Comparative iTRAQ proteomics identified proteins associated with sperm maturation between yak and cattleyak epididymis.

BACKGROUND: During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. In recent years, two-dimensional gel electrophoresis has been employed in proteomics studies conducted in rat, boar and human. However, there has not been a complete information regarding the proteins associated with sperm maturation in the epididymis. In this study, we employed iTRAQ proteomics to investigate proteins associated with sperm maturation between yak and cattleyak epididymis. RESULTS: After a successful sampling and protein extraction, the iTRAQ coupled with LC-MS/MS mass spectrometry and bioinformatics analysis were performed. We identified 288 differentially abundant proteins (DAPs) between yak and cattleyak epididymis; 151 were up-regulated while 137 were down-regulated in cattleyak relative to yak. Gene Ontology analysis identified that down-regulated DAPs in cattleyak were mostly enriched in the acetylation of protein component, along with negative and positive regulatory activities. iTRAQ proteomics data showed that the top up-regulated DAPs were mainly enriched in cell communication, cell adhesion, cytoskeleton organization, stress response, post-translational modifications and metabolic functions while the down-regulated DAPs were predominantly associated with sperm maturation, long-term sperm storage, sperm forward motility, sperm-oocyte fusion and regulatory functions. CONCLUSION: These results provide insight into the molecular mechanisms underlying male cattleyak sterility.

Next-generation sequencing analysis reveals segmental patterns of microRNA expression in yak epididymis.

MicroRNAs (miRNAs) have emerged as potent regulators of gene expression and are widely expressed in biological systems. In reproduction, they have been shown to have a significant role in the acquisition and maintenance of male fertility, whereby deletion of Dicer in mouse germ cells leads to infertility. Evidence indicates that this role of miRNAs extends from the testis into the epididymis, controlling gene expression and contributing to regional variations in gene expression. In this study, RNA sequencing technology was used to investigate miRNA expression patterns in the yak epididymis. Region-specific miRNA expression was found in the yak epididymis. In all, 683 differentially expressed known miRNAs were obtained; 190, 186 and 307 differentially expressed miRNAs were identified for caput versus corpus, corpus versus cauda and caput versus cauda region pairs respectively. Kyoto Encyclopedia of Genes and Genomes results showed endocytosis as the most enriched pathway across region pairs, followed by protein processing in the endoplasmic reticulum, phagosome, spliceosome and biosynthesis of amino acids in region pair-specific hierarchical order. Gene ontology results showed varied enrichment in terms including cell, biogenesis, localisation, binding and locomotion across region pairs. In addition, significantly higher miR-34c expression was seen in the yak caput epididymidis relative to the corpus and cauda epididymidis.

Region-specific gene expression in the epididymis of Yak.

Immature spermatozoa undergo series of events in the epididymis to acquire motility and fertilizing ability. These events are a direct result of exposure to, and interaction with, the luminal environment created by the epididymal epithelium. The three conventional regions of the epididymis namely; caput, corpus and cauda have been identified to play specific roles in the epididymal maturation process of the spermatozoa; their respective roles have been associated with specific gene expression patterns that account for the composition of the luminal fluid that bathe the spermatozoa as they transit through the epididymal lumen and ensure their maturation. The identification of genes expressed in a region-specific manner provides valuable insight into the functional differences among the regions. Microarray technology has previously been employed in region-specific gene expression studies using the epididymis as a model in different species such as mouse, rat, boar and human. However, to characterize gene expression in the different regions of the epididymis, RNA-seq analysis was used in our study to examine gene expressions in the caput, corpus, and cauda of yak epididymis. Comparative transcriptomic analysis was performed between region pairs in the order; caput vs corpus, caput vs cauda and corpus vs cauda. DEGs among the various region pairs were detected and functional analysis were performed for the detected DEGs. Overall, the caput vs cauda epididymidis pair produced the highest number of DEGs (49.4%) while the corpus vs cauda pair produced the least number of DEGs (19.3%). The caput segment demonstrated relatively high expression of Sal1, LCN6, PTDS, DEFB109, DEFB 119, DEFB 123, SPAG11, PROC, CST3, ADAM28, KCNJ12 and SLC13A2; corpus epididymis demonstrated relatively high expression of MAN2B2, ELP, ZFYVE21, GLB1L, BMP4, DEFB125, PPP1R10, RIOX2, TKDP1, DEFB106A, NPBWR1 and SLC28A1; and the cauda epididymis, demonstrated relatively high expressions of MCT7, PAG4, OAS1, TGM3 and PRSS45. Gene Ontology results showed that DEGs in the caput vs corpus and corpus vs cauda pairs were mostly enriched in the cell/cell part GO term. On the other hand, DEGs in the caput vs cauda pair was were mostly enriched in the cellular process term. KEGG pathway annotation was also performed for DEGs among the various groups. AMPK signaling pathway, which is characterized by the ratio between cellular AMP and ATP and also determines cellular energy state, was selected from among the top five KEGG pathways for DEGs in the caput vs corpus pair. Our results showed that some down-regulated DEGs in the caput and corpus pair such as HN4a, eEF2K and CFTR were present and played significant roles in the AMPK signaling pathway. In the corpus vs cauda pair, our results showed that up-regulated DEGs such as XDH, TRMP2 and ENTPD were involved in the purine metabolism KEGG pathway, which was among top five KEGG pathways for DEGs in this pair. Pentose phosphate pathway functions in antioxidation to protect both the spermatozoa and epididymis from oxidative damage; it was among top five KEGG pathways for DEGs in the caput vs cauda pair. Our results also showed that down-regulated genes in the caput vs cauda pair such as TALDO1 was found to be involved in the Pentose phosphate pathway. The significance of the upregulated and downregulated genes on the pathways were elucidated. SAL1, which showed high expression in the caput, had previously not been demonstrated in the epididymis, needs further investigation to establish its unique role in the yak epididymis.

Comparative RNA-Seq Analysis of Differentially Expressed Genes in the Epididymides of Yak and Cattleyak.

BACKGROUND: Cattleyak are the Fl hybrids between (♀) yak (Bos grunniens) and (♂) cattle (Bos taurus). Cattleyak exhibit higher capability in adaptability to a harsh environment and display much higher performances in production than the yak and cattle. The cattleyak, however, are females fertile but males sterile. All previous studies greatly focused on testes tissues to study the mechanism of male infer-tility in cattleyak. However, so far, no transcriptomic study has been conducted on the epididymides of yak and cattleyak. OBJECTIVE: Our objective was to perform comparative transcriptome analysis between the epididymides of yak and cattleyak and predict the etiology of male infertility in cattleyak.Methods: We performed comparative transcriptome profiles analysis by mRNA sequencing in the epidi-dymides of yak and cattleyak. RESULTS: In total 3008 differentially expressed genes (DEGs) were identified in cattleyak, out of which 1645 DEGs were up-regulated and 1363 DEGs were down-regulated. Thirteen DEGs were validated by quantitative real-time PCR. DEGs included certain genes that were associated with spermatozoal matura-tion, motility, male fertility, water and ion channels, and beta-defensins. LCN9, SPINT4, CES5A, CD52, CST11, SERPINA1, CTSK, FABP4, CCR5, GRIA2, ENTPD3, LOC523530 and DEFB129, DEFB128, DEFB127, DEFB126, DEFB124, DEFB122A, DEFB122, DEFB119 were all downregu-lated, whereas NRIP1 and TMEM212 among top 30 DEGs were upregulated. Furthermore, protein processing in endoplasmic reticulum pathway was ranked at top-listed three significantly enriched KEGG pathways that as a consequence of abnormal expression of ER-associated genes in the entire ER protein processing pathway might have been disrupted in male cattleyak which resulted in the down-regulation of several important genes. All the DEGs enriched in this pathway were downregulated ex-cept NEF. CONCLUSION: Taken together, our findings revealed that there were marked differences in the epididymal transcriptomic profiles of yak and cattleyak. The DEGs were involved in spermatozoal maturation, mo-tility, male fertility, water and ion channels, and beta-defensins. Abnormal expression of ER-associated genes in the entire ER protein processing pathway may have disrupted protein processing pathway in male cattleyak resulting in the downregulation of several important genes involved in sperm maturation, motility and defense.