Functions Of MIRNA-2954 And Its Regulation Relationships With Relevant Genes In Chicken Testis Development

The differentiation and development of gonads is an important process of animal growth and development.Among them,the differentiation and development of testis are the important basis for male individuals to mature and maintain normal reproductive physiology.Many factors that are gender-biased at different levels consist of complex regulatory networks and play an important regulatory role in the development of gonads.In the chicken,there is little information about the regulation process and mechanism of testis or ovary development,which is still to be further studied.In the previous studies,our study group screened out miRNA-2954 which was expressed with male preference and verified that FOXD1 and YY1 were its two target genes.Based on these basis,we further analyzed the gene expression spectrum of FOXD1 in chicken testis,conducted its tissue and cell localization,and investigated the regulation relationships of miRNA-2954,target genes FOXD1 and YY1 and downstream genes related with testis development in Sertoli cell line,to study whether the miRNA-2954 function through two target genes in testicular development.The research results are as follows: 1.We conducted quantitative PCR in six different kinds of tissues in adult chickens and gonads in the embryonic stage to study tissues and time expression profiles of FOXD1,and we confirmed the male-biased expression pattern of FOXD1 that it is highly expressed in brain,kidney,gonad of male adult chicken and testis at E6.5,E12.5,E18.5.2.The immunohistochemistry and immunofluorescence results of the testicular tissue sections at E6.5-18.5 showed that FOXD1 was expressed in the Sertoli cells of chicken testis;and immunofluorescence results also showed that the expression of FOXD1 in testis is the same as that of Sertoli-specific expression marker SOX9.The immunohistochemistry and immunofluorescence of the chicken Sertoli cells showed that FOXD1 was mainly expressed in nucleus.3.Using trypsin/collagenase digestion method to prepare chicken testis progenitor cells,and we conducted the progeny,transfection,cryopreservation and resuscitation of the Sertoli cells;through Oil Red O staining we observed a large number of fat particles in the cytoplasm of Sertoli cells.We successfully established a complete system for Sertoli cell culture in our laboratory.4.We successfully conducted over-expression and inhibition of miRNA-2954 in chicken testicular Sertoli cells,via quantitative PCR we detected the expression of target genes YY1 and FOXD1 together with testis-related genes like DMRT1,SOX9,AMH,AR after transfection and the results show that the miRNA-2954 inhibits the expression of these genes,which is consistent with previous results of target genes inhibition in DF-1 cells.5.We performed siRNA interference in FOXD1,YY1 in chicken testicular Sertoli cells,and used quantitative PCR to detect the expression of genes related with differentiation and development of testis,except DMRT1,all genes detected are regulated by the FOXD1,which is consistent with the results of miRNA-2954 over-expression;however,the inhibition of YY1 caused the up-regulation of DMRT1,SOX9,AMH,and quantitative analysis indicated that the inhibition of YY1 may have negative feedback regulation effect on miRNA-2954.The above results suggest that the target genes FOXD1 plays an important role in chicken testicular development,confirm the inhibitory effect of miRNA-2954 on FOXD1 and YY1,and through interference of target genes,indicate that miRNA-2954 function through mediated target genes FOXD1,YY1 to regulate the expression of downstream related genes,and may also be regulated by YY1 via a negative feedback in order to maintain homeostasis.