Identification Of Gender-biased MiRNAs In Medaka Brains And Targeting Of Related MiRNAs To Cyp19a

Fish is the lower vertebrates that most widely distributed on the Earth.Different from the higher vertebrates,sex determination of fish is not only influenced by genetic factors,but also by environment(hormone,temperature,salinity,oxygen,etc.)and hormone,so the mechanism of sex determination is complex.However,there is a conserved factor in the control of ovarian differentiation,which is the gonadal aromatase.Meanwhile,brain of teleost is characterized by highly aromatase activity,which is related to the m RNA expression of cyp19a1 b.Moreover,the brain of teleost shows a degree of sexual plasticity throughout their lifetime,so we speculate that there is some connection between brain and gonadal differentiation.Mi RNAs participate in the reproductive process by regulating the expression of target genes.Therefore,it is possible that miRNAs regulate cyp19 a gene between the gonadal and brain differentiation to affect the expression of aromatase.Medaka(Oryzias latipes)is considered as a good model for studying the gonadal development of vertebrates due to its small size(2-3 cm),short sexual maturation cycle(3 months),and sex plasticity.Furthermore,medaka has an XY sex chromosome,and it is the first species that is defined as a sex determining gene(dmy).In addition,other research in medaka provide the basis for this study,for example,the database of gonadbiased miRNAs has been established and published complete genome.Besides,dazl,dnd,piwi,vasa have been shown important roles on germ cell migration,at the same time,transgenic fish that relevant to gonad differentiation have been established,including cyp19 a transgenic medaka.In conclusion,these advantages of medaka are used to explore the development of gonads in vertebrates.In this study,we first established the miRNAs database of different expression between male and female brains.The target gene prediction and GO analysis were performed for miRNAs.Twelve miRNAs were subsequently selected for q PCR verification in 1-and 3-month male and female brains.Cyp19 a,an aromatase gene with expression bias in both brain and ovary,was selected as the target gene.Then,medaka were treated with the aromatase inhibitor exemestane to detect the changes of cyp19 a and miRNAs.And miR-181b-5p,miR-375-3p,miR-26 and miR-10b-3-3p,which have significantly different expression levels in sex-biased brain,were selected as target miRNAs to investigate whether these miRNAs and cyp19 a have a regulatory relationship.Database revealed 56 miRNAs with significantly different expression in male and female brains,q PCR showed that miRNAs were significantly different in adult brains,but not in young medaka.RT-PCR showed that cyp19a1 a was highly expressed in the ovary of the medaka and weakly in the testis,while no obvious signal was detected in other tissues,the expression of cyp19a1 b was high in brain and ovary,weak in eyes,kidney and testis,which is consistent with the sequence alignment and phylogenetic tree analysis that fish have formed two aromatase genes,cyp19a1a(ovarian aromatase)and cyp19a1b(brain aromatase).In the stage of embryo,cyp19a1 a and cyp19a1 b were detected until 7 days after fertilization.In addition,after 21 days of treatment with aromatase inhibitors,the phenotype were altered;the result of HE found that the gonad structure was still ovary,but there were obvious degeneration and cavitation compared with the wild type gonad,q PCR detected the expression of miRNAs was increased compared with the normal group.The expression of female-related genes decreased,while the expression of genes related to male increased.In situ hybridization showed that cyp19a1 a was mainly expressed in early oocytes and granulosa cells in wildtype,but cyp19a1 a was detected mainly in early oocytes in the EM-treated group.The difference in cyp19a1 b between the two groups is mainly reflected in the lower expression level in the treatment group.Luciferase revealed that miR-375 targets cyp19a1 a and cyp19a1 b,while miR-181b-5p and miR-10b-3-3p and miR-26 have no direct regulation.The results of this study supplemented the miRNAs database of brain in medaka,indicating that aromatase inhibitor EM can affect the expression of cyp19 a and miRNAs,thus affecting the maintenance of ovary,meanwhile,providing reference for the study of brain and gender maintenance and differentiation.