Analysis Of Y Chromosome Gene Expression Patterns And Genome-wide DNA Methylation Differences In Cattle,Yak And Yattle

The yattle is a hybrid of the cattle and the yak,which has obvious heterosis in meat production and milk production.However,yattle male infertility makes its heterosis unable to be cross-fixed,and it is a “blocking tiger” for the improvement of new breeds in yak.Previous histology and molecular biology studies have found that the process of spermatogenesis is blocked and gene expression in spermatogenic processes is disordered in yattle testes.The mammalian Y chromosome male-specific region(MYS)gene plays a crucial role in spermatogenesis.Bovine MSY contains three major regions,the X-degenerate region that comprises single-copy genes,and the Y-transitional region and Y-ampliconic region that contains largely amplified gene families.DNA methylation can regulate gene expression.During mamMalian spermatogenesis,abnormal DNA methylation will cause the spermatogenic gene expression to be disordered,leading to male sterility.It is unclear whether the Y chromosome gene expression pattern and the difference in genome-wide DNA methylation are associated with male sterility in yattle.In this study,testicular tissue of adult cattle(n=8),yak(n=8)and yattle(n=8)were collected.The copy number variations(CNVs),mRNA and protein expression of MSY genes and genome-wide DNA methylation level were investigated using using qPCR,RT-qPCR,Western Blot,whole genome bisulfite sequencing(WGBS)and other techniques.The results were obtained as follow.1.Histologic section showed that spermatogonial cells and spermatocytes are significantly reduced,and almost no round sperm cells and lengthened sperm cells were present in the lumen of seminiferous tubule.Meanwhie,there are many vacuoles in yattle.The cell types in the seminiferous tubules of the cattle and yak testicular tissues are normal.The parental and maternal origin of the test samples were confirmed by Sanger sequencing of ZFY-10,UTY-19,and mtDNA D-loop.2.The mRNA expression levels of 10 X-degenerate region genes of MSY in adult cattle,yak and yattle testicular tissues were analyzed by RT-qPCR.The results showed that RBMY,UBE1 Y,and EIF1 AY were specifically expressed in testis tissue,while the remaining genes were widely expressed in various tissues.The UTY,OFD1 Y,and USP9 Y were significantly higher expressed in yattle than cattle and yaks testes(P<0.004),while the expression levels of DDX3 Y,EIF1AY,RBMY,ZFY,EIF2S3 Y,ZRSR2Y and UBE1 Y in the testis of yattle were not significantly differentbetween to cattle and yak(P>0.05).The results suggest that the high expression of UTY,OFD1 Y and USP9 Y may be associated with yattle infertility.3.The copy number variation(CNV)of MSY multi-copy genes TSPY1,PRAMEY,TSPY3,ZNF280 BY and HSFY were analyzed using qPCR.TSPY1,TSPY3 and PRAMEY can be divided into 2,2 and 4 subtypes according to the difference of coding sequences.qPCR analysis using the type-specific and Y-chromosome-specific primers and DNA templates revealed that the TSPY1-T2 subtype DNA sequence on the Y chromosome of the yak contains only 1 copy on average,while the cattle contained average 126 copies of TSPY1-T2.The TSPY3-T2 sequences were absent in yak Y chromosomes.Copy number of PRAMEY-T2 and PRAMEY-T4 was amplified in yak.The copy number of ZNF280 BY in yak was significantly higher than cattle(P<0.001).These results suggest that the genomic structure of the Y chromosome differed between cattle and yak.4.The mRNA expression patterns of MSY multi-copy genes TSPY1,PRAMEY,TSPY3,ZNF280 BY and HSFY were analyzed uisng RT-qPCR.The results showed that HSFY was specifically expressed in the testis and muscle,and TSPY1,TSPY3,PRAMEY and ZNF280 BY were specifically or predominantly expressed in the testis.Compared with cattle and yak,TSPY1-T2,PRAMEY-T2,HSFY and ZNF280 BY were almost not expressed in yattle,and their expression patterns were consistent with the PRM1 and PRM2.The PRM1 and PRM2 genes are used as positive markers for spermatozoa.The expression of PRAMEY-T3 in the testis tissue of yak is significantly lower than that of cattle and yattle(P<0.05).The expression of PRAMEY-T3 was not significantly different between cattle and yattle(P>0.05);PRAMEY-T4 was not significantly different among yattle,yak and cattle(P>0.05).However,the specific antibodies against PRAMEY-T3 and PRAMEY-T4 were preparated for Western blot analysis Showed that the expression of PRAMEY-T3 was increased in yattle,while PRAMEY-T4 was not significantly different among yattle,yak and cattle.Combined with the phenotype and mRNA expression pattern of sperm cells in the yattle testis tissue,these results suggested that the Y chromosome multi-copy genes TSPY1-T2,ZNF280 BY,HSFY and PRAMEY-T2 may be mainly involved in spermatogenesis after meiosis.These genes were almost not expressed may be as the result that the yattle cannot produce sperm properly.5.Our transcriptome(RNA-Seq)analysis found that the genes related to spermatogenesis were highly differentially expressed between the testis tissues ofyattle,cattle and yak.In order to further explore the epigenetic regulation mechanism of related gene expression disorder of yattle infertility,the DNA methylation of mature testis tissue of cattle and F1 yattle was analyzed using WGBS.The results showed that in the differential methylation region(DMR),the genomic DNA methylation level was significantly increased in yattle,and the CpG island,promoter and repeat region showed hypermethylation status in yattle testes.Total 419 promoter genes were hypermethylated and these genes were mainly enriched in GO pathways of DNA methylation involved in gametogenesis,piRNA metabolism,spermatogenesis and so on.Combined with RNA-seq data,it was found that the promoter hypermethylation of the piRNA-producing genes was significantly assocatied with lower mRNA expression in yattle testes than in cattle and yak(P<0.001).Basing on the pachytene piRNA plays an important role in the transformation of the spermatozoa into sperm cells,the piRNA production may be related to the failure of the yattle to produce sperm.Therefore,the hypermethylation of the key genes of spermatogenesis in the testis of yattle will affect spermatogenesis genes expression and piRNA production,which may be related to yattle infertility.In summary,the gene expression patterns revealed that the X-degenerate region genes and multi-copy genes of the Y chromosome play different roles in yattle infertility.The genome-wide DNA methylation analysis revealed that genome-wide DNA hypermethylation were occurs in the testes of yattle,and promoter hypermethylation mainly involves spermatogenesis and piRNA production pathway genes,and inhibits gene expression.The results of this study reveal the relationship between Y chromosome genes and genomic DNA methylation and male sterility in yak,which will provide theoretical basis and scientific data for revealing the molecular mechanism of yattle male sterility.