A Preliminary Study Of ApoC3 Expression In Oocyte And The Regulatory Mechanism In Polycystic Ovary Syndrome

BackgroundPolycystic ovary syndrome(PCOS)is a reproductive endocrine and metabolic disease characterized by hyperandrogenemia,persistent anovulation or oligo-ovulation,and polycystic ovary changes,which is the main cause of infertility and spontaneous abortion.The incidence of PCOS is up to 6-20% in women of reproductive age.The etiology of PCOS is complex and related to genetic,immunological,environmental,psychological and other factors,but the specific mechanism is uncertain.Ovulation disorder is considered as an important pathophysiology underlying PCOS.Ovary is the site of egg production,the abnormal local regulation of ovary make an essential effect on the occurrence and development of PCOS,which could result in ovulatory dysfunction and oocyte formation disorder.The high expression of apolipoprotein C3(ApoC3)in the serum of patients with PCOS was found through proteomics technology in our previous study.ObjectiveThe aim of this study was to further investigate the expression of ApoC3 in the ovarian tissues of PCOS,and discuss the role of ApoC3 in PCOS ovulation disorder and its regulatory mechanism.Methods1.A total of twenty-four patients undergoing laparoscopic surgery in the obstetrics and gynecology department of Guangdong Women and Children hospital were collected and divided into twelve in the PCOS group and twelve in the control group.Follicular fluid samples was obtained from women who undergoing assisted reproductive technology in reproduction center of Guangdong Women and Children hospital,and divided into fourty cases in the PCOS group and thirteen cases in the control group.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of ApoC3 in follicular fluid of patients with PCOS.2.PCOS mouse animal model was induced by dehydroepiandrosterone for 3 weeks and divided into control group and PCOS group(18 mice in each group).On the 10 th day of modeling,vaginal secretions were preformed on vaginal cytological smear.Serum levels of ApoC3 and sex hormones in both groups were detected weekly by ELISA,and the correlation between ApoC3 and sex hormone was analyzed.Haematoxylin-eosin staining(HE)was used to observe the morphological changes of ovary.The expression of ApoC3 in ovarian tissue of PCOS mouse was experimented by western blot(WB)and real-time reverse transcription polymerase chain reaction(RT-PCR).3.Morphological changes of ovarian tissue in healthy postnatal(P)mice on 7th(P7),14th(P14),21st(P21),28th(P28),35th(P35),and 42nd(P42)were observed,and dynamic changes of ApoC3 in mouse plasma and ovaries on P7-P42 were detected by ELISA and IHC.4.RNA sequencing was used to analyze the differences in transcriptome levels of ovarian tissue between the PCOS mouse and control mouse.Genome single-cell RNA sequencing was preformed to obtained different cell populations.Results1.Compared with the control group,ApoC3 expression of ovarian tissue and follicular fluid in PCOS patients was increased(P<0.05).2.DHEA-induced PCOS mice were successfully simulated the endocrine and metabolic characteristics of PCOS.The result of WB and RT-PCR showed that compared with control,the expression of ApoC3 in ovarian tissue of PCOS mice were increased after the second week of modeling(P<0.05).Both IHC and IF staining indicated that ApoC3 was located in the cytoplasm of mouse oocytes,and ApoC3 in oocytes of PCOS mouse increased apparently compared to control group as disease progression(P<0.05).3.ApoC3 in plasma of PCOS mouse were positively correlated with luteinizing hormone(LH),testosterone(T)and anti-mullerian hormone(AMH).4.after observing the variation of ApoC3 in ovarian tissue of healthy mouse from P7-P42,we can see a weakly positive expression of ApoC3 on P7.ApoC3 was gradually upregulated in oocytes,and reaching a peak on P21 with age,and then declining.Correlation analysis showed that the physiological rise of ApoC3 in plasma was proportional to the levels of LH and AMH.5.RNA sequencing for ovarian tissue of PCOS mouse and single-cell sequencing of ovarian tissue from PCOS patients: the progression of PCOS is strictly related to lipid metabolic process,cellular response totumor necrosis factor,necroptosis and c GMP-PKG signaling pathway.Single-cell sequencing: Seven kinds of cell populations were displayed by UMAP visualization analysis,including granulosa cells,oocytes,luteum cells,endothelial cells,theca cells,stromal cells,and immune cells.Conclusions1.ApoC3 is overexpressed in ovarian tissues and follicular fluid of patients with PCOS.2.ApoC3 is located in oocyte of PCOS mouse and likely related to progression of disease and ovulation disorders.3.ApoC3 might associate with oocyte maturation disorders in mouse ovary.4.Oocyte development disorder in PCOS may be closely rcorrelated to lipid metabolic process,cellular response totumor necrosis factor,necroptosis and c GMP-PKG signaling pathway.