The Research On The Age-related Changes In The Mitochondria Of Human Granulosa Cells And The Regulation Mechanism

Background and objectives:In the modern society,more and more women have to delay marriage,childbearing and their first pregnancy for professional or financial reasons.The maternal aging causes reduced fertility,and the clinical pregnancy rate is still very low for women using assisted reproductive technology(ART)in their late 30s or 40.The reasons for fertility impairment are mainly the decreased follicle number and the poor oocyte quality.The dialogue between the oocytes and granulosa cells(GCs)is necessary for the oocytes development,and mitochondria,as the powerhouse of cells,produce the energy required for cellular functions in GCs.In this research,we are aimed to establish a stable method for preparing GCs for culture,and then study the age-related changes in the mitochondria of GCs and the regulation mechanism during the progress.Methods:1.GCs were isolated using density gradient centrifugation method from discarded follicular fluid in the ART process.Cell viability was estimated by trypan blue staining,and cell purity was tested by flow cytometry.The cellar ultrastructure of GCs was observed by transmission electron microscopy(TEM).Immuohistochemical staining and immunofluorescence assay were used to detect the expression of follicle stimulating hormone receptor.Cell proliferation rate was detected by CCK-8 assay.2.The mitochondrial ultrastructure of GCs was observed by TEM,and real-time quantitative polymerase chain reaction was applied to quantify the mitochondrial DNA(mtDNA)copy number,4977-bp deleted DNA and mRNA expression of mitochondrial ATP synthases ATP5A1,ATP51 and HIBADH.MMP was detected by flow cytometry and fluorescence microscopy,respectively.Reactive oxygen species(ROS)was tested by flow cytometry.A luminometer was used to measure the ATP levels and western blot to analyze the oxidative phosphorylation(OXPHOS)complex.3.Western blot was used to analyze the expression of HIBADH protein in GCs and the HIBADH or OXPHOS complex protein in KGN cells when HIBADH gene was over-expressed or silenced in KGN cells,respectively.Automatic microplate reader was applied to test the ROS,ATP and MMP.Results:1.Cell viability of isolated GCs was above 99%,and contamination rate of white blood cells was below 6.73%.Positive expression of FSHR in GCs was above 99%,and GCs reached the maximum level in proliferating stage in the 3rd day after cultured in vitro.2.In the young group,mitochondria were mostly round or oval,with a few intact parallel tubular-vesicular cristae and homogenous matrix density,while elongated mitochondria were mainly observed in the old group,which had numerous cristae and more high-density matrix particles.Abnormal mitochondria were more common in aging women.mtDNA relative copy number was positively correlated with maternal age and we found no one with 4977-bp deleted mitochondria.MMP in the old group was significantly lower than the young group.Intracellular ROS levels between the groups did not differ significantly.The intracellular ATP level in the young group was 1.75-fold higher than that of the advanced-age group.The protein expression of ATP5A1,as one of five proteins of OXPHOS,decreased with aging.ATP5A1,HIBADH mRNA expression was negatively correlated with aging,and HIBADH mRNA expression was correlated with ATP5A1,ATP5I mRNA expression.3.In GCs,expression of HIBADH protein decreased with aging.In KGN cells,cell proliferation,ATP and expression of OXPHOS complex protein-IV MTCO1,V-ATP5A1 increased when HIBADH gene was over-expressed,and decreased when HIBADH gene was silenced.Conclusions:1.An efficient and stable method is established to isolate,culture and identify GCs.2.GCs dysfunction with aging is mainly linked to impaired mitochondrial function,especially OXPHOS function.3.HIBADH gene plays an important role for the regulation of mitochondrial OXPHOS function in granulosa cells.