Effects Of Testosterone On PCOS Susceptible Gene Expression And Reproductivity In Zebrafish

Chapter ?.Create zebrafish PCOS modelBACKGROUND:Polycystic ovary syndrome(PCOS)is a gynecological endocrine and metabolic abnormality disease that occurs in women of childbearing age,is characterized by hyperandrogenism(HA),oligo-ovulation(OA),and polycystic ovaries(PCO).However,traditional animal models such as mice,rats,rabbits,and rhesus monkeys are not suitable for PCOS endocrine-related studies due to the long reproductive cycle and insensitivity to sex hormones.Maybe these animals are more similar to human ovaries in structure and function,but are also more expensive and time-consuming,so they are not suitable for basic research.Every existing animal model have advantages and disadvantages,can't fully simulate the characteristics of PCOS,so it is crucial to select an animal model according to the experimental design.Zebrafish has high genetic homology with humans,and with short sexual maturation cycle,in vitro fertilization and development,transparent embryos.It has been widely used in the study of human nervous system,immune system and reproductive system diseases.Now using zebrafish as animal model for research of PCOS is relatively deficient,but zebrafish were selected to study genetics and fertility research by many scholars,making zebrafish a good animal model in reproductive disease.OBJECTIVE:Zebrafish PCOS model was constructed in order to examine the mechanism of PCOS suscetibility genes and reproductive behavior related to PCOS.MATERIALS AND METHODS:By immersing adult zebrafish(5-10 months old)with testosterone,determinating testosterone concentration by high performance liquid chromatography(HPLC)in ovary and testis,then immobilizing,dehydrating.embedding and HE staining,morphological changes of zebrafish ovaries were observed.Using ovulation rate,ovulation number,hatching rate and GSI,we also detected the changes of reproduction between control and testosterone group.RESULTS:Ovarian testosterone levels increased in a dose-dependent with different treatment concentrations.Compared with the control group,testosterone treatment group showed lower rate of mature follicles(P<0.01),higher rate in immature follicles(P<0.01)and ovarian follicles in testosterone treatment group were decreased(P<0.01).After treatment with testosterone,the ovulation rate,total ovulation number(P<0.01)and hatching rate(P<0.05)was decreased,and the GSI was significantly increased(P<0.01).CONCLUSION:In this study,by using testosterone treatment,we constructed a zebrafish PCOS model and successfully simulated the characteristics of PCOS:hyperandrogen,oligo-ovulation,polycystic ovaries change and other fertility decline problems,for example,the quality of embryos decreased and ovarian fertility potential increased.Which partially accorded with the characteristics of PCOS,so we believed that we have successfully established a PCOS fertility-related model.Chapter ?.Expression of PCOS susceptible geneBACKGROUND:The etiology of PCOS is complicated,including genetic factors,environmental factors,social-psychological factors,and lifestyle.Although the etiology of PCOS has not been fully elucidated,genetic is considered to be the main causes of PCOS.Through genome-wide association study(GWAS),more than ten PCOS gene susceptibility regions such as TOX3,LHCGR,FSHR,DENND1A,HMGA2,YAP1,INSR,SERBP1,RAB5B,SIRT1,THADA,ERBB3,SUMO1P1,SUOX were identified.We are interested in TOX3 and DENND1A.Studies have shown that both TOX3 and DENND1A are closely related to PCOS.However,due to the lethal effect of these two genes on mouse knockout embryos,no good knockout mice have been obtained so far.Therefore,it is difficult to further study the pathogenesis of TOX3 and DENND1A.Based on the high homology of zebrafish and human genes,and the strong vitality of zebrafish,this study focused on zebrafish,using zebrafish as an animal model for the detection of PCOS susceptibility genesOBJECTIVE:To detect the expression of susceptibility genes tox3 and dennd1a in wild zebrafish and PCOS model zebrafish,and to lay a foundation for the construction of gene knockout and overexpression zebrafish PCOS model.MATERIALS AND METHODS:Using reverse transcription-polymerase chain reaction(RT-PCR)and real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)techniques,we detected the gene expression in wild zebrafish and PCOS model zebrafish.We also determined the expression pattern of dennd1a and tox3 gene in zebrafish embryos using whole-mount in situ hybridization(WISH)assays.RESULTS:In embryos and juveniles,the expression level of dennd1a and tox3 increased along with age,and in adult animals,the expression was found in different organs.qRT-PCR and WISH showed that gene expression especially in the nervous system and male reproductive system.The expression of aennd1a and tox3 in PCOS model was decreased than that in normal control group in ovary(P<0.5),brain(P<0.5),eye(P<0.01).In male fish treated with testosterone,the aennd1a and tox3 mRNA expression was upregulated(P<0.5).CONCLUSION:The gene expression of dennd1a and tox3 played an important role in early embryonic development,especially in the central nervous and male reproductive system.The difference in expression between the model and the control group indicates that these two genes are related to PCOS hyperandrogenism.And the expression of aennd1a and tox3 mRNA were dimorphic.Chapter ?.PCOS model's sexuality and reproductivityBACKGROUND:The main concern of PCOS patients are fertility-related issues.Although current studies focus on the organic level of PCOS such as oligo-ovulation,no one is concerned about whether PCOS women also have infertility issues caused by psychological factors such as decreased sexual desire.In order to find out the reason of infertility,we designed mating-like behavior experiments.At present,the common animal models such as mice and rats are difficult to operate,time-consuming and costly in fertility-related experiments,so they didn't have advantages of fertility experiments.However,zebrafish have short sexual maturation cycle and in vitro fertilization,which are especially suitable for fertility experiments.Therefore,zebrafish were selected to study mating-like behavior.OBJECTIVE:To find whether the PCOS fertility problems is related to simply functional ovulation disorder or a decline in sexual interest.To evaluate the effects of PCOS zebrafish on reproductive behavior under different lifestyles(single female,mixed male and female).MATERIALS AND METHODS:Behavioral experiment:Constructing zebrafish PCOS model,video recording,analyzing zebrafish mating-like behavior,scoiring objectively,evaluating zebrafish sexual interest differences;After building the model,using different lifestyle treatment of single female or male and female mixed,testing the difference of fertility recovery between the two lifestyles of PCOS zebrafish.Fertility experiment:After treatment,the ovulation rate and hatching rate were counted,and the GSI level was calculated.RESULTS:Compared with the control group,the relative sexual ability of females in PCOS group was decreased,which was manifested in swimming speed and relative position with males(P<0.01).After 3,14,30 and 60 days of different lifestyle treatments,the relative sexual response of single-female fish was higher than that of male and female grouped fish,statistically significant differences were considered when P<0.05.About fertility:There was no significant difference in hatch rate between different lifestyle treatments.However,with the prolongation of different treatment,the mating rate of single female PCOS fish was gradually higher than mixed PCOS fish(P<0.05).CONCLUSION:PCOS fish's infertility is related not only to the oligo-ovulation,but also to the decrease of relative sexuality.Moreover,single state fish showed better sexual response recovered than in social state fish.The mating rate of single PCOS fish was higher than mixed fish as time pass by,which indicated that the harmful effects of male-female mixed on PCOS fish,also indicated that the increase of androgen in mixed PCOS fish was really not conducive to the recovery of PCOS..