The Expression Of CTRP3 In The Ovary And Its Function On Ovarian Follicular Development

Polycystic ovarian syndrome(PCOS)is a common cause of female infertility.The highly complex heterogeneous phenotypes of PCOS consist of oligo-or amenorrhea,clinical or biochemical hyperandrogenism or insulin resistance,and polycystic appearance of the ovaries.Though the quiescent primordial follicles of PCOS patients can be activated into developmental stages,most of the growing follicles are getting atresia at the early stage of antral follicle and cannot develop into dominant follicles,which lead to formation of a lot of early antral follicles with compacting granulosa cell layer and the cavity of polycystic ovaries.C1q/TNF-related protein-3(CTRP3)is a novel adipokine with roles in multiple cellular processes,including metabolism regμlation,cell differentiated,cardiovascular protection and anti-inflammation.Recent data showed that serum and omental adipose tissues CTRP3 are lower in women with PCOS,and metformin treatment increases the serum CTRP3 level in these women and in omental adipose tissues explants,indicating that it may be a beneficial adipokine to regμlate the pathophysiology of PCOS.The abnormal follicular development of polycystic ovary is the main characterization of PCOS.Here we mainly study the expression of CTRP3 in the healthy ovary and DHEA-induced PCOS model mice ovary,and investigate the effect of CTRP3 on follicular development by in vitro ovarian explants culture and preantral follicle culture system and in vivo ovarian intrabursal injecton.RT-PCR and Western blot show that the m RNA and protein of CTRP3 are expressed in mouse ovarian tissues,and immunochemistry show that CTRP3 protein is detected in the oocyte and granulosa cells of growing follicles,and not detected inthe primordial follicles.The expression of CTRP3 protein reaches its maximal expression at antral follicular stage.The expression of CTRP3 protein is slightly detected at granulosa cells of corpus luteum,and not detected at theca cells.Immunofluorescence shows that CTRP3 also widely expressed in granulosa cells of human ovarian tissues.The expression of CTRP3 in ovary is in an age-dependent manner.Increased expression of CTRP3 transcripts in age-dependent manner also detects in the somatic cells obtained from mice during prepubertal,but litter changes in oocyte,and the expression level is higher detected in the granulosa cells compared with in the oocytes,and negligible in the theca cells.Increased expression of CTRP3 transcripts also detects during follicles development,reaching highest levels in preovulatory follicles.PMSG stimulates the expression of CTRP3 transcripts,and the maximal level of CTRP3 expression is detected after PMSG injection 48 h later,however,the expression of CTRP transcripts is decreased after treatment with hCG.After treatment with hCG,the expression of CTRP3 is suppressed to a very lower level.Administration of DHEA induced the formation of follicular cysts.The number of antral and preantral follicles is increased compared with vehicle control,while the number of corpus luteum is decreased.Vaginal smears show DHEA-treated mice persist in diestrus and no ovulation happen.The estrogen level is decreased in DHEA-treated mice,while no significant difference is detected in progesterone level between DHEA-treated and vehicle mice.The steroid metabolism related genes,such as CYP11A1,Star,HSD-3β and CYP17A1 are down-regμlated in DHEA-treated mice.These data indicate that the PCOS mouse model is successful constructed by DHEA.qRT-PCR analyses show the transcript of CTRP3 is down-regμlated in PCOS model mouse ovaries.Consistently,CTRP3 protein is decreased in in the compact granulosa cells of cystic follicles in DHEA-treated mice by immunoflurence analysis.High concentration of androgen induces the expression of CTRP3 in primary granuosa cells,and metformin,pioglitazone,and IGF-1 upregrulate the expression ofCTRP3 in granulosa cells.In vitro ovarian explants and follicle cultures show recombinant CTRP3 protein accelerates FSH-induced growth of ovarian explants from prepubertal mice containing preantral follicles and isolated preantral follicles.The data of follicle counting of serial sections of ovarian explants show that CTRP3 mainly promote the preantral follicular development.The transcript of Cyclin D2,are increased in primary granulosa cells and ovarian explants after treatment with CTRP3.In cultured ovarian explants,combined CTRP3 and FSH treatment increases Akt and mTOR phosphorylation compared with FSH alone,while there are no change of the phosphorylation of FOXO3 a and ERK1/2 between two groups,suggesting that Akt/mTOR signal pathway involving in CTRP3-regμlated follicle development.In vivo,ovarian intrabursal administration of CTRP3 protein has no obvious effect on ovarian weight increase.However,intrabursal administration of the CTRP3 antibody produces a delay of follicle development induced by PMSG.While,CTRP3 antibody previously adsorbed with the CTRP3 protein decreased the inhibition effect of CTRP3 antibody on follicle development.Conclusion: Taken together,CTRP3 is expressed in oocyte and granulosa cells,especially in granulosa cells of antral follicles.The expression of CTRP3 is decreased in the ovaries of DHEA-treated PCOS mouse model.CTRP3 accelerates the effect of FSH on ovarian explants and preantral follicles growth by activation of Akt/mTOR signal pathway,which could promote the expression Cyclin D2,which may involve in promote the proliferation of granulosa cell.This study suggests that CTRP3 may involve in the development of PCOS by regμlating the preantral and antral follicle development.