Insights Into The Mechanism Of Bovine Spermiogenesis Based On Comparative Transcriptomic Studies

The spermiogenesis process affects the quality of sperm,which in turn affects the reproductive function of sire.Poor quality semen may even induce male sterility.At present,because reproduction efficiency has become the focus of industry concern,and the social problem of male infertility has become increasingly prominent.As an important stage of spermatogenesis and maturation,sperm deformation has gradually become the focus of scientific research.If the expression of some important functional genes is abnormal during this process,it will affect the morphology and structure of the sperm,and even endanger the function of the sperm,leading to male sterility.Therefore,in-depth dig for the regulation mechanism of spermiogenesis and mining of important functional genes have become the focus of exploration and prevention of the causes of male reproductive disorders.Transcriptome analysis was performed on round spermatids,elongated spermatids and caudal epididymal sperm of the bovine.In order to find the differentially expressed genes(DEGs)that play an important role in spermatozoon deformation,transcriptome sequencing data of mice with relatively clear genetic background were introduced as reference.The homologous genes of mouse and bovine with the same expression trend were obtained by means of homologous comparative analysis between species.The specific research methods are as follows: first,we performed bioinformatics analysis on the original cattle sequencing data and the mouse RNA-seq data,calculated the expression of the genes involved in the process,and screened out the DEGs;in addition,Real-time Quantitative polymerase chain reaction(RT-q PCR)verification of bovine RNA-seq data and principal component analysis(PCA)clustering results with mouse data indicate that the sequencing data is accurate,reliable and reproducible.It shows that our data can provide a solid foundation for the accuracy of the analysis results of later enrichment analysis and protein-protein interaction.After the difference analysis,it was found that during the whole sperm deformation process,the up-regulated genes gradually decreased,and the down-regulated genes gradually increased.It can be seen that in the process of spermiogenesis,as the chromosome shrinks,transcription gradually decreases,but it does not stop completely.The selected DEGs were analyzed by Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Among them,genes in 14 items including acetyltransferase activity may be involved in the regulation of chromosome remodeling during spermiogenesis;the genes of 25 entries including sperm flagellum may be involved in the formation of the tail during sperm deformation;the genes of 2 items including acrosomal vesicle may be involved in sperm head formation.PPI analysis of DEGs in bovines and mice during metamorphosis period revealed 43 core regulatory proteins with more than 5 directly linked proteins such as RHOD,IFNG,STAT5 A,CXCL16and F2RL1.In the regulatory network,once the core protein expression changes,it will have an important impact on the normal biological functions of the entire network.The PPI network was further analyzed by MCODE(Molecular Complex Detection),and 11 core regulatory networks were obtained.Then,we compared the homology trend of DEGs in the metamorphic stage of bovine sperm and mouse sperm in the same period,and obtained a total of 578 differential genes with homology and the same trend.PPI analysis of these 578 genes was performed,and the regulatory network that regulates sperm ubiquitination,acetylation,deacetylation and glycosylation with UBC,KAT5,HDAC1 and PARP1 as the core.Interestingly,the ART3 gene belonging to the NAD+ ADP-ribosyltransferase activity(GO:0003950)entry may have the same effect as PARP1,and they may repair DNA strand breaks caused by the replacement of histones by protamine through glycosylation.In order to further explore the role of ART3 in the process of spermiogenesis,we performed immunofluorescence localization of the ART3 gene on the testes of mice and cattle,and found that the positive signals on the mouse round and elongated spermatid showed polar distribution,especially on the elongated spermatid,the positive red signal was only distributed on the outer periphery of the cells near the lumen.Because the head of the elongated spermatid faces the basal layer of the curved fine tube,and the tail faces the lumen.It is speculated that ART3 protein is related to sperm tail formation.Then,in order to further reveal the biological role of ART3 on the cell membranes of round,elongated spermatids and epididymal sperm,we obtained 122 proteins that may interact with ART3 through immunoprecipitation experiments and mass spectrometry analysis.Combined with the immunofluorescence results of ART3 expressed on the sperm cell membrane,we further screened out16 proteins that can also be localized on the cell membrane from the Unitprot database by consulting the Unitprot database.These 16 proteins may be co-localized with ART3,then the possibility of their interaction will be further enhanced.Finally,we injected 3-MBA,an inhibitor of ART3,into the testis of mice.The results showed that after the injection of 3-MBA,the mice reduced the number of sperm produced,affected the quality of sperm,and caused a significant increase in the rate of sperm abnormalities.The function of sperm is affected,and the ratio of forward movement related to the head and the vitality related to the sperm tail are significantly decreased.At the same time,6 hours after the injection of 0.906 mg/m L into the seminiferous tubules of mice,the vacuoles in the seminiferous tubules gradually increased,and with the passage of time,the vacuoles gradually increased and increased,and the area of vacuolation reached on the 5th day.The largest;the arrangement of spermatids in the seminiferous tubules becomes scattered and irregular,and the spermatogenic cells gradually decrease.This result verifies the previous speculation on the function of ART3,that ART3 is related to the formation of sperm head and tail.The acquisition of these results provides important data support for revealing the secret of spermiogenesis.