The Role And Mechanism Of ER-phagy Receptor CCPG1 In The Development Of Mouse Ovarian Follicle

Objective In this study,the endoplasmic reticulum stress and the unfolded protein response inducer tunicamycin was used to establish a model of premature ovarian failure in mice,and the role and mechanism of ER-phagy receptor CCPG1 in follicular development and atresia was investigated,as well as the interactions between ER stress,endoplasmic reticulum unfolded protein response and ER-phagy receptor CCPG1 were explored.Methods The ovaries of 21-day-old ICR mice were harvested 24 hours after a single intraperitoneal injection of tunicamycin.To investigate whether the mouse model of premature ovarian failure was successfully established,the ovarian histology was detected by HE,and transmission electron microscopy,and the autophagic and apoptotic proteins were detected by western blot.The methods of western blot,IF and flow cytometry were used to investigate the effect of tunicamycin on ovarian granulosa cells in vitro,the changes of endoplasmic reticulum stress,unfolded protein response transcription factors,autophagy and apoptosis related proteins were detected by western blot;RNA interference,lentiviral infection,CHIP-seq and Co-IP were used to investigate the molecular mechanism of CCPG1,ER stress,unfolded protein response and autophagy,and to screen the target proteins by proteomic sequencing and western blot,to further explore the mechanism of apoptosis induced by CCPG1 abnormal expression.ResultsIn vivo experiment:1.Results suggested that follicular atresia and premature ovarian failure were caused by5μg/g of tunicamycin treated mice.2.The results of transmission electron microscopy（TEM）showed that the follicles of all stages of the mouse ovaries were obviously atresia,and a large number of granulosa cells died in the follicles.3.Western blot showed that expression of ER stress and unfolded protein response transcription factors,proteins related to autophagy and apoptosis were significantly increased in ovarian tissues.These data were supported by the results from physiological aging ovaries.In vitro experiments:1.Western blot,IF and flow cytometry results showed that ER stress and UPR^ER were increased,and the cells were nucleated,at the same time,the expression of ER-phagy increased and apoptosis occurred.2.After treatment with tunicamycin,the expression of ATF4 protein was down-regulated by RNAi.Western blot and IF results showed that the expression of ATF4,CCPG1 and MAP1LC3 protein were correlated,ATF4 regulates LC3A gene expression after nucleation,and Co-IP results show that LC3A and CCPG1 interact with each other.3.The expression of CCPG1 protein was altered by RNAi and lentivirus infection.Western blot and IF results showed that both ER stress and autophagy-related protein expression were affected,and transcription factors were translocated into the nucleus,apoptosis was increased and protein aggregation bodies showed that misfolded proteins failed to degrade and accumulated in the cells.4.After downregulation of CCPG1 protein by RNAi,the results of proteomic sequencing showed that the cells not only underwent apoptosis but also necrotic apoptosis.STAT1 and STAT3 were screened out to participate in the necrotic apoptosis of CCPG1-regulated cells.At the same time,the expression of apoptosis-related protein was significantly increased after the expression of CCPG1.5.The expression of CCPG1 protein was down-regulated by RNAi.STAT1 and STAT3showed synergistic effects on the regulation of necroptosis by STAT1 and STAT3.The results of immunofluorescence showed that STAT1 and STAT3 could not translocated into nucleus,CO-IP was performed using antibodies to STAT1 and RIPK1,respectively,and the results showed that STAT1 and CCPG1 had direct interactions,and RIPK1 and STAT1 and STAT3had direct interactions.6.After tunicamycin treatment,the expression of necrotic apoptosis-related protein was significantly decreased,and the expression of necrotic apoptosis-related protein was significantly increased after endoplasmic reticulum stress inhibitor treatment.Conclusion1.Endoplasmic reticulum stress-induced apoptosis involved in follicular atresia,and endoplasmic reticulum stress links to ER-phagy through ATF4-MAP1LC3A-CCPG1signaling pathway,ER-phagy,which is activated during ER stress,can inhibit ER stress-induced follicular atresia.2.CCPG1 is a key protein in the maintenance of follicular development and both increased and decreased expression of CCPG1 will lead to abnormal cellular protein homeostasis,which in turn causes necroptosis,and necroptosis is regulated by the STAT1 and STAT3.