| The Reeves' pond turtle(Mauremys reevesii)is a typical species with temperature-dependent sex determination(TSD).The offspring are all females based on the fertilized egg hatching temperature 32 °C;The offspring are all males based on26 °C;The offspring are females and male 1:1 based on 29 °C.Although the TSD mechanism has been studied for decades,it is necessary for us to think about whether TSD species have controversial sex chromosome differentiation,and how external signals affect the expression of sex determination related genes.In this study,M.reevesii was used as the research object,the sex determination system and epigenetic studies on early development and adult function maintenance of gonad by the morphological characteristics of the embryo gonad,RNA interference,epigenetic analysis,high-throughput sequencing and other molecular biology techniques.The main contents are as follows:1.The fertilized eggs hatching experiments were carried out to elucidate the morphological developmental characteristics of M.reevesii embryos based on male hatching temperature(MPT,26 ?),female producing temperature(FPT,32 ?)and Pivotal temperature(PvT,29 ?).The comparative results showed that the embryonic gonad development began at the stage 15,and there was no significant difference in the development of the male and female gonads from the stage 15 to the stage 21.According to previous studies,the period of stage 15 to stage 21 was determined to be the temperature sensitive stage of gonad development.In the stage23,testis and ovary showed the different developmental features: testis were oval;ovary were slender and were associated with the differentiation of the fallopian tubes.It showed that the gonads had been completely differentiated.2.The sex determination system of M.reevesii was studied by the molecular gender identification method.The male and female M.reevesii individuals were obtained from four-aged turtles hatched under PvT in our laboratory.The differencein male and female copy number of 18 genes related to sex chromosomes(ADARB2,ATP2A2,ENOX2,LPAR4,GPATCH8,CCDC92,MED13 L,SP4,PEBP1,GAD2,ITGB1,ARMC4,DMRT1,SOX9,AMH,CYP19A1,WT1 and SF1)were compared by real-time PCR(qPCR).Pelodiscus sinensis(ZZ/ZW)as the control group.The results showed that the copy number of 17 genes in male were twice than in female.The 17 genes were located on the M.reevesii Z chromosome,and one gene was located on the autosome chromosome.The sex-determining related genes AMH and WT1 of PvT offspring were verified by fluorescence in situ hybridization(FISH),and there were two hybridization signals in male individuals;only one hybrid signal was present in female individuals.M.reevesii was first confirmed to be ZZ/ZW sex determination system.M.reevesii was a typical species with TSD mechanism,the sex ratio of the offspring was still regulated by temperature,it is speculated that although the turtle had sex chromosome differentiation,the sex determination is mainly influenced by epigenetics,and the sex determination mechanism of M.reevesii may be GSD+TE type.3.Effects of DNA methylation at the temperature-sensitive stage of gonadal development on the expression of sex-determining genes.DNA methylation levels of CYP19A1,FOXL2,DMRT1,SOX9 and AMH proximal promoter regions of in stage17,stage 19 and stage 21 embryo gonads were compared by bisulfite modified sequencing between MPT and FPT.The mRNA expression levels of above five sex-related genes between MPT and FPT in three stages were also detected by qPCR.Correlation analysis showed that the mRNA expression level of sex-related genes was highly negatively correlated with the CpG methylation status of the promoter region(CYP19A1: r =-0.98,P < 0.001;FOXL2: r =-0.95,P < 0.01;DMRT1: r =-0.87,P <0.001;SOX9: r =-0.82,P < 0.01;AMH: r =-0.94,P < 0.001).The expression levels of CYP19A1 and FOXL2 under FPT were significantly higher than those of MPT(P <0.0001),and the expression levels of DMRT1,SOX9 and AMH under MPT were significantly higher than those of FPT(P < 0.0001).In order to screen candidate enzymes,the expression levels of the DNA methylase genes(DNMT1,DNMT3 A,DNMT3B,TET1,TET2 and TET3)and histone demethylase genes(KDM5B,KDM1 A,KDM6B,KDM8,KDM3 B,NO66,JMJD6 and PHF2)were detected by qPCR in stage17,stage 19 and stage 21 embryo gonads under MPT,PvT and FPT.Correlation with above 14 genes mRNA expression and 5 sex-related genes expression showedthat the expression levels of NO66 and KDM6 B decreased significantly with rising temperature(P < 0.0001),and the expression levels of NO66 and KDM6 B were positively correlated with the expression levels of male sex-related genes DMRT1,SOX9 and AMH(NO66-DMRT1: r = 0.96,P < 0.001;NO66-SOX9: r = 0.87,P <0.001;NO66-AMH: r = 0.9,P < 0.001;KDM6B-DMRT1: r = 0.94,P < 0.001;KDM6B-SOX9: r = 0.85,P < 0.01;KDM6B-AMH: r = 0.88,P < 0.001).The results suggested that the histone demethylase genes KDM6 B and NO66 were regulated by temperature,and their histone demethylation could promote male gonad development.Given that the role of the KDM6 B gene had been elucidated,the role of NO66 gene was worthy of further study.4.Effect of histone demethylase gene NO66 on gonadal development of M.reevesii embryos.NO66 mRNA specific shRNA was used to transfect the stage 14 embryo by the lentivirus vector,and constructed the NO66 expression knockdown model.Low expression of NO66 can cause the feminization of MPT embryos.Compared with the control group,the methylation degree of the promoter region of DMRT1 gene was significantly up-regulated in the stage 21 transfected embryo(P <0.001),while the expression level was significantly down-regulated(P < 0.001),and the expression levels of SOX9 and AMH were also significantly down-regulated(P <0.001),however,the expression levels of CYP19A1 and FOXL2 were significantly up-regulated(P < 0.001),and the expression of KDM6 B was not significantly reduced.The results suggested that the demethylase gene NO66 could also promote the development of male gonads by regulating the expression of the male sex related gene DMRT1,and speculated that its role was either downstream of KDM6 B or both.Due to the high mortality of NO66 and KDM6 B double-knockdown model embryos,no effective data had been obtained so far,and further experiments such as immunoprecipitation are needed to verify the correlation between NO66 and KDM6 B.5.High-throughput transcriptome sequencing compared mRNAs and miRNAs in testicular and ovarian tissues.Differentially expressed genes(DEGs)were screened by comparing transcriptomics analysis.KEGG analyzed the pathways related to gonad maintenance and speculated that the sex-determining gene molecular signal cascade regulatory network.The miRNAs and differential expression characteristics of M.reevesii were also annotated and analyzed,and the mapping to the corresponding target genes were predicted.Based on the miRNAs and target genesregulatory networks,we focused on the enrichment analysis of sex-related gene targets,and initially constructed the key molecular mechanisms and regulatory networks for the gametogenesis and functional maintenance of adult turtles.This study firstly confirmed that M.reevesii with TSD mechanism had ZZ/ZW sex determination system.It was speculated that the sex determination mechanism may be GSD+TE type.The influence of epigenetics on early embryonic gonadal development and adult gonad function maintenance were explored.The role of gonad function maintenance;Using genetic manipulation techniques,the promotion of NO66 on the development of gonads in MPT embryos was demonstrated.This study provided the direct evidence for epigenetic regulation of TSD and had important scientific implications for the further study of the molecular mechanisms of TSD. |
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