Mechanism Study Of Protein C Receptor In Follicular Development And Polycystic Ovary Syndrome

The number of infertile patients is increasing every year,making infertility the third critical disease after cancer and cardiovascular disease.The World Health Organization(WHO)estimates that between 48 million couples and 186 million individuals live with infertility globally.According to the European Society of Human Reproduction and Embryology(ESHRE),one in six couples worldwide experience some form of infertility problem at least once during their reproductive lifetime.With the rapid development of assisted reproductive technology(ART),we are able to manage infertility caused by tubal factors and egg-sperm binding problems to some extent.However,in the case of infertility caused by oogenesis,effective intervention is very limited.Therefore,it's high time we should explore the key factors in oogenesis,which can provide mature healthy oocytes and assist with this kind of infertility.Follicles are the basic functional units of the ovary,comprised of an immature oocyte and surrounding somatic cells(granulosa cells(GCs)and theca cells).Reproduction requires controlled development of the oocyte and the somatic cells.Once a follicle is activated,the single layer of flattened GCs first proliferate and differentiate into cuboidal GCs,awakening the dormant oocyte.In the growing follicles,GCs undergo at least 10 clonal divisions to support follicle growth and oocyte maturation,reaching a total amount of several thousand cells at the mature antral stage.At the preovulatory stage,the well-differentiated GCs also exert a variety of specialized functions to ensure successful ovulation,such as secreting large amounts of hormones and growth factors,adapting hormone receptivity to the endocrine milieu,nursing the oocyte,and communicating with the enclosed oocyte and the surrounding thecal cells.After ovulation,GCs quit cell cycle,cease division,and finally differentiate into steroidogenesis luteal cells,which can sustain embryo implantation and development during early pregnancy.Protein C receptor(PROCR),first identified and separated on the surface of endothelial cells as the receptor of protein C,plays a role in anti-coagulation by transforming its ligand into activated protein C(a PC),which can inactivate factor Va and factor VIIIa.PROCR has been reported to mark stem cells in several adult tissues,such as the mammary gland,vascular endothelial cells,and hematopoietic system.Our latest study has illustrated its role as a progenitor cell marker in ovarian surface epithelium(OSE): PROCR+ OSE cells proliferate rapidly upon OSE rupture and contribute to the ovulatory rupture repair.As a single-pass transmembrane protein,PROCR is reported to activate several important intracellular signaling,such as PI3K/AKT signaling and ERK signaling,resulting in increased proliferation,migration,and survival in different tissues.However,the role of PROCR in follicular development has not been studied and deserves exploring.In the first part of this study,we explored the expression of PROCR in the ovary as well as the function of PROCR+ GCs.RNA in situ hybridization of wild-type mouse ovary section and immunofluorescence of Procr-rt TA;Tet O-H2B-GFP mouse ovary section were utilized to identify the cellular localization and expression dynamics of PROCR in the ovary.Here,we found that PROCR was localized to granulosa cells of all follicular developmental stages: primordial,primary,secondary,antral,and preovulatory follicles.To better understand the function of PROCR+ GCs,we performed Ed U staining and direct cell counting in isolated primary mouse GCs.The results indicated that PROCR+ GCs showed more active proliferation than PROCR-GCs.Having observed the different proliferation ability between PROCR+ and PROCR-mouse GCs,we next focused on genes which have been reported to be associated with steroidogenesis.Previous studies have found that genes encoding steroidogenic enzymes(such as Cyp19a1)and genes encoding hormone receptors(such as Esr,Fshr,and Lhcgr)were upregulated in GCs during folliculogenesis,whereas an important transcription factor Foxl2 was decreased in GCs along follicle development.QPCR analysis revealed that PROCR+ GCs expressed lower levels of Cyp19a1,Esr2,Fshr,Lhcgr,but higher level of Foxl2 than PROCR-GCs.In order to confirm the gene expression findings,the culture medium of primary GCs was collected to determine the alterations of hormone secretion.Consistently,ELISA results showed that levels of E2 and progesterone were significantly increased in the culture medium of PROCR-GCs compared with PROCR+ GCs,which indicates a higher differentiation degree of PROCR-GCs compared with PROCR+ GCs.In the second part,we explored the role of PROCR+ GCs in folliculogenesis.Procr-Cre ERT2;Rosa26-m Tm G and Procr-Cre;Rosa26-m Tm G mice were used in lineage tracing experiment.PROCR+ GCs and their progenies were labeled with m GFP expression in these transgenic mice,which can reflect the contribution of PROCR+ GCs during follicular development in vivo.By tracing at different timing,we found that PROCR+ GCs had the proliferative ability in different developmental stages(E13.5,P1,8w).Also,the lineage tracing results showed that GFP+ GCs were detected in all follicular developmental stages: primordial,primary,secondary,antral,and preovulatory follicles.Of note,the portion of GFP+ GCs was significantly increased as follicles developed.These results suggest that PROCR+ GCs contribute to granulosa cell expansion during folliculogenesis and might be essential in this process.In the third part,targeted ablation of PROCR+ cells was achieved using Procr-Cre ERT2;Rosa26-DTA mice to verify the physiologic role of PROCR+ granulosa cells in folliculogenesis.By administration of tamoxifen(TAM)at 3 weeks,diphtheria toxin(DT)was conditionally expressed in PROCR+ cells and we examined the ovary function one month later.The whole-mount imaging and section imaging revealed that Procr-Cre ERT2;Rosa26-DTA ovaries manifested polycystic ovarian morphology.Compared with the control(Rosa26-DTA),there were more immature follicles(secondary follicle,preantral follicle)and fewer mature follicles(antral follicle,preovulatory follicle)in Procr-Cre ERT2;Rosa26-DTA ovaries.Notably,there was no corpus luteum in Procr-Cre ERT2;Rosa26-DTA ovaries,indicating anovulation.ELISA results showed that plasma testosterone level was significantly increased after ablation of PROCR+ cells,whilst Cyp19a1,which encodes for the enzyme P450 aromatase,was shown to be downregulated by q PCR.Also,the Procr-Cre ERT2;Rosa26-DTA mice trended toward more super-ovulated oocytes compare with controls,with granulosa cells sticky to its surface.However,these oocytes failed to fertilize in the following procedure of in vitro fertilization(IVF),indicating an immature status.In addition,the plasma anti-M llerian hormone(AMH)and LH/FSH ratio were tested by ELISA,exhibiting a significant increase.These are all similar to the clinical features of polycystic ovary syndrome(PCOS).In addition,we collected granulosa cells of the IVF patients,and analyzed PROCR+ GCs in PCOS patients and the control.Consistently,FACS results showed that the proportion of PROCR+ GC was significantly declined in PCOS patients,indicating disruption of PROCR+ GC may result in PCOS.By targeted ablation of PROCR+ cells,we discover the vital role of PROCR+ GCs in folliculogenesis and the pathogenesis of PCOS.Also,we create novel animal model,which closely mimics both reproductive and metabolic features of PCOS.In the last part,to gain molecular insights of the proliferative ability of PROCR+ GC,PROCR was knockdown by sh RNA in KGN cells(a human ovarian granulosa cell-like tumor cell line).Knockdown of PROCR inhibited the proliferation as shown by decreased Ed U staining as well as reduction in direct cell counting.Furthermore,knockdown of PROCR up-regulated the expression of GC differentiation-associated genes,such as CYP19A1,ESR2,FSHR,and LHCGR.Consistently,ELISA results showed that levels of E2 and progesterone were significantly increased in the culture medium of PROCR-knockdown KGN cells.These observations suggest that knockdown of PROCR suppresses proliferation but stimulates differentiation of GCs.To understand the molecular mechanisms underlying,we next screened expression of key molecules related to GC proliferation and folliculogenesis.Western blot analyses indicated that knockdown of PROCR in KGN cells compromised phosphorylation of AKT and ERK,whereas addition of a PC,the agonist of PROCR,enhanced the activation of AKT and ERK signaling pathway.Together,PROCR controls PI3K/AKT and ERK signaling pathways to regulate GC and follicle development.To summarize,we reveal for the first time that PROCR plays a key role in ovarian follicle development via promoting proliferation and suppressing differentiation of granulosa cells.Ablation of PROCR+ cells disrupts folliculogenesis and leads to a polycystic ovary syndrome phenotype.Our findings not only shed new light on the role of PROCR in follicular development,but also extend knowledge of potential genes associated with PCOS in human patients.Meanwhile,we create novel PCOS animal model,which covers the shortage of pervious tools.