeling factor LC3 and P62 in two groups of granulosa cells by qRT-PCT,Western blotting,immunohistochemistry:the expression of Drp1,LC3 and P62 in the ovarian granulosa cells of older mice group significantly increased(P<0.01);the expression of Opa1 significantly increased in protein level(P<0.05)instead of mRNA level.5.The mRNA and protein levels of PINK and Parkin in ovarian granulosa cells were detected by qRT-PCT and Western blotting:The expression of PINK(P<0.001)and Parkin(P<0.05)in the ovarian granulosa cells of older mice group increased significantly.In summary,our study showed that the mitochondrial autophagy of the granulosa cells in the human super-old age group is impaired,and the oocytes have a bad outcome after fertilization,indicating that the impaired granusola cell function will affect the quality of the oocyte and reduce fertility.The results of mice experiment showed that the mitochondrial fission fusion of ovarian granulosa cells in older mice group was affected,the mitochondrial morphology was destroyed,at the same time,the level of mitochondrial autophagy increased;the reduced mitochondrial membrane potential will initiate the mitochondrial PINK1-Parkin autophagy pathway,which will increase the level of mitochondrial autophagy.The above changes may lead to mitochondrial dysfunction,leading to increased apoptosis of granulosa cells in elderly mice,which in turn accelerates the oocyte apoptosis in the follicles.This may be one of the reasons that the reduction of ovarian reserves leads to a decrease in fertility.