Preliminary Study On Differential Proteomics Of Spermatozoa In The Caput,Corpus And Cauda Of Buffalo Epididymis

In mammals,spermatozoa acquire the ability to be fertilized during epididymal transit,which is known as sperm maturation.Spermatozoa that have been deformed in the testis show substantial silence during genetic transcription and translation,and its maturation derives from developmental endogenous environmental regulation or regulation by protein modifications.Sperm maturation involving morphological and functional modifications is closely associated with the quality of spermatozoa.The current studies on sperm maturation tend to focus on epididymal tissues or single-site spermatozoa,while a few studies are conducted on the dynamic development of proteins during the whole procedure of sperm maturation.Buffalo(Bubalus bubalis)is known as an outstanding dairy-meat animal in the southern subtropical areas of China,however,with a low reproductive efficiency,while the quality of male gametes is considered as a major factor affecting their reproduction.Therefore,investigating the molecular regulatory mechanism of the sperm maturation in buffalo is essential into improving the quality of male gametes.In this study,we investigated the spermatozoa in the caput,corpus and cauda of buffalo epididymis by biological techniques including high-throughput protein profiling,transmission electron microscopy observation,fluorescent probe labeling and flow cytometric analysis to demonstrate micro-level transformations in sperm maturation,screening and identification of critical proteins,analyzing significant contributors to sperm maturation in the epididymis,and providing a new perspective for the study of buffalo reproductive mechanisms.The main conclusions are summarized as follows:1.Ultrastructural observation and analysis of spermatozoa in different sites of buffalo epididymisSpermatozoa from the caput,corpus and cauda of the epididymis were isolated with a purity of 95% by percoll density gradient centrifugation;sperm motility was detected by CASA with 8.35% in the caput,20.21% in the corpus and 65.60% in the cauda;Transmission electron microscopy showed that spermatozoa in different regions of the epididymis shared common deficient patterns in the acrosome,mitochondrial sheath,outer dense fiber,axial filament,and axial fiber.Flow cytometric analysis revealed that spermatozoa in the cauda of the epididymis showed the highest ratio of mitochondrial membrane potential,and the sperm plasma membrane integrity rate gradually increased from the caput to the cauda;Flow cytometer and fluorescence microscopy showed that the sperm acrosome integrity rate gradually increased from the caput to the cauda region of epididymis.2.Quantitative proteomic analysis of spermatozoa from the caput,corpus and cauda of epididymis in buffaloThe identification of spermatozoa from the caput,corpus and cauda of epididymis by TMT quantitative proteomics resulted in 2796 proteins,in which,taking spermatozoa from the caput of epididymis expressing proteins as the control group,we found a common total of 130 differential proteins(Foldchange>1.3)in the corpus and cauda,with 53 up-and 77down-regulated.Most of the differential expressed proteins were involved in reproduction-related biological processes,and cellular component analysis showed that most of the differential expressed proteins were associated with exosomes,vesicles,and endoplasmic reticulum.KEGG enrichment indicated that the signaling pathways involved in differentially expressed proteins were predominately in Endoplasmic reticulum protein processing,Peroxisomes,Antigen processing presentation,Metabolism,and Hormone synthesis.To further investigate the complexity of interaction among these proteins,we constructed a differential protein interaction network by STRING online tool,with sub-networks primarily consisting of Metabolism,Localization,Translation,and Oxidative Stress.Finally,we confirmed that HSD17B4 and PRDX4 were highly expressed in the corpus and cauda of epididymal sperm relative to the caput by immunofluorescence and Western Blot,and that PRDX4 was localized in the caput and cauda of sperm,and HSD17B4 was localized in the caput of sperm.In conclusion,the current research analyzed the supra-microstructural and differential characteristics of spermatozoa from different regions of the buffalo epididymis,constructing a protein landscape of the differential expression in buffalo epididymal spermatozoa,identifying several sperm maturation-related pathways and crucial proteins by bioinformatics analysis.Therefore,the present study serves as a fundamental basis for elucidating the molecular regulatory mechanisms of the sperm maturation in buffalo.