| Flounder(Paralichthys olivaceus)is one of the important economic marine fishes in China.It has high protein content,and high nutritional value.In addition,the growth rate of flounder has obvious gender differences.Single-sex culture can effectively enhance economic efficiency.Testis development is the basis of sexual reproduction,and Spermatogenesis is the key to the normal development of the testis.Spermatogenesis refers to the process in which diploid spermatogonia produce mature haploid spermatozoa through a series of complex biochemical reactions such as meiosis.Although our understanding of spermatogenesis has made great progress,and research on related genes and chemicals has been continuously discovered and identified.However,at present,studies on gonadal development and spermatogenesis of Paralichthys olivaceus focus on the expression analysis of sex hormones and sex differentiation-related genes.In addition,researches on genes related to gonadal development and spermatogenesis have mostly focused on the expression rules and functional analysis of these coding genes,and there have been few reports on the regulation of their post-transcriptional levels,and this field is the current hotspot of genetics research.In the early stage of this laboratory,through high-throughput sequencing and other technologies,the libraries of miRNA in the nests of flounder and ovary were established,and a number of miRNA were screened out that were differentially expressed in male and female gonads.Among them,miR-202-5p was a miRNA that was richly expressed in gonads and had significant differences between male and female expressions.On this basis,this study used fluorescence quantitative PCR and in situ hybridization to construct the expression profile of miR-202-5p in different tissues and gonadal development of flounder.The results showed that miR-202-5p was specifically and highly expressed in the testis of the flounder,followed by the ovary,and the expression was lower in other tissues.In situ hybridization(ISH)and fluorescence in situ hybridization(FISH)were used to detect the expression localization of miR-202-5p in testis and ovary.The results of in situ hybridization showed that miR-202-5p had a strong hybridization signal only in oocytes in stage IV.However,in other stage oocytes,the hybridization signal was weak.The signals of vasa were detected in early stage oocytes such as I,II,and III.In IV and V stage oocytes,the hybridization signal of vasa gradually weakened;in the testis,miR-202-5p showed a similar expression pattern to vasa,which was detected in spermatogonia and primary spermatocytes.Consistent with the results of in situ hybridization,the results of fluorescence in situ hybridization revealed that in the testis,the fluorescence signal of miR-202-5p is mainly concentrated in spermatogonia and spermatocytes,which is similar to the expression pattern of vasa.While in the ovary,the fluorescence signal of vasa gradually weakened with the development of oocytes.Mi R-202-5p showed a different expression pattern from vasa,and only a strong fluorescence signal of miR-202-5p was detected in stage IV oocytes.This testis-specific expression pattern suggests that miR-202-5p may be involved in testis development and spermatogenesis.In order to further explore the functions of miR-202-5p in testis development and spermatogenesis,we used in vivo injection of retinoic acid to study the relationship betweeen retinoic acid(RA)and miR-202-5p,as well as the genes related to spermatogenesis and testis development,such as cbx2,ccnd1,sox9,smad1/3,bmp3/5 and tgf-β3.The results showed that the expression of miR-202-5p increased after retinoic acid injection.The expression of ccnd1 and cbx2 decreased,while the expression of sox9,smad1/3,bmp3/5 and tgf-β3 increased.The prediction and identification of Mi RNAs is the key basis for studying the function of miRNAs.In this study,the bioinformatics method was used to predict the complementary relationship between the cbx2 and ccnd1 genes and miR-202-5p seed sequences.The prediction results showed that the 3′UTR of ccnd1 is completely complementary to the seed sequence of miR-202-5p;the 3′UTR of cbx2 and miR-202-5p seed sequence have only one position non-complementary,and the remaining positions are complementary.When miRNAs are not completely complementary to target genes,miRNAs can still bind to the 3′UTR region of the target genes to suppress the regulation of post-transcriptional translation.In order to further explore the relationship between miR-202-5p,cbx2 and ccnd1,this study used dual luciferase reporter gene technology to identify the targeting relationship between miR-202-5p,cbx2 and ccnd1.Preliminary identification results show that cbx2 and ccnd1 are direct target genes of miR-202-5p,suggesting that miR-202-5p may play an important role in testis development and spermatogenesis by regulating genes such as cbx2 and ccnd1.Finally,this study further verified the relationship between miR-202-5p,cbx2 and ccnd1 by overexpressing or inhibiting miR-202-5p in testis tissues cultured in vitro.The results showed that in the experimental group transfected with miR-202-5p mimics,the expression of miR-202-5p increased and the expression of cbx2 and ccnd1 decreased.In the experimental group transfected with miR-202-5p antagomir,the expression of miR-202-5p decreased,while the expression of cbx2 and ccnd1 increased.In order to rule out interference from testis somatic cells,this study also detected the expression changes of miR-202-5p,cbx2 and ccnd1 by transfecting primary spermatogenic cells cultured in vitro.After transfection with miR-202-5p mimics,the expression of cbx2 and ccnd1 decreased as miR-202-5p expression increased.After transfection with mir-202-5p antagomir,the expressions of cbx2 and ccnd1 increased with the decrease of mir-202-5p expression.It is further shown that cbx2 and ccnd1 are the target genes regulated by miR-202-5p in testis tissues and cells of flounder.In summary,this study provides new insights for further exploring the mechanism of miR-202-5p in spermatogenesis and testis development of flounder. |
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