| Diversified sex determination mechanisms exist in vertebrates.Among them,temperature-dependent sex determination(TSD)mechanism is widespread in reptiles and fish,and is currently the most widely studied type of environmental sex determination(ESD)mechanism.Sexual fate was originally considered to be that the bipotential gonads are guided to sex-specific pathways after the gradual cascade of downstream molecules,leading the gonads to the eventual sexual fate.Subsequent studies have shown that sexual fate is not only determined by the competition of female/male antagonistic signaling pathways during embryonic period,but also needs to be actively maintained in adulthood to fight against the opposite sex.This suggests the plasticity of sexual fate.Sexual fate is no longer considered a static state of sex determination during embryonic,but a dynamic process affected by genetic or environmental factors throughout the life.In the wild,sex reversal occurs in fish,amphibians,and reptiles.As a unique freshwater alligator in China,the Chinese alligator do not have sex chromosomes like the other 22 crocodiles species remaining in the world.The sexes of Chinese alligator are completely determined by the incubation temperature of temperature sensitive period(TSP),and remain irreversibly fixed thereafter.How does TSD,as the only sex determination mechanism of crocodiles,maintain good stability in the long-term evolutionary process? In previous research,our research group has analyzed the molecular regulation mechanism of sex determination and differentiation during embryonic stage of Chinese alligator.In this study,we collected ovary/testis tissues of adult Chinese alligators in two seasons,but mainly analyzed the situation in summer.We combine m RNA sequencing,bisulfite sequencing and small RNA sequencing technology,from the three levels of gene expression potential,gene expression and post-transcriptional regulation,to reveal the epigenetic mechanism of sex maintenance and gonadal development in adult Chinese alligator.The main research contents are as follows:(1)At the transcriptome level,we analyze the differential expressed genes(DEGs)among the gonads of adult Chinese alligator.We identified 5,845 sex-biased DEGs(comprising 3,092 male-and 2,753 female-biased DEGs)in summer,when the alligators are actively breeding.Among them,genes involved in the oocyte meiosis pathway and steroid hormone biosynthesis pathways showed large-scale biased expression.We believe that these two pathways play a role in the female/male maintenance and gonadal development respectively in adult Chinese alligators.In addition,we have also constructed an antagonistic pathways for maintenance and gonadal development.(2)At the methylome level,the global DNA methylation pattern is completely different in the ovaries and testes of adult Chinese alligator.The methylation level of genes in testes is significantly higher than that in ovaries.Differentially methylated region(DMR)analysis also shows that among the annotable DMR and DMR-related genes,hyper-methylated DMR and hyper-methylated genes in the testis account for more than 90%,and most of the DMRs occur in genebodies.(3)Joint analysis of the methylome and transcriptome shows that genes with sex-biased DMRs were significantly more likely to be sex-differentially expressed.The methylation of promoter play a negative role in regulating gene expression to a certain extent.In the male-biased DEGs in testis of adult Chinese alligators,the relationship between the level of gene body methylation and the gene expression is unique: the hyper-methylation of the gene body enhances the expression level of male-biased DEGs.(4)At the level of small RNA transcriptome,we identified 813 mature miRNAs(comprising 130 conserved and 683 unique miRNAs)in Chinese alligator gonads.There are 134 differential expressed genes(DEmiRs),among which 95 are conserved miRNAs.Family analysis and target gene prediction of conserved DEmiRs are performed.We found that female-biased DEmiRs are mainly distributed in the mir-10,mir-8 and mir-23 families;male-biased DEmiRs are mainly distributed in the mir-17,mir-19,and mir-34 families.Moreover,members of a miRNA family always target on one or more genes.This study analyzes the genetic and epigenetic mechanism of sex maintenance and gonadal function development in adult Chinese alligators.It is an important supplyment to fully understand the sex determination and maintenance mechanism of Chinese alligator,and lay a theoretical foundation for the future scientific programs of Chinese alligator and the in-depth understanding of the vertebrate sex determination mechanism evolution. |
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