Xingxu Zhao

Reproductive aspects and storage of semen in Camelidae

The characteristics of male and female reproductive tracts and reproductive physiology in camelids are described. An account is given on methods of collection, characteristics and storage of semen, and fertility after artificial insemination (AI) with fresh, liquid-stored and frozen-thawed lamoid and camel semen.

Molecular Cloning, Bioinformatics Analysis and Expression of Insulin-Like Growth Factor 2 from Tianzhu White Yak, Bos grunniens

The IGF family is essential for normal embryonic and postnatal development and plays important roles in the immune system, myogenesis, bone metabolism and other physiological functions, which makes the study of its structure and biological characteristics important. Tianzhu white yak (Bos grunniens) domesticated under alpine hypoxia environments, is well adapted to survive and grow against severe hypoxia and cold temperatures for extended periods. In this study, a full coding sequence of the IGF2 gene of Tianzhu white yak was amplified by reverse transcription PCR and rapid-amplification of cDNA ends (RACE) for the first time. The cDNA sequence revealed an open reading frame of 450 nucleotides, encoding a protein with 179 amino acids. Its expression in different tissues was also studied by Real time PCR. Phylogenetic tree analysis indicated that yak IGF2 was similar to Bos taurus, and 3D structure showed high similarity with the human IGF2. The putative full CDS of yak IGF2 was amplified by PCR in five tissues, and cDNA sequence analysis showed high homology to bovine IGF2. Moreover the super secondary structure prediction showed a similar 3D structure with human IGF2. Its conservation in sequence and structure has facilitated research on IGF2 and its physiological function in yak.

Analysis of copy number variations by SNP50 BeadChip array in Chinese sheep

The sheep (Ovis aries) plays a major socio-economic role in the world. Copy number variations (CNVs) are increasingly recognized as a key and potent source of genetic variation and phenotypic diversity, but little is known about the extent to which CNVs contribute to genetic variation in Chinese sheep breeds. Analyses of CNVs in the genomes of eight sheep breeds were performed using the sheep SNP50 BeadChip genotyping array. A total of 111 CNV regions (CNVRs) were obtained from 160 Chinese sheep breeds. These CNVRs covered 13.75Mb of the sheep genome sequence. A total of 22 Go terms and 17 candidate genes were obtained from the functional analysis. Ten CNVRs were selected for validation, of which 7 CNVRs were further experimentally confirmed by quantitative PCR. Four candidate genes were selected to confirm the results of the functional analysis. These results provide a resource for furthering understanding of ruminant biology, and for further improving the genetic quality of sheep breeds.

Identification and verification of potential piRNAs from domesticated yak testis

PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA–protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18–40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18–30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM–receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity.

Yak IGF2 Promotes Fibroblast Proliferation Via Suppression of IGF1R and PI3KCG Expression

Insulin-like growth factor 2 (IGF2) recapitulates many of the activities of insulin and promotes differentiation of myoblasts and osteoblasts, which likely contribute to genetic variations of growth potential. However, little is known about the functions and signaling properties of IGF2 variants in yaks. The over-expression vector and knockdown sequence of yak IGF2 were transfected into yak fibroblasts, and the effects were detected by a series of assays. IGF2 expression in yak muscle tissues was significantly lower than that of other tissues. In yak fibroblasts, the up-regulated expression of IGF2 inhibits expression of IGF1 and insulin-like growth factor 2 receptor (IGF2R) and significantly up-regulates expression of IGF1R. Inhibition of IGF2 expression caused the up-regulates expression of IGF1, IGF1R and IGF2R. Both over-expression and knockdown of IGF2 resulted in up-regulation of threonine protein kinase 1 (Akt1) expression and down-regulation of phosphatidylinositol 3-kinase, catalytic subunit gamma (PIK3CG). Cell cycle and cell proliferation assays revealed that over-expression of IGF2 enhanced the DNA synthesis phase and promoted yak fibroblasts proliferation. Conversely, knockdown of IGF2 decreased DNA synthesis and inhibited proliferation. These results suggested that IGF2 was negatively correlated with IGF1R and PIK3CG and demonstrated an association with the IGFs-PI3K-Akt (IGFs-phosphatidylinositol 3-kinase- threonine protein kinase) pathway in cell proliferation and provided evidence supporting the functional role of IGF2 for use in improving the production performance of yaks.

Expression and cellular localization of double sex and mab-3 related transcription factor 1 in testes of postnatal Small-Tail Han sheep at different developmental stages

Double sex and mab-3 related transcription factor 1 (Dmrt1), an evolutionarily conserved gene, is a sex-related gene expressed in male gonads, that is involved in the regulation of sex differentiation, testicular development and reproductive function maintenance. Until now, functional studies on the Dmrt1 gene in sheep (Ovis aries) have been lacking. In this study, testis, heart, liver, spleen, lung, kidney and longissimus dorsi muscle tissues were collected from Small-Tail Han sheep at 0, 2, 5, 12 and 24months after birth (mab). Dmrt1 expression and cellular localization were detected in various testicular tissues by quantitative real time PCR (qRT-PCR), western blot and immunohistochemistry methods. The morphological structures of testicular tissues at different developmental stages were observed by hematoxylin & eosin (HE) staining. The Dmrt1 mRNA expression levels in 12 and 24 mab sheep were significantly higher than those in 0 and 2 mab sheep (P<0.05), and Dmrt1 protein expression showed a similar trend. The qRT-PCR results in various tissues at 12 mab showed that Dmrt1 mRNA was predominantly expressed in testes. Immunohistochemical staining in testes at different developmental stages showed that Dmrt1 protein immunoreactive responses were mainly localized in Sertoli cells and gonocytes at 0, 2 and 5 mab, while they were localized in spermatocytes, sperm cells and some spermatogonia and Sertoli cells at 12 and 24 mab. We speculate that the Dmrt1 gene plays a vital role in postnatal sheep spermatogenesis, perhaps by regulating the maturation and functional maintenance of Sertoli cells, the proliferation and differentiation of gonocytes in prepubertal sheep testes, and the mitosis and meiosis of germ cells in adult sheep, but the specific mechanisms underlying these phenomena must be further studied and verified. ABBREVIATIONS

Identification of novel and differentially expressed microRNAs in ovine ovary and testis tissues using Solexa sequencing and bioinformatics

MicroRNAs (miRNAs) are small, single stranded, non-coding RNA molecules, about 19–25 nucleotides in length, which regulate the development and functions of reproductive system of mammal. To discover novel miRNAs and identify the differential expression of them in ovine ovary and testis tissues, the study constructed two libraries by using next-generation sequencing technologies (Solexa high-throughput sequencing technique). As a result, 9 321 775 and 9 511 538 clean reads were obtained from the ovary and testis separately, which included 130 562 (90 genes of ovary) and 56 272 (85 genes of testis) of known miRNAs and 486 potential novel miRNAs reads. In this study, a total of 65 conserved miRNAs were significantly differentially expressed (P<0.01) between the two samples. Among them, 28 miRNAs were up-regulated and 3 miRNAs were down-regulated on ovary compared with testis. In addition, the known miRNAs with the highest expression level (5 miRNAs) and 30 novel miRNAs with the functions related to reproduction were validated using the real-time quantitative RT-PCR. Moreover, the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expressed miRNAs were involved in ovary and testis physiology, including signal transduction, gonad development, sex differentiation, gematogenesis, fertilization and embryo development. The results will be helpful to facilitate studies on the regulation of miRNAs during ruminant reproduction.

Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensional electrophoresis (2-DE) analysis in the project of breeding a new variety of sheep. Differential expression proteins were detected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed through liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 480 total protein spots displayed in 2-D gels, 6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center for Biotechnology Information databases (NCBI) indicated that differentially expressed proteins correspond to two proteins, identified to be enolase and transcription factor AP-2-alpha (TFAP-2α). The two proteins were up-regulated in the irradiated sperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation in sheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheep reproduction and in clarifying the mechanisms involved in irradiation or space breeding.

Comprehensive Analysis of MicroRNA–Messenger RNA from White Yak Testis Reveals the Differentially Expressed Molecules Involved in Development and Reproduction

Testis development is a vital and tightly regulated process in mammals. Understanding the biological mechanisms underlying testis development will benefit the animal reproduction industry. Expression changes in microRNA and messenger RNA in response to dynamic regulation effects have been associated with this process. However, very little is known about the roles of these molecules in yak development. Using whole-genome small RNA and messenger RNA sequencing, we performed a comprehensive analysis of the microRNA–messenger RNA interaction network expression in the testicles of Tianzhu white yaks during three developmental stages. Using Short Time-series Expression Miner analysis we identified 589 differentially expressed microRNAs (DERs) and 3383 differentially expressed messenger RNAs (DEGs) in the three age groups. A total of 93 unique DEGs are primarily involved in reproduction and testis development. Subsequently, four integration networks were constructed according to the DEGs and DERs in three biological processes. Nineteen DEGs were potentially regulated by 60 DERs, of which miR-574 and target gene AURKA played a crucial role in yak testis development and reproduction. The results of this study provide a basis for further exploration of the microRNA–messenger RNA interactions in testis development and reproduction and aid in uncovering the molecular mechanisms of spermatogenesis in male mammals.

Comparative Profiling of MicroRNAs Reveals the Underlying Toxicological Mechanism in Mice Testis Following Carbon Ion Radiation

This study investigated the toxicity of heavy ion radiation to mice testis by microRNA (miRNA) sequencing and bioinformatics analyses. Testicular indices and histology were measured following enterocoelia irradiation with a 2 Gy carbon ion beam, with the testes exhibiting the most serious injuries at 4 weeks after carbon ion radiation (CIR) exposure. Illumina sequencing technology was used to sequence small RNA libraries of the control and irradiated groups at 4 weeks after CIR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses implicated differential miRNAs in the regulation of target genes involved in metabolism, development, and reproduction. Here, 8 miRNAs, including miR-34c-5p, miR-138, and 6 let-7 miRNA family members previously reported in testis after radiation, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate miRNA sequencing data. The differentially expressed miRNAs described here provided a novel perspective for the role of miRNAs in testis toxicity following CIR.