Sex-biased MiRNAs In Chicken Gonad And Their Potential Roles For Early Embryonic Gonadal Development

Sexual dimorphism is pervasive throughout many species from insects,fish and birds to mammals,and most of the differences in embryos and adults between the sexes arise and persist via the regulation of sets of molecules(including protein-coding genes and non-coding RNAs)in a sex-specific developmental manner.These differences result from two processes during embryonic development,sexual determination and differentiation.The theory of sex determination with multiple parallel primary pathways initiated by genes(Sry)and other factors encoded by the sex chromosomes has been recognized in mammals,but still remains unclear in chicken.Several studies indicate that sexual dimorphic microRNAs(miRNAs)in chicken gonads are likely to have important roles in sexual development,but a more global understanding of the roles of miRNAs in sexual differentiation is still needed.To this end,we constructed the expression profiles of miRNAs in 2 groups(male and female chicken gonads at E5.5)by Solexa sequencing,and the sex-biased miRNAs were identified by qRT-PCR and their potential roles in gonad differentiation were clarified by KEGG and GO analysis.Next,the roles of male-biased miR-107 in regulating the targets(NR5al and AMH)and downstream genes were validated.Finally,based on the previous study,the regulation relationship among miR-92 and its targets(ATRXand DDX3X)were validated.1.Sexually dimorphic microRNA expression profiling from chicken embryonic gonads.1).A total of 860 known mature miRNAs have been found by Solexa deep sequencing.Altogether,491 of the 860 miRNAs(57%)were equally-expressed in the two libraries,while 165(19%)and 204(24%)of the remaining miRNAs were highly expressed in female and male gonads,respectively.Among the sex-biased miRNAs with a fold-change>2(log2(fold-change)>l),only 26 female-biased and 42 male-biased miRNAs can be found in the chicken microRNA database(miRBase).To confirm the results of the miRNA sequencing analysis,we chose miRNAs with the criterion log2(fold-change)>2 and validated them by qRT-PCR.Among the 36 tested miRNAs,18(12 male-biased and 6 female-biased miRNAs)shared similar trends by qRT-PCR and deep sequencing.2).The predicted target genes of sex-biased miRNAs were analyzed by KEGG and GO.According to GO analysis of multiple biological processes,the predicted targets for sex-biased miRNAs were associated with the developmental process.Several miRNAs were suggested to be widespread or perform general functions.Certainly,some miRNAs might perform more specific roles,such as miR-7,miR-2188,miR-1787,miR-2954 and miR-3533.3).According to KEGG analysis of multiple biological processes,we chose the pathways which might be correlated with sexual differentiation(“Wnt signaling pathway","GnRH signaling pathway”,and“oocyte meiosis pathway”).Most of the sex-biased miRNAs might stimulate oocyte meiosis pathway,and down-regulated in both Wnt and GnRH pathways.2.Identification of male-biased miR-107 as a direct regulator for NR5al1).According to the results of dual luciferase reporter assays,MiR-107 directly bound the NR5al-3'-UTR and consequently inhibited the luciferase expression.However,miR-107 showed no appreciable inhibitory effect on dual luciferase construction,and did not directly bound the AMH-3'-UTR.The results indicate that NR5al is the direct target of miR-107,but not AMH.2).In this study,the over-expression of miR-107 decreased the expression of NR5al and CYP19A1 in both mRNA and protein levels,and the inhibition of miR-107 did not cause any expression difference of NR5al.3).AMH was not directly or indirectly regulated by miR-107,and showed different trends with NR5al and CYP19A1 after the overexpression or inhibition of miR-107,suggesting that miR-107 functions as an inhibition regulator in reducing NR5a1 in chicken gonads and mediating the post-transcriptional regulation of estrogen signaling pathways.3.Identification of miR-92 as a direct regulator for ATRXand DDX3X1).ATRX and DDX3X were predicted to be the targets of miR-92 in multiple online tools(miRDB,Targetscan and miRecords).To verify the regulation relationships of miR-92 with ATRX and DDX3X,we performed dual luciferase reporter assays.MiR-92 directly bound the ATLRX-3'-UTR and DDX3X-3'-UTR and consequently inhibited the luciferase expressions.The results indicate that ATRX and DDX3X are the direct targets of miR-92.2).In this study,the over-expression of miR-92 decreased the expression of ATRX,and the inhibition of miR-92 increased the expression of ATRX,suggesting that miR-92 functions as a regulator by targeting ATRX in chicken gonads and mediating the post-transcriptional regulation of AMH signaling pathways in Mullerlan duct development.3).The over-expression of miR-92 decreased the expression of DDX3X,and the inhibition of miR-92 increased the expression of DDX3X,suggesting that miR-92 play roles in Wnt signaling pathways in chicken gonadal development.4).There is no regulation relationships between ATRX and DDX3X,indicating that miR-92 might perform different roles in early chicken gonadogenesis by mediating the post-transcriptional regulation of ATRX and DDX3X.In conclusion,a large number of miRNAs show sexually dimorphic expression patterns,and potentially participate in chicken embryonic gonadal development.Additionally,male-biased miR-107,which specifically mediates the avian ovary-development by post-transcriptional regulation of estrogen signaling pathways,directly inhibits NR5al and its downstream CYP19A1,but not AMH.Male-biased miR-92 might perform different roles in different pathways by mediating the post-transcriptional regulation of ATRX and DDX3X.