Identification And Biological Function Analysis Of Microrna Related To Ovarian Development Of Yellow River Carp(Cyprinus Carpio)

Fish is a large number of vertebrates,and an important source of protein for human beings.It is a common phenomenon that there is a difference in growth rate between the two sexes.The research on the reproductive mode of fish is of great significance in fish culture.It is important to study the molecular mechanism of the differentiation and development of fish ovary in fisheries and to understand the mechanism of biological sex determination.The types of sex determination and differentiation in fish is diversity,which are influenced by genetic and environmental factors.MicroRNAs(miRNAs)are small regulatory RNAs that regulate the stability and translation of cognate mRNAs at the post-transcriptional level.Growing evidence has demonstrated that miRNAs participate in the regulation of many biological processes related to sex determination and gonadal development in reproductive processes such as germ cell formation,gametes formation,fertilization and embryo development.Yellow River carp is an important and widespread fresh water cultured fish.Its growth has obvious gender dimorphism,females grows much faster than males.There are few studies on the regulatory mechanism of miRNA during the differentiation and development of carp ovary.In this study,the effects of environmental endocrine disruptors(ATR and LET)on gonadal differentiation of Yellow River carp were analyzed.By analyzing the expression changes of sex-related genes after ATR and LET treatment,the molecular mechanism affecting ovarian differentiation was analyzed.The expression of miRNAs in five stages of ovary development of Yellow River carp was identified by Illumina Solexa sequencing,the differentially expressed mi RNAs were identified in five stages of ovarian development,the target genes of miRNA were also predicted,and the function of mi RNA was analyzed by KEGG pathway and GO enrichment analysis.The effects of environmental endocrine disruptors(ATR and LET)on the expression of miRNA during gonadal differentiation of Yellow River carp were analyzed.The molecular mechanism of miRNA involvement or influence on ovarian differentiation under the interference of exogenous hormones was further analyzed through analyzing the effects of ATR and LET on the expression of miRNA in two critical stages of development.And the last,eight high expression and differentially expressed miRNAs were selected,the possible regulatory functions of these eight miRNAs were analyzed which may provide basis to reveal the regulation mechanism of miRNA in the process of ovarian differentiation and development of Yellow River carp.The main results are as follows: 1.Analyzed the effects of environmental endocrine disruptors(ATR and LET)on gonadal differentiation.The results showed that ATR and LET could not only affect the expression of aromatase gene,but also the expression of upstream genes such as Foxl2 and Dax,ultimately affect the differentiation and development of gonad.The effects of different concentrations of ATR and LET on the sex ratio and the expression of gene-related genes in juvenile Yellow River carp were observed.The results showed that(1)continuous exposure to LET and high concentration of LET can promote male-biased sex ratio,and high concentration of ATR can promote female-biased sex ration.(2)histological results showed that LET could inhibit oocyte growth in female and promote the development of testes in males,the results of ATR are the opposite.(3)Long-term and high concentrations of LET treatment could down-regulate the expression of female-biased genes such as Cyp19 A,Cyp19B,Er?,Dax and Foxl2,and up-regulate the expression of male-biased genes such as Dmrt1 and Ar?.ATR had the opposite results.These findings suggest that continuous exposure to LET may perturb sex differentiation in carp,the molecular mechanism not only involve inhibition of cytochrome P450 aromatase activity,but also the changes of upstream genes in sex differentiation.2.Identification,expression and functional analysis of miRNA in five key stages of ovary development,we enriched the number of miRNA in carp,identified the miRNAs which play an important roles in the gonadal differentiation and ovarian development of the Yellow River carp,such as miR-101 b,miR-181 a and so on.The target genes of these miRNAs such as cyp19,Foxl2,Gsdf were found by target gene prediction,and the pathway these target genes involved in was found to be related to gonadal differentiation and ovarian development,such as TGF-? signaling pathway,Wnt signaling pathway.The length distributions of the high-quality reads was analyzed by high-throughput sequencing of which the vast majority were 22 nt in length,but miRNAs in the juvenile ovary library ranged from 18 to 29 nt,with a peak at 25–26 nt.These mi RNAs corresponded to Piwi-interacting RNAs(piRNAs).Totals of 517,946,942,989 and 737 conserved miRNAs were identified from the five libraries(primordial gonad,juvenile ovary,and adult ovary,and neurula and complete yolk sac absorption stage embryos)respectively.145,139,143,143 and 145 existing carp miRNAs were identified respectively.3,278,3,257,3,188,4,480 and 3,192 novel miRNAs were obtained.We also analyzed the specially expressed,co-expressed and differentially expressedmiRNAs in the five developmental stages.Target-gene prediction showed that the predicted target genes included genes such as cyp19?foxl2?ATM?gsdf?dmrt?smad4 and so on which have been shown to be key factors in early ovary differentiation.Kyoto Encyclopedia of Genes and Genomes(KEGG)functional annotation identified 239 annotated signaling pathways,including pathways involved in reproductive biology,including transforming growth factor-?(TGF-?)signaling,Wnt signaling,mitogenactivated protein kinase(MAPK)signaling,p53 signaling and so on.The results demonstrated that these miRNAs are closely related to ovarian differentiation and development,which may be involved in the regulation of gonadal differentiation and early differentiation of gonad in Yellow River Carp.3.Analyzed the effects of environment endocrine disturbance(ATR and LET)on miRNA expression during the course of sex differentiation in the Yellow River carp.The differentially expressed miRNAs at critical stages of gonadal development under ATR and LET treatments were identified.The targets of these miRNAs,such as cyp19,sf1 and dmrt1 were identified by target gene prediction.The pathway involved in these target genes is related to gonadal differentiation and ovarian development,such as TGF-? signaling pathway and Wnt signaling pathway.Ovary of two critical stage of development were exposed to ATR and LET to identify miRNA expression,differentially expressed miRNAs,predict targets as well as KEGG analyse.In primordial gonads,ATR and LET treatment resulted in a large number of changes in the expression of miRNAs.ATR and LET exposure not only affected the number of identified miRNAs,but also affected the expression level of miRNAs.The effects of ATR and LET treatment for 8 h and 24 h on the expression of miRNAs in juvenile testis and ovaries were also different.Target-gene prediction showed that the predicted target genes included genes such as foxl2,stat1,sf1,dmrt1,gsdf,smad3,smad4,sox9 and ATM which have been shown to be key factors in gonad differentiation.Kyoto Encyclopedia of Genes and Genomes(KEGG)functional annotation identified 288 annotated signaling pathways,including pathways involved in reproductive biology,including transforming growth factor-?(TGF-?)signaling,Wnt signaling,mitogenactivated protein kinase(MAPK)signaling,p53 signaling and so on.We speculate that the up and down-regulated expressed miRNAs after treatment with ATR and LET plays an important role in the gonadal differentiation and development of Yellow River carp.The possible molecular mechanism is to regulate the genes and signaling pathways which involved in ovary differentiation,such as the steroid bio-synthesis pathway,by altering the expression of miRNA in the primordial gonads.4.Analyzed the function of eight highly and differentially expressed miRNAs during ovarian differentiation and development of Yellow River carp,and verified the relationships between the eight miRNAs and their corresponding target genes.Based on the miRNA expression profile,eight highly and differentially expressed miRNAs were selected which predicted targets were genes related to sex determination.RT-PCR was performed to detect the expression level of the miRNAs and its corresponding targets.The seven miRNAs were shown to be negatively correlated with target genes mRNA expression in different tissues of juvenile carp.Dual luciferase assay confirmed the direct binding of the miRNAs to target genes mRNA 3'UTR.Cultured juvenile carp ovaries transfected with the seven miRNA mimics exhibited decreased target genes expression in protein.To our knownlege,this is the first time that miRNA-target pairs that has been reported to regulate sex differentiation and gonad development in Yellow River carp,and further our understanding of the regulation.In conclusion,we analyzed the species,quantity and structural characteristics of miRNA and its possible functions in five key stages of ovarian differentiation of Yellow River carp,and specific expression was found in each stage.Target gene analysis and pathway analysis showed that these mi RNAs were involved in ovarian development and differentiation.The results showed that they could significantly inhibit the expression of target genes and protein level.LET and ATR could significantly change the ratio of sex differentiation of Yellow River carp.LET and ATR exposure significantly changed the expression level of some mi RNA.Except cyp19,the targets genes of these miRNAs are mainly concentrated in genes and signaling pathways which related to sex determination and gonadal differentiation.The results suggest that the mechanism of the effects of exogenous hormone analogues such as LET on gonadal differentiation is not only to change the expression pattern of cyp19 and its corresponding miRNA,but also to influence the early stage of gonadal differentiation gene and its corresponding miRNA expression pattern.These conclusions are important to reveal the function of miRNA in the process of ovarian differentiation and the molecular mechanism of exogenous effects on ovarian differentiation.