The Influence Of Aging-induced Circadian Clock Disturbances On Granulosa Cell And Oocyte Quality And Its Related Mechanisms

The biological clock system regulates the physiology and behavior of the body to ensure that development,survival and reproduction activities adapting to the diurnal changes in the environment.The circadian clock system is composed of a multi-level regulatory network,including the peripheral circadian clock and the central circadian clock.Its self-regulating ability gradually declines with age,accompanied by the decline of the body’s metabolic homeostasis and changes in physiological behavior.The biological clock system plays an important role in female reproduction,including endocrine regulation and follicle development.Among them,the ovarian circadian clock is a peripheral circadian clock,which is regulated by neuroendocrine signals from the central circadian clock of the suprachiasmatic nucleus of the hypothalamus to maintain the function of the normal female reproductive system.Studies have shown that circadian rhythm disturbances are associated with increased risk of infertility and adverse pregnancy outcomes in elderly women.The decline of ovarian function and oocyte quality is one of the main reasons for the decline of fertility in elderly women,which is manifested by the dysfunction of granulosa cells and the progressive decline in the quality of oocytes.The effects of ovarian circadian clock on endocrine function of granulosa cells and oocyte quality and its mechanism are rarely studied.Therefore,based on our earlier discovery that advanced age leads to circadian rhythm disorder,the present study will explore how circadian rhythm disorder affects granulosa cell function and oocyte quality and its related mechanisms,and further investigate whether it can regulate ovarian biological clock to improve the endocrine function of granulosa cells and oocyte quality in elderly women to improve their pregnancy outcomes.Objective:In the present,we investigates the changes of ovarian circadian clock caused by age to further elucidate the effect of circadian clock disorder caused by advanced age on granulosa cells function and oocyte quality,and to conduct in-depth research on its possible molecular mechanism.Methods:1.Detect the expression and oscillation pattern of circadian clock genes in different samples by RT-q PCR.2.Through primary culture of granulosa cells in vitro,Elisa hormone assay,cell immunofluorescence staining,RNA-seq,WB and other techniques to detect the effect of rhythm gene Nr1d1 on the granulosa cell function and oocyte quality in female mice and the related mechanisms.Results:1.The luteinized granulosa cells of clinically elder and appropriate age women were collected.RT-q PCR results showed that the expression of rhythm genes BMAL1,CLOCK,PER1,and PER2 were all down-regulated in the elderly women group,while the expression of NR1D1 was significantly up-regulated.And abnormal age-related ovarian clock gene rhythm disorder was probably related to the oocyte quality.2.Collect follicular phase granulosa cells from age-appropriate and aged female mice.RT-q PCR results showed that the oscillation pattern of circadian clock genes in the aged mice group was disordered.The expression of rhythm genes Bmal1,Per1,and Rorαwas down-regulated,and Nr1d1 is significantly up-regulated.The results of luteinized granulosa cells were consistent.3.Treatment of mouse granulosa cells and cumulus oocyte complexes with the rhythm gene Nr1d1 agonist GSK4112,the results suggested that the up-regulation of Nr1d1 expression affected the endocrine function of granulosa cells,as shown in the following:the levels of E₂and P in the cell culture supernatant were increased,and the expression of luteinization markers like St AR,Cyp11a1 and Lhcgr were up-regulated which showed signs of luteinization.4.Premature luteinization of granulosa cells promoted the premature"closing"of the gap junction between the cumulus and oocyte,and significantly reduces the rate of polar bodies.It impaired the assembly of the oocyte spindle and chromosome arrangement and the proportion of oocytes with abnormal spindle shape and chromosome arrangement increased significantly.Mitochondrial dysfunction in oocytes after GSK4112 treatment was manifested as:significantly increased ROS level,decreased mitochondrial membrane potential,decreased mt DNA and ATP production,and intracytoplasmic mitochondria were mostly distributed in clusters and clumps,which seriously affected the quality of oocytes.5.The differentially expressed genes screened by RNA-seq data of mouse granulosa cells were used for enrichment analysis,and verified by RT-q PCR and Western Blot.The results showed that the mitochondrial biosynthesis gene Pgc1αand lipid transport related gene Slc27a6,Cpt1α,etc.in the GSK4112 treatment group were up-regulated at both the m RNA and protein levels.This result suggested that the up-regulation of Nr1d1 expression increased mitochondrial biosynthesis and lipid transport,thereby increasing the entry of sex hormone precursors cholesterol and lipids into the inner mitochondrial membrane,and leading to granulosa cell lipids overloaded,which promoted the luteinization of granulosa cells in advance.Meanwhile it activated the PTEN/PI3K/AKT signaling pathway,which further accelerated the differentiation of granulosa cells,leading to premature luteinization,thereby affecting the meiosis of oocytes and ultimately affecting the quality of oocytes.Conclusion:1.Advanced age leads to disorder of ovarian clock genes,which are manifested as down-regulation of rhythm genes BMAL1,CLOCK,PER1,and PER2,while the expression of transcriptional repressor NR1D1 is significantly up-regulated,and the oscillation amplitude of Nr1d1 in the granulosa cells of aged female mice is more variable.2.The up-regulation of the expression of the rhythm gene Nr1d1affects the endocrine function of granulosa cells,which is manifested as premature luteinization of granulosa cells,promoting the"closing"of the gap junction between the cumulus and the oocyte,leading to the blocking of oocyte meiosis and affecting the oocyte maturation,manifested by a marked decrease in polar body rate,the damage of oocyte spindle assembly and chromosome alignment,and mitochondrial dysfunction,severely affecting oocyte quality.3.The highly expressed rhythm gene Nr1d1 upregulates the expression of mitochondrial biosynthesis gene Pgc1αand lipid transport-related genes Slc27a6,Cpt1α,etc.in granulosa cells,which increase mitochondrial biosynthesis and lipid transport and lead to lipid overload in granulosa cells,inducing premature luteinization in granulosa cells.And by activating the PTEN/PI3K/AKT signaling pathway,it further accelerates the luteinization process of granulosa cells,which together results in premature luteinization,thereby hindering the meiotic of oocytes,and ultimately affecting the quality of oocytes.In conclusion,the high expression of the rhythm gene Nr1d1 caused by advanced age leads to lipid overload in granulosa cells by increasing mitochondrial biosynthesis and lipid transport,affecting the endocrine function,resulting in premature luteinization of granulosa cells.The changes in endocrine function of granulosa cells lead to early closure of the gap junction between the cumulus cells and oocyte and further affects the meiosis of the oocyte,resulting in abnormal spindle assembly and chromosome arrangement disorder,which ultimately affects the quality of the oocyte.This mechanism may be one of the important reasons for the increased aneuploidy rate of oocyte in older women.