TserangDonko Mipam

Comparative analysis of testis transcriptomes associated with male infertility in cattleyak

Cattleyak exhibit equivalent adaptability to harsh environment as yak and much higher performances than yak. However, male infertility of cattleyak due to spermatogenic arrest greatly restricts their effective utilization in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest, there is little information available in regard to the differences in transcriptomic profiling between cattleyak and yak testes. In this work, histologic observation indicated that spermatogonia were the main type of germ cells present in cattleyak testis, whereas all types of germ cells in differentiation were present in yak testis. Transcriptomic profiling identified 2960 differentially expressed genes (DEGs) in which 679 were upregulated and 2281 were downregulated in cattleyak. Significantly enriched gene ontology terms comprised a large number of DEGs associated with male infertility of cattleyak. The upregulation of STRA8 and NLRP14 may be associated with the accumulation of undifferentiated spermatogonial cells and serious cellular apoptosis in cattleyak. However, downregulated SPP1, SPIN2B, and PIWIL1 were associated with cell cycle progression and spermatogonial genome integrity, whereas CDKN2C, CYP26A1, OVOL1, GGN, MAK, INSL6, RNF212, TSSK1B, TSSK2, and TSSK6 were involved in meiosis. Furthermore, scores of genes associated with sperm components were also downregulated in cattleyak. Wnt/β-catenin signaling pathway was involved in the top-listed three significantly enriched pathways, and the downregulation of Wnt3a, PP2A, and TCF/LEF-1 may have contributed to the arrest of spermatogonial differentiation in cattleyak. The data suggest that spermatogenic arrest of cattleyak might occur at the stage of spermatogonial differentiation and get aggravated during meiosis, which results in minimal number of sperms with morphologic abnormalities and structural deficiency lacking fertilization ability.

SNPs detected in the yak MC4R gene and their association with growth traits.

MC4R (melanocortin 4 receptor) is expressed in the appetite-regulating areas of the brain and takes part in leptin signaling pathways. Sequencing of the coding region of the MC4R gene for 354 yaks identified the following five single nucleotide polymorphisms (SNPs): SNP1 (273C>T), SNP2 (321 G>T), SNP3 (864 C>A), SNP4 (1069G>C) and SNP5 (1206 G>C). SNP1, SNP2 and SNP3 were synonymous mutations, whereas SNP4 and SNP5 were missense mutations resulting in amino acid substitutions (V286L and R331S). Pairwise linkage disequilibrium (LD) analysis indicated that two pairs of SNPs, SNP2 and SNP5 (r(2)=0.81027) and SNP4 and SNP5 (r(2)=0.53816), exhibited higher degrees of LD. CC genotype of SNP4, CGACG and CTCCC haplotypes for all SNPs were associated with increased BW of animals that were 18 months old and with the average daily gain. The secondary structure and transmembrane region prediction of the yak MC4R protein suggested that SNP4 was correlated with influential changes in the seventh transmembrane domain of the MC4R protein and with the functional deterioration or even incapacitation of MC4R, which may contribute to the increased feed intake, BW and average daily gain of the yaks with CC genotypes. The data from this study suggested that 1069G>C SNP of the MC4R gene could be used in marker-assisted selection of growth traits in the Maiwa yak breed.

Abundant variations of MC4R gene revealed by Phylogenies of Yak (Bos grunniens) and other mammals

MC4R gene was proved to play important roles in body weight regulation in many mammals and exhibit higher homology among different species. The mutations MC4R significantly correlated to the restricted feeding weight, fat deposition and energy balance. In this work, ORF sequences of MC4R gene of Bos grunniens were cloned and phylogenetic relationships of yak and other mammals were analyzed on the basis of MC4R genes. Totally 290 variable sites were examined in 25 sequences from 22 different mammals, and 23 haplotypes were defined with a haplotype diversity of 0.9900. All the sequences were clustered into phylogenetic clades representing different orders or families. The individuals of Bos grunniens, Bos taurus and Ovis aries which belonged to the family of Bovidae were more divergent from the other orders or families and bovid animals may have branched out from the phylogenetic tree earlier than other mammals analyzed during 450 million years of vertebrate evolution. Amino acid sequences inferred from MC4R genes exhibited 54 variable sites, while high conservation of MC4R was observed within the same order or family. We concluded that coding region of MC4R gene displayed abundant variations among different mammal phylogenetic clades, whereas, the conservation of MC4R within order or family could be explained that MC4R gene may have been subjected to substantial constraints or strong purifying selection during several million years of mammal evolution.

Comparative iTRAQ proteomics revealed proteins associated with spermatogenic arrest of cattleyak.

UNLABELLED Male infertility of cattleyak due to spermatogenic arrest greatly restricts their effective utilization in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest, there is no information regarding the differences of protein composition between cattleyak and yak testis. Comparative investigation of testis proteomes between cattleyak and yak using iTRAQ proteomics identified 256 differentially abundant proteins with fold change values higher than ±1.5. Most of the differentially abundant proteins were involved in extracellular matrix organization, response to stimulus, metabolic and cellular process, in which a large number of the cattleyak predominant proteins were associated with various stresses, cell adhesion and germ cell migration. Such upregulated proteins as integrins and their ligands in the extracellular matrix involved in ECM-receptor interaction pathway may help germ cells to endure pulling forces and impede their migration. In contrast, down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Such Downregulated proteins as the subunits of mitochondrial cytochrome Bc1 complex involved in the Alzheimers disease and oxidative phosphorylation pathways may lead to mitochondrial dysfunction and cell death in cattleyak testis. BIOLOGICAL SIGNIFICANCE Spermatogenic arrest of cattleyak involves defects of both germ cells and their micro-environment in testis. In this study, Dozens of proteins possibly associated with spermatogenic arrest of cattleyak were identified by comparative iTRAQ proteomics, in which a large number of the cattleyak predominant proteins may act in response to various stresses (especially inflammatory stresses), enhance cell adhesion and impeded germ cell migration, while down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Therefore, germ cells in testis of cattleyak may be constantly in a stress state (similar to inflammation) due to accumulation of some toxic intermediate products resulted from metabolic disturbances. Metabolic defects of germ cells and their deleterious micro-environment in testis of cattleyak may be the root of all other problems during spermatogenesis.

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.

Comparative testis proteome of cattleyak from different developmental stages.

Cattleyak (hybrid of cattle and yak) exhibit higher capability in adaptability and production than cattle and yak, while the infertility of F1 males greatly restricts the effective utilization of this hybrid and little progress has been made on investigating the mechanisms of the cattleyak infertility. Cattleyak individuals at three development stages (10, 12 and 14-month old) were sampled in this work and the isobaric tag for relative and absolute quantification method was employed to identify differences between their testicular proteomes. The proteomic analysis identified 318 proteins differentially expressed with significance at 12-month stage and 327 at 14-month compared with 10-month stage, respectively. Compared with the testicular proteome from 10-month cattleyak, the gene ontology (GO) annotations of the differentially expressed proteins at 12 months did not indicate significant differences from those at 14 months, which confirmed the histological observation that germ cell reduction was more obvious and spermatogenic arrest may become more serious in 12-month-old cattleyak. On the other hand, 56 differentially expressed proteins were coexpressed at 12 and 14-month stage compared with 10-month stage, in which 32 proteins were upregulated and 24 downregulated. GO analysis revealed that most of the differently expressed proteins were involved in the molecular function of catalytic activity, transporter activity, oxidoreductase activity and protein binding. Further analysis indicated that the differently expressed proteins including testis-expressed protein 101 precursor, RNA-binding motif protein, X chromosome, putative RNA-binding protein 3, heparin-binding proteins, tudor domain-containing protein 1, glutathione S-transferases (GSTA2, GSTP1), heat shock-related 70 kDa protein 2, estradiol 17-β-dehydrogenase11, 2,4-dienoyl-CoA reductase and peroxiredoxin-2 were possibly associated with testis development and spermatogenesis, which could be selected as candidate proteins in future study to examine the mechanisms of cattleyak infertility.

Comparative testis proteome dataset between cattleyak and yak

Cattleyak are hybrid between cattle and yak, which exhibit equivalent adaptability on the Qinghai-Tibetan Plateau as yak and much higher capability in economic traits. However, the F1 males of cattleyak are infertile due to spermatogenic arrest and this greatly restricts the effective utilization of this hybrid. In this data article, differentially expressed proteins (DEPs) were identified from testis proteome of cattleyak and yak using high-performance liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS). All the DEPs were subjected to functional classification by Gene Ontology (GO) analysis and gene-pathway annotation by Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative testis proteome dataset here can shed new light on the molecular characteristics of male infertility of cattleyak on proteome level, “Comparative iTRAQ proteomics revealed proteins associated with spermatogenic arrest of cattleyak” [1].

Transcriptome profile and unique genetic evolution of positively selected genes in yak lungs

The yak (Bos grunniens), which is a unique bovine breed that is distributed mainly in the Qinghai-Tibetan Plateau, is considered a good model for studying plateau adaptability in mammals. The lungs are important functional organs that enable animals to adapt to their external environment. However, the genetic mechanism underlying the adaptability of yak lungs to harsh plateau environments remains unknown. To explore the unique evolutionary process and genetic mechanism of yak adaptation to plateau environments, we performed transcriptome sequencing of yak and cattle (Bos taurus) lungs using RNA-Seq technology and a subsequent comparison analysis to identify the positively selected genes in the yak. After deep sequencing, a normal transcriptome profile of yak lung that containing a total of 16,815 expressed genes was obtained, and the characteristics of yak lungs transcriptome was described by functional analysis. Furthermore, Ka/Ks comparison statistics result showed that 39 strong positively selected genes are identified from yak lungs. Further GO and KEGG analysis was conducted for the functional annotation of these genes. The results of this study provide valuable data for further explorations of the unique evolutionary process of high-altitude hypoxia adaptation in yaks in the Tibetan Plateau and the genetic mechanism at the molecular level.

Genetic Diversity, Molecular Phylogeny and Selection Evidence of Jinchuan Yak Revealed by Whole-Genome Resequencing

Jinchuan yak, a newly discovered yak breed, not only possesses a large proportion of multi-ribs but also exhibits many good characteristics, such as high meat production, milk yield, and reproductive performance. However, there is limited information about its overall genetic structure, relationship with yaks in other areas, and possible origins and evolutionary processes. In this study, 7,693,689 high-quality single-nucleotide polymorphisms were identified by resequencing the genome of Jinchuan yak. Principal component and population genetic structure analyses showed that Jinchuan yak could be distinguished as an independent population among the domestic yak population. Linkage disequilibrium analysis showed that the decay rate of Jinchuan yak was the lowest of the domestic yak breeds, indicating that the degree of domestication and selection intensity of Jinchuan yak were higher than those of other yak breeds. Combined with archaeological data, we speculated that the origin of domestication of Jinchuan yak was ∼6000 yr ago (4000–10,000 yr ago). The quantitative dynamics of population growth history in Jinchuan yak was similar to that of other breeds of domestic and wild yaks, but was closer to that of the wild yak. No significant gene exchange between Jinchuan and other domestic yaks occurred. Compared with other domestic yaks, Jinchuan yak possessed 339 significantly and positively selected genes, several of which relate to physiological rhythm, histones, and the breed’s excellent production characteristics. Our results provide a basis for the discovery of the evolution, molecular origin, and unique traits of Jinchuan yak.