Study On The Characteristic And Function Of Human Cumulus Cells Subpopulation

Infertility constitutes a significant challenge for the contemporary reproductive medicine,with the global prevalence of 15%.In our country,the prevalence rate is increasing year by year,which has seriously affected the physical and mental health of the women of the reproductive-age and is prone to a series of medical and social problems.The causes of female infertility include ovulatory disorders,ovulated oocyte defects,abnormal fertilization,luteal phase deficiency,and so on.Ovulated oocyte defects and ovulatory disorders are the most common cause of female infertility in women.Ovarian follicles are central to female reproductive system.Granulosa cell and oocyte are important cells of ovarian follicle.Bidirectional granulosa cell-oocyte signaling plays an important role in determining an oocyte’s developmental fate.Traditionally,During growth and maturation of the ovarian follicle,granulosa cells divide and differentiate into a multilayer of Mural Granulosa Cells(MGCs)and a cluster of Cumulus Cells(CCs).The cumulus cells wrap around the oocytes and have extensive communication with the oocytes,with different effects at different stages of folliculogenesis.During follicle development,the level of Expression of Anti-mu¨llerian hormone(AMH)in the cumulus cells increases by reducing the growth of follicle-stimulating hormone(FSH)sensitivity plays a role in inhibiting the collection of the original follicles and regulating the development of the follicles.During ovulation,oocytes secrete growth differentiation factor 9(GDF9),which plays a role in regulating ovulation by promoting the up-regulation of PTGS2 expression in cumulus cells.In addition,Polycystic ovarian syndrome(PCOS)is a complex endocrine condition characterized by oligo/anovulation,high androgen levels,and polycystic ovaries.Studies have shown that the dysfunction of cumulus cells is an important cause of ovulation disorders in PCOS patients,but the link and specific regulatory mechanism of their participation are still unclear.This study attempts to explore the molecular expression characteristics of ovulation cells in normal follicles and the regulatory mechanism of key genes in the ovulation process and provides potential targets for the diagnosis and treatment of ovulation disorders diseases.First,we performed Single-cell RNA sequencing(scRNA-seq)on freshly collected GC samples from two healthy women.We identified three reproducible clusters of cells visualized on the cell layouts.Combined the documents and reports on the GC transcriptomes before and after using recombinant human Chorionic Gonadotropin(rhCG)to trigger ovulating.It is interesting to find that hCG upregulated genes,including CD24,prostaglandin synthases(AKR1C1,PLA2G4 A,PTGES,and PTGS2),and prostaglandin transporters(SLCO2A1 and ABCC4),were highly expressed in one GC cluster.Considering the specific overexpression of CD24 in this GC subpopulation,we named it CD24(+)GCs.To further explore the in vivo regulation of granulosa cellular CD24,AKR1C1,PLA2G4 A,PTGES,PTGS2,SLCO2A1,and ABCC4 expression,we used the pregnant mare serum gonadotropin(PMSG)-primed/hCG-triggered immature mouse superovulation model.Experimental results showed that mouse cumulus GC Cd24 a,Pla2g4a,Ptgs2,and Slco2a1 transcripts increased after the administration of hCG.In tumor-related studies,the researchers found that CD24 was associated with epithelial growth factor receptor(EGFR)to activate the EGFR-ERK1/2 pathway to promote tumor cell invasion.CD24 expression in cancer is related to tumor aggressiveness,especially cell invasion and cancer cell stemness.It is known that acute upregulation of the EGFR-ERK1/2 pathway is an essential component of the ovulatory cascade as it transmits the luteinizing hormone(LH)signal from the periphery of the follicle to the cumulus-oocyte complex.Therefore,we believe that CD24(+)GCs may play an important role in the follicles ovulation process.By applying co-immunoprecipitation assays,immunofluorescence,actinomycin protein translational inhibition and other methods,we proved that CD24 and EGFR are physically connected in GCs lines(COV434 and KGN),and CD24 protein can increase the stability of EGFR protein and the activation of downstream signal.By in vitro culture of the normal person cumulus cells,by using hCG,CD24 interference slow virus and MEK inhibitors,we found that CD24-EGFR-ERK1/2 signaling pathway is involved in the hCG-induced upregulation of prostaglandin synthase(ARK1C1,PTGS2,PTGES,and PLA2G4A)and prostaglandin transporter(SLCO2A1 and ABCC4)expression in GCs.Then,in the human ovary granule lines of low CD24 expression,we found that the up-regulation CD24 can promote EGFR nuclear import by applying nucleosome separation,immunofluorescence,Western blot,and so on.Finally,through flow cytometry analysis and Real-time PCR methods,we found that the fraction of CD24(+)GC subpopulation decreased significantly in PCOS patients compared to the control patients.We then examined the relative mRNA abundances of prostaglandin synthases and prostaglandin transporters using GCs collected from PCOS over the control patients who underwent IVF-ET.As shown in this study decreased mRNA abundances of CD24,PTGS2,SLCO2A1,PTGES,ARK1C1,PLA2G4 A,and ABCC4 were observed in GCs of PCOS patients compared with those of the control patients.Conclusion: applying scRNA-seq technique,we have analyzed hundreds of Cumulus cells in two healthy women,discovered that there is heterogeneity between cumulus cell and cumulus cell can be divided into three GCs subpopulations depending on the molecular expression characteristics.We discussed the role of CD24(+)GC characteristics and its signature molecule CD24 in the ovulation and we found that CD24 participated in the ovulation process by activating the EGFR-ERK1/2 pathway and increasing the expression of prostaglandin synthase(ARK1C1,PTGS2,PTGES and PLA2G4A)and prostaglandin transporter protein(SLCO2A1 and ABCC4).Finally,we found that the fraction of CD24(+)GC subpopulation decreased significantly in PCOS patients compared to the control patients.The expression of prostaglandin synthase and prostaglandin transporter protein decreased in GCs of PCOS patients compared with those of the control patients.We find that CD24 and its regulated signal pathways are involved in the normal ovulation process.The proportion of CD24(+)GC in cumulus cells may become a diagnostic indicator of ovulation disorder diseases and the CD24-EGFR-ERK1/2 pathway can be used as a potential intervention target for ovulation disorder diseases.