| Interspecific hybrid male sterility is a common occurrence in nature and plays an important role in species reproductive isolation.Yattle(cattle-yak or DZO),the offspring of interspecific cross between domestic yak(Bos grunniens)and cattle(Bos taurus),is a unique animal model for investigating interspecific hybrid male sterility.yattle females are completely fertile while the males are sterile.In recent years,molecular studies have demonstrated that the expressions of genes were dysregulated during meiosis in yattle testis,as compared to those in cattle or yak.Other studies have revealed that epigenetic factors/events,such as DNA methylation,histone modification and non-coding RNA,are also involved in spermatogenesis.Recently,studies have revealed that mi RNA is mainly regulated during the mitosis and prophase of meiosis of mammalian spermatogonia,and pi RNA is mainly regulated during the meiosis of spermatocytes,indicating that mi RNA and pi RNA can affect mammalian reproduction.Therefore,exploring the effects of mi RNA and pi RNA on the spermatogenesis of the yattle testis may provide new insights and ideas for male sterility in the yattle.This study selects the testicular tissues and other tissues of F1 adult male sterile yattle(n=8),yak(n=8)and cattle(n=8),mainly using TUNEL cell apoptosis detection,RT-q PCR,vector Construction,cell transfection,dual luciferase report,small RNA transcriptome sequencing(s RNA-seq)(yattle,n=2;yak,n=2;cattle n=2)and other technologies,targeting mi RNA and pi RNA pairs The influence of male sterility in Yattle was explored,and the following experimental results were obtained:(1)Using s RNA-seq technology to perform mi RNA analysis on three types of bovine testis tissues,raw reads of 1.993,2.069,and 1.768 Gbp were obtained for cattle,yak,and yattle,respectively.The ratio of mi RNAs to s RNAs in the testis of Yattle is about 35%,which is significantly higher(P<0.05)than that of yak(～9%)and cattle(～8%);27 mi RNAs in Yattle are more significant than those of cattle and yak Down-regulation(|log2(foldchange)|>1,P<0.01),8 mi RNAs are all significantly up-regulated;RNA-Seq and RT-q PCR show that they are bta-mi R-200a,bta-mi R-449a,bta-mi R-34c,bta-mi R-15b were significantly down-regulated compared with yak and cattle(P<0.05),and bta-mi R-101,bta-mi R-145,and bta-mi R-30-5p were all significantly up-regulated(P<0.05);The down-regulated bta-mi R-449a,bta-mi R-34c,and bta-mi R-200a target the up-regulated NOTCH1,NANOS2,USP9X,respectively,and the differential mi RNA target genes are mainly enriched in the differentiation process of spermatogonial stem cells into spermatocytes;The enrichment of GO and KEGG pathways indicates that target genes are mainly enriched in ribosomal and HIF-1signaling pathways.(2)Using s RNA-seq technology to analyze the difference between the pi RNA transcriptomes of the three types of bovine testis tissues,cattle,yattle,and yak obtained1.967×10~7,1.768×10~7,2.036×10~7clean reads respectively;comparison of pi RNAs genome sources The analysis showed that the expression of 29～31nt pi RNAs derived from genomic DNA in the pachytene stage decreased,and the expression of 26-27nt pi RNAs derived from the transposon in the pachytene stage increased;the pi RNA clusters 2680,5129 and 5758 in pachytene stage of yattle The expression level of pi RNA clusters 3649,6797 and 3647 in the pre-pachytene stage of yattle is lower than that of yak and cattle;RT-q PCR of pi RNA pathway related genes and transposable factors shows that most of the pi RNA pathways are related Genes(PIWIL1,DDX4,MAEL,PLD6,TDRD1,TDRD9,RNF17,FKBP6,AGO2,and MOV10L1)were significantly down-regulated in the testes of yattle(P<0.05;P<0.01;P<0.001).The expression levels of LINE1,LINE-RET,and SINE-CORE m RNA in the testis of yattle were significantly lower than those of yak and cattle(P<0.05).(3)HE staining of testicular paraffin section showed that cattle spermatogenesis was blocked.Compared with yak and cattle with normal spermatogenesis,there were no round spermatozoa and long spermatozoa cells in the concentric spermatotubules of yattle.TUNEL cell apoptosis analysis showed that the apoptosis rates of testis cells in yattle,cattle and yak were 1.05%,0.24%and 0.40%,respectively.RNA-seq and RT-q PCR of apoptosis-related genes showed that only CASP9 was significantly different in yattle(P<0.05),and most apoptosis-related genes were not significantly different in yattle.(4)UTX and USP9X were amplified by RT-q PCR.The results showed that UTX and USP9X were significantly upregulated in yattle testicles(P<0.05).s RNA-seq and RT-q PCR showed that bta-mi R-200a(P<0.05)was significantly down-regulated in yattle and targeted at USP9X(P<0.05).Targeted verification by dual luciferase reporting system showed that the relative fluorescence value(Ratio)of fireflies in the experimental group WT-200a was significantly different from that in the other 5 groups(P<0.05;P<0.01;P<0.001),indicating that bta-mi R-200a can effectively bind and decomposition the 3’UTR terminal of USP9X,highlighting the apparent modification role of mi RNA on yattle spermatogenesis.In summary,the analysis of the mi RNA and target gene differences in the testicular tissues of yattle,yak,and cattle in this study showed that the down-regulated mi RNAs expression of yak is related to the up-regulation of spermatogenesis-related target gene expression;USP9X is bta-mi R-200a Target gene;pi RNA difference analysis showed that the decrease of pi RNAs derived from genomic DNA in the pachytene stage is related to the down-regulation of pi RNA pathway-related gene expression,and the increase of pi RNAs derived from the transposon in the pre-pachytene stage is related to the down-regulation of transposable factor expression.These research results provide a theoretical and scientific basis for the epigenetic research of male sterility in yattle. |
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