Effect Of Lipid Metabolism Related Molecules Col6a5 And Gpr1 On Hyperandrogenic Mice And Its Mechanism

Polycystic ovarian syndrome(PCOS)has a serious impact on women's quality of life due to its high incidence and diverse clinical phenotypes.Hyperandrogenism is now considered to be an important pathological factor in PCOS.About 70% of patients with PCOS have hyperandrogenism,and hyperandrogen can cause metabolic abnormalities,ovarian dysfunction,and these symptoms can be alleviated by improving hyperandrogenism.Abnormal lipid metabolism also has a high incidence in PCOS patients,and it can affect PCOS-related symptoms such as abnormal hormone levels and insulin resistance.However,it is yet to be explored whether correcting lipid metabolism abnormalities can alleviate PCOS-related symptoms caused by hyperandrogen.In this study,mice were used as models to carry out the following three experiments:The first part: explore the effects of hyperandrogen on fat and ovarian lipid metabolism.The 25-day-old C57BL/6 mice were randomly divided into two groups.The mice in the experimental group were subcutaneously implanted with a silicone tube containing dihydrotestosterone(DHT),and sustained hyperandrogen stimulation for 60 days.During the period,measured weight and made vaginal smear.On day 60,serum and tissue samples were collected from a part of mice in each group,and the remaining mice in each group were removed from the silicone tube for another 30 days.Tissue morphology was observed,gene expressions were examined,and the transcriptome was analyzed.The results showed that:(1)Continuous exposure to DHT induced cell hypertrophy and lipid accumulation in fat and ovarian tissue,and cell hypertrophy was associated with lipid accumulation;(2)Abnormal expression of lipid metabolismrelated genes in two tissues was organizationally specific;(3)When DHT was removed to relieve ovarian symptoms,the adipose tissue symptoms was not improved during the observation period of this experiment.The second part: explore the regulatory targets of hyperandrogen induced fat and ovarian lipid metabolism abnormalities.In the experiment,the DHT-induced gene with high expression of fat and ovary simultaneously was first determined,and one of them was selected as a target gene.Ovarian stromal cells and 3T3-L1 adipocytes were treated with DHT,cell fat content was measured,lipid components was analyzed,and gene expressions were analyzed.The expression of target genes in ovarian stromal cells and 3T3-L1 adipocytes was inhibited by siRNA and Crisper-cas9 system,respectively,and the effect on DHT effect was observed.Finally,hypertrophy was observed by injecting lentivirus into the tail vein of DHT-induced hyperandrogenic mice to inhibit target gene expression.The results showed that:(1)Col6a5 was highly expressed in DHT-treated mice fat tissues and ovaries;(2)DHT induced fat accumulation of ovarian stromal cells and 3T3-L1 adipocytes;(3)Cholesterol was the main components of lipids accumulated DHT-induced in ovarian stromal cells.(4)Col6a5 expression inhibition attenuated DHT-induced lipid accumulation and cell hypertrophy.The third part: explore the effects of hyperandrogens on follicular development and its regulatory targets.First,we investigated lipid-related molecules that affect ovarian function,and selected on of them as a target molecule.Primary granulosa cells and luteinized granulosa cells were isolated and cultured,and cells were treated with specific antagonist peptides and antibodies to observe cell differentiation and hormone synthesis when the target molecule activity was inhibited.Superovulation mice were injected with a target molecular antagonist peptide to observe serum hormone levels and follicular development.The mouse was continuously administered subcutaneously with dehydroepiandrosterone(DHEA)in neutral oil for 20 days.Observe the effect of DHEA on follicular development,hormone synthesis,and the effect of target gene deletion on DHEA effect.The results showed that:(1)Gpr1 as a lipid metabolismrelated molecule could affect ovarian activity;(2)The suppression of Gpr1 activity promoted ovarian granulosa cell luteinization and hormone synthesis;(3)DHEA sustained exposure increased the number of developmental follicles,Gpr1 gene deletion alleviated this effect of DHEA.In summary,the following conclusions can be drawn:(1)Androgen has a significant effect on lipid metabolism and follicular development,ovarian dysfunction in hyperandrogenic mice depends on the hyperandrogen-stimulation.(2)DHT continues exposure induces lipid accumulation and cell hypertrophy in ovarian stromal cells andadipocytes through different lipid metabolism pathways.(3)Col6a5 expression suppression alleviates abnormal lipid metabolism in ovary and adipose tissue through cholesterol conversion and lipid metabolism,respectively.(4)Continuous stimulation of DHEA induces an increase in developmental follicles.(5)Gpr1 inhibition can attenuate DHEA-induced ovarian dysfunction by affecting granulosa cell activity,raising serum estradiol levels.