Difference Analysis And Validation Of Transcriptome Of Spermatids During Spermiogenesis In Dairy Cows

After mitosis,meiosis,and spermiogenesis,spermatogonial cells firstly form round spermatids and eventually developed to be tadpole like mature sperm.During spermiogenesis,the morphology of cells and organelles changes tremendously,but the mechanism of this process is still unknown.In order to explore the genes involved in the morphogenesis of sperm,this study used dairy cows as materials,performed differential expression analysis of the transcriptomes of round,elongated sperm cells and epididymis tail sperm,and verified the differential genes by qPCR.The results of transcriptome differential expression analysis showed that a total of 6665 genes were differentially expressed during the development of round spermatids into epididymal sperm(p < 0.05).A total of 994 genes were differentially expressed between round and enlongated sperm,of which 246 were up-regulated and 748 were down-regulated.There were 4771 differentially expressed genes between enlongated sperm and epididymis tail sperm,of which 1613 were up-regulated and 3158 were down-regulated.A total of 4634 genes were differentially expressed between the round spermatids and epididymal tail sperm,of which 2036 were up-regulated and 2598 were down-regulated.According to the annotation,6665 genes expressed differently were analyzed by GO enrichment,and ten types of functional items that were significantly associated with spermiogenesis were selected,including histones,mitochondria,microtubules,centrosomes,acrosome,Golgi apparatus,chromosome condense,Flagella,Transcription,and Sperm.Statistical analysis of Alternative splicing(AS)events about the transcriptomes of round,elongated spermatid,and epididymal sperm of dairy cows revealed that five distinct types of alternative splicing events have occurred in these three stages of cells,ie intron retention,exon skipping,alternative splicing at the 5’ or 3’ end,the alternative splicing of transcription initiation region,and the alternative splicing of end-transcription region.The alternative splicing species in the spermatozoa of the epididymis were the same as those in the round and elongated sperm cells,but in the number of occurrences,there was no significant change in the alternative splicing except that multiple introns and boundary-fuzzy multi-intron retention.In addition,the number of alternative splicing events was significantly reduced in epididymis sperm.In all cell samples,TSS and TTS are the two most important alternate forms of splicing during the process of spermiogenesis.5 differently expressed genes were selected from the genes enriched entries,including Pld6,Ccin,Spem1,Pdilt,and Tssk1 B.Using β-actin as a housekeeping gene,q PCR was performed with these five genes.The results showed that Pld6 and Spem1 were up-regulated in the process of round spermatids developed to elongated spermatids and down-regulated during elongated spermatids developed to epididymis tail sperm.Ccin,Pdilt,and Tssk1 B genes were down-regulated during spermiogenesis.The results of q PCR about these five differentially expressed genes were all as the same as RNA-seq,demonstrating the reliability of sequencing data.The structure and function of the PLD6 protein were predicted and analyzed by bioinformatics analysis.The results showed that Pld6 gene was enriched in the mitochondrial fusion term,and it is a transmembrane protein with a transmembrane region from10-32 th amino acids in cattle.The tertiary structure of its protein was horn-like with a conservedβ-sheet active site H(X)K(X4)D(HKD)inside.The protein structure of PLD6 in cattle is similar to which in mouse.Both of them have the closest evolutionary trend.Therefore,according to the studies about mouse,it is concluded that,during the process of bovine spermaiogenesis,increased expression of Pld6 gene induces the cluster of mitochondria in the midpiece of sperm tail,which promotes the formation of normal mitochondrial sheaths and plays an important role in ensuring bovine sperm viability and fertilizing ability.In the subsequently analysis of function differential genes,the function of gene in mouse can provide important evidence for differential gene analysis in cattle.In this experiment,dairy cows were used as experimental materials to study gene expression in the process of spermaiogenesis,which provided a theoretical basis for investigating gene transcription and expression mechanisms in the process of sperm production in dairy cows and played a positive role in improving the reproductive performance of male dairy cows.