Study On The Expression And Function Of AQP7 And AQP9 In Granulose Cells And Oocytes Of Patients With PCOS

The incidence of polycystic ovary syndrome(PCOS)is 5% ~ 10% in women of childbearing age in China.It is a complex disease complicated with glucose metabolism disorder and reproductive dysfunction.At present,the pathogenesis of polycystic ovary syndrome is not clear.The study suggests that PCOS may be caused by the interaction of genetic variation and adverse environment,which is a complex polygene genetic disease,and its ovulation disorder is one of the problems in the field of reproduction.Although assisted reproductive technology is already a routine method of treating PCOS infertility,follicular dysplasia caused by glucose metabolism and endocrine disorders can often causes PCOS patients to undergo in vitro fertilization and embryo transfer(in vitro fertilization and embryo transfer,IVF-ET)during the process of egg maturity and poor quality of embryos,resulting in egg utilization and embryo planting rate decreased.And PCOS patients often appear menstrual disorders,infertility and obesity,etc.,and suffering from cardiovascular disease and type 2 diabetes incidence also increased significantly in the late,a serious threat to the patient’s physical and mental health.Therefore,it is a urgent problem to solve the problem of follicular dysplasia in PCOS patients,which is the hotspot in the field of reproduction.Aquaporins(AQPs)are a family of small molecule proteins present on the cell membrane that increase the permeability of water to the cell membrane and are important for maintaining the water balance inside and outside the cell.Researchers have found that 11 AQPs(AQP0-AQP10)are abundantly expressed in a variety of mammalian tissue cells and participate in a variety of reproductive processes such as spermatogenesis,follicular development,blastocyst formation,embryo implantation and so on.The role in the field of reproduction has been more and more attention.It includes three subfamilies: the first class of classical aquaporins: AQP0-2,AQP4-6 and AQP8,which are highly selective for water and are mainly responsible for the transport of water molecules;the second is integrated water channel,including AQP3,AQP7,AQP9 and AQP10,can transport water,urea and glycerol and some other small molecules,it is also known as glycerol-aquaporin.The third category is super aquaporins,including AQP11 and AQP12,and its structure and function is not clear.Early studies have shown that AQP1-4 mRNA expression is different in follicular granulocytes at different developmental stages.Recent studies have shown that only normal AQP1,7,9,11 and 12 mRNA expression in granulosa cells,and AQP7 mRNA expression level and fertilization rate was positively correlated,suggesting that AQP7 expression may be normal follicle formation and healthy oocyte markers.Tan Yajing and other scholars found that AQP7 in mice and normal oocytes have expression,and can improve the viability of vitrified frozen oocytes after resuscitation.The studies have shown that obese women have lower levels of AQP7 expression in obese women than in poor women,and mRNA levels are negatively correlated with BMI.It was also reported that the expression of AQP7 mRNA and protein in omental tissue of PCOS patients was significantly higher than that of control group,and it was positively correlated with fasting insulin level.Therefore,it was speculated that AQP7 overexpression in PCOS adipose tissue might be related to insulin sensitivity and maintenance of systemic glucose of the steady state.The above results suggest that AQP7 plays an important role in glycerol transport and energy metabolism.However,the expression of AQP7 in granulosa cells and oocytes in PCOS patients and their association with insulin has not been reported.Studies have shown that AQP9 is expressed in sinus follicles and granulosa cells during follicular development.AQP9 expression in PCOS patients is still controversial: There are studies reported AQP9 mRNA expression is detected in PCOS patients and control group of granulocytes.But there was no significant difference between the two groups;However,there are some scholars reported that AQP9 mRNA expression levels in PCOS Group was significantly lower than the normal group.In animal experiments: recent studies have found that AQP9 plays an important role in maintaining the homeostasis of follicular cells during vitrification and freezing of ovarian tissue.Some scholars have reported AQP3,8 and 9mRNA expression in mouse mature oocytes;It has also been reported that AQP9 mRNA disappeared gradually during rat oocyte maturation process.The above results suggest that AQP9 may play an important role in the process of oocyte maturation.However,the expression of AQP9 in granulosa cells and oocytes in PCOS patients and their relevance to insulin remains to be further confirmed.In summary,the study of the expression and function of AQP7 and AQP9 in PCOS patients with follicular development,not only contribute to the pathophysiology of PCOS pathophysiological research,but also for clinical diagnosis and treatment to provide new ideas.Purpose:In this study,Real-time qPCR and Western-Blot were used to detect the expression of AQP7 and AQP9 in granulosa cells of PCOS group and control group,to investigating their characteristic of expression in granulosa cells in each groups.The effects of AQP7 and AQP9 on oocyte maturation were investigated by detecting the localization of AQP7 and AQP9 in oocytes.To investigate the effect of insulin on the expression of AQP7 and AQP9 in granulosa cells by adding different concentrations of insulin and different treatment time to intervene in cultured human granulosa cells.Methods:1.The ovarian tissues were collected and some sections were taken by HE staining.Then observe and compare the ovaries structure of each group under microscope.The other parts of ovarian tissues were treated with immunohistochemical method to compare AQP7 and AQP9 distribution and integrated optical density(integrated option density,IOD).2.Grouping: divided into PCOS group and control group.The expression of AQP7 and AQP9 in granulosa cells and cumulus granulosa cells were detected by different methods.The expression of AQP7 and AQP9 gene was detected by Real-time qPCR.The expression of AQP7 and AQP9 protein was detected by Western-Blot technique.3.Immunofluorescence technique was used to detect the localization of AQP7 and AQP9 in oocytes of different periods,and to explore its role in oocyte development.4.To investigate the regulation of insulin on the expression of AQP7 and AQP9 in granulosa cells,we detect the expression of AQP7 and AQP9 gene and protein in the granulosa cells by adding different concentrations of insulin and different treatment time respectively.Results:1.The expression of AQP7 protein and mRNA in ovarian tissue and granulosa cells of PCOS group were significantly higher than those in control group(P <0.05).The AQP7 protein is localized to the cytoplasm and cell membrane of oocytes in various periods.2.The expression of AQP9 in ovarian tissue of PCOS group was significantly higher than that in control group(P <0.05).The expression of AQP9 in PCOS group was significantly lower than that in control group(P <0.05).The AQP9 protein is localized to the cytoplasm and cell membrane of oocytes in various periods.3.Compared with the control group,the expression of AQP7 and AQP9 mRNA and protein decreased with the increase of insulin concentration.The expression of insulin was the most significant when the concentration of insulin was 100nmol/L,the difference was statistically significant(P <0.05).4.The expression of AQP7 and AQP9 was the lowest at 12 h after treated with 100nmol/L insulin in normal cultured granulosa cells for 6 ~ 24h(P <0.05).Conclusion:1.AQP7 was highly expressed in ovarian tissue and granulosa cells of PCOS patients and expressed in oocytes.It suggested that AQP7 might be involved in the growth and development of follicles in PCOS patients.2.AQP9 is highly expressed in ovarian tissues of PCOS patients and is lower in granulosa cells,and is expressed in all stages of oocytes,suggesting that AQP9 may be involved in the growth and development of follicles in PCOS patients.3.The expression of AQP7 and AQP9 was negatively correlated with the concentration of AQP7 and AQP9 at 100nmol/L insulin for 12 h,which suggests that insulin may affect the follicular development by regulating the expression of AQP7 and AQP9.