DNA methylome of primary spermatocyte reveals epigenetic dysregulation associated with male sterility of cattleyak.

DNA cytosine methylation modification in the germline is of particular importance since it is a highly heritable epigenetic mark. Although cytosine methylation has been analyzed at the genome-scale for several mammalian species, our knowledge of DNA methylation patterns and the mechanisms underlying male hybrid sterility is still limited in domestic animals such as cattleyak. Here we for the first time show the genome-wide and single-base resolution landscape of methylcytosines (mC) in the primary spermatocyte (PSC) genome of yak with normal spermatogenesis and the inter-specific hybrid cattleyak with male infertility. A comparative investigation revealed that widespread differences are observed in the composition and patterning of DNA cytosine methylation between the two methylomes. Global CG or non-CG DNA methylation levels, as well as the number of mC sites, are increased in cattleyak compared to yak. Notably, the DNA methylome in cattleyak PSC exhibits promoter hypermethylation of meiosis-specific genes and piRNA pathway genes with respect to yak. Furthermore, major retrotransposonson classes are predominantly hypermethylated in cattleyak while those are fully hypomethylated in yak. KEGG pathway enrichment indicates Rap1 signaling and MAPK pathways may play potential roles in the spermatogenic arrest of cattleyak. Our present study not only provides valuable insights into distinct features of the cattleyak PSC methylome but also paves the way toward elucidating the complex, yet highly coordinated epigenetic modification during male germline development for inter-specific hybrid animals.

Testis transcriptome profiling identified lncRNAs involved in spermatogenic arrest of cattleyak.

Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P < 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak.

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.

Testis transcriptome profiling identified genes involved in spermatogenic arrest of cattleyak.

BACKGROUND: Cattleyak are the hybrid offspring between cattle and yak and combine yak hardiness with cattle productivity. Much attempt has been made to examine the mechanisms of male sterility caused by spermatogenic arrest, but yet there is no research systematically and precisely elucidated testis gene expression profiling between cattleyak and yak. METHODS: To explore the higher resolution comparative transcriptome map between the testes of yak and cattleyak, and further analyze the mRNA expression dynamics of spermatogenic arrest in cattleyak. We characterized the comparative transcriptome profile from the testes of yak and cattleyak using high-throughput sequencing. Then we used quantitative analysis to validate several differentially expressed genes (DEGs) in testicular tissue and spermatogenic cells. RESULTS: Testis transcriptome profiling identified 6477 DEGs (2919 upregulated and 3558 downregulated) between cattleyak and yak. Further analysis revealed that the marker genes and apoptosis regulatory genes for undifferentiated spermatogonia were upregulated, while the genes for differentiation maintenance were downregulated in cattleyak. A majority of DEGs associated with mitotic checkpoint, and cell cycle progression were downregulated in cattleyak during spermatogonial mitosis. Furthermore, almost all DEGs related to synaptonemal complex assembly, and meiotic progression presented no sign of expression in cattleyak. Even worse, dozens of genes involved in acrosome formation, and flagellar development were dominantly downregulated in cattleyak. CONCLUSION: DEGs indicated that spermatogenic arrest of cattleyak may originate from the differentiation stage of spermatogonial stem cells and be aggravated during spermatogonial mitosis and spermatocyte meiosis, which contributes to the scarcely presented sperms in cattleyak.

Bovid microRNAs involved in the process of spermatogonia differentiation into spermatocytes.

The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak.

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.

Isolation and characterization of spermatogenic cells from cattle, yak and cattleyak.

Cattleyak forms the first generation in the cross-breeding of cattle (Bos taurus) and yak (Bos grunniens), the purpose of which is to increase the yak's performance in meat and milk production. The female cattleyak is fertile while the male remains sterile due to spermatogenic arrest. The spermatogenic cells (including spermatogonia and spermatocytes) of cattle, yak and cattleyak have not been successfully isolated so far. In this work, spermatogenic cells were isolated from these bovid species with the STA-PUT method that has been previously used for germ cell sorting in human and mouse, and the isolated cells could be used to investigate the mechanisms involved in male sterility observed in cattleyak. The characteristics and size of the isolated cells were investigated through microscopic examination, and the cell types were identified by RT-PCR amplification of the marker genes. The purity of spermatogonia and spermatocytes isolated from each bovid species was found to be higher than 85%. The spermatogonium diameter of cattle (10.10 ±â€¯1.04 µm) and yak (14.90 ±â€¯2.30 µm) were significantly larger (P < 0.01) than that of cattleyak (8.60 ±â€¯0.92 µm). The spermatocyte diameter of cattle (19.40 ±â€¯1.50 µm) and yak (20.50 ±â€¯2.42 µm) were also significantly larger (P < 0.01) than that of cattleyak (17.70 ±â€¯2.05 µm). Therefore, the STA-PUT was again validated to be a convenient, economical and efficient method for isolation of spermatogenic cells as it yields more cells within a short time frame.

Differentially expressed microRNAs between cattleyak and yak testis.

Cattleyak are interspecific hybrids between cattle and yak, exhibiting the same prominent adaptability as yak and much higher performances than yak. However, male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted their effective utilization in yak breeding. In past decades, much work has been done to investigate the mechanisms of spermatogenic arrest, but little is known about the differences of the post-transcriptional regulators between cattleyak and yak, which may contribute to the impaired spermatogenesis. MiRNAs, a class of endogenous non-coding small RNA, were revealed to play crucial roles in regulating gene expression at post-transcriptional level. In the present study, we identified 50 differentially expressed (DE) known miRNAs and 11 novel miRNAs by using Illumina HISeq and bioinformatic analysis. A total of 50 putative target sites for the 13 DE known miRNAs and 30 for the 6 DE novel miRNAs were identified, respectively. GO and KEGG analyses were performed to reveal the functions of target genes for DE miRNAs. In addition, RT-qPCR was performed to validate the expression of the DE miRNAs and its targets. The identification of these miRNAs may provide valuable information for a better understanding of spermatogenic arrest in cattleyak.