Comprehensive analysis of differentially expressed mRNAs, circRNAs, and miRNAs and their ceRNA network in the testis of cattle-yak, yak, and cattle.

Cattle-yak is a hybrid offspring resulting from the crossbreeding of yak and cattle, and it exhibits substantial heterosis in production performance. However, male sterility in cattle-yak remains a concern. Reports suggest that noncoding RNAs are involved in the regulation of spermatogenesis. Therefore, in this study, we comprehensively compared testicular transcription profiles among cattle, yak, and cattle-yak. Numerous differentially expressed genes (DEGs), differentially expressed circRNAs (DECs), and differentially expressed miRNAs (DEMs) were identified in the intersection of two comparison groups, namely cattle versus cattle-yak and yak versus cattle-yak, with the number of DEGs, DECs, and DEMs being 4968, 360, and 59, respectively. The DEGs in cattle-yaks, cattle, and yaks were mainly associated with spermatogenesis, male gamete generation, and sexual reproduction. Concurrently, GO and KEGG analyses indicated that DEC host genes and DEM source genes were involved in the regulation of spermatogenesis. The construction of a potential competing endogenous RNA network revealed that some differentially expressed noncoding RNAs may be involved in regulating the expression of genes related to testicular spermatogenesis, including miR-423-5p, miR-449b, miR-34b/c, and miR-15b, as well as previously unreported miR-6123 and miR-1306, along with various miRNA-circRNA interaction pairs. This study serves as a valuable reference for further investigations into the mechanisms underlying male sterility in cattle-yaks.

The Effect of the Feeding System on Fat Deposition in Yak Subcutaneous Fat.

Fat deposition is very important to the growth and reproduction of yaks. In this study, the effect of the feeding system on fat deposition in yaks was explored by transcriptomics and lipidomics. The thickness of the subcutaneous fat in yaks under stall (SF) and graze feeding (GF) was evaluated. The transcriptomes and lipidomes of the subcutaneous fat in yaks under different feeding systems were detected by RNA-sequencing (RNA-Seq) and non-targeted lipidomics based on ultrahigh-phase liquid chromatography tandem mass spectrometry (UHPLC-MS), respectively. The differences in lipid metabolism were explored, and the function of differentially expressed genes (DEGs) was evaluated by gene ontology (GO) and Kyoto encyclopedia of genes and genome (KEGG) analysis. Compared with GF yaks, SF yaks possessed stronger fat deposition capacity. The abundance of 12 triglycerides (TGs), 3 phosphatidylethanolamines (PEs), 3 diglycerides (DGs), 2 sphingomyelins (SMs) and 1 phosphatidylcholine (PC) in the subcutaneous fat of SF and GF yaks was significantly different. Under the mediation of the cGMP-PKG signaling pathway, the blood volume of SF and GF yaks may be different, which resulted in the different concentrations of precursors for fat deposition, including non-esterified fatty acid (NEFA), glucose (GLU), TG and cholesterol (CH). The metabolism of C16:0, C16:1, C17:0, C18:0, C18:1, C18:2 and C18:3 in yak subcutaneous fat was mainly realized under the regulation of the INSIG1, ACACA, FASN, ELOVL6 and SCD genes, and TG synthesis was regulated by the AGPAT2 and DGAT2 genes. This study will provide a theoretical basis for yak genetic breeding and healthy feeding.

Single-Cell RNA Sequencing Reveals Atlas of Yak Testis Cells.

Spermatogenesis is a complex process that involves proliferation and differentiation of diploid male germ cells into haploid flagellated sperm and requires intricate interactions between testicular somatic cells and germ cells. The cellular heterogeneity of this process presents a challenge in analyzing the different cell types at various developmental stages. Single-cell RNA sequencing (scRNA-seq) provides a useful tool for exploring cellular heterogeneity. In this study, we performed a comprehensive and unbiased single-cell transcriptomic study of spermatogenesis in sexually mature 4-year-old yak using 10× Genomics scRNA-seq. Our scRNA-seq analysis identified six somatic cell types and various germ cells, including spermatogonial stem cells, spermatogonia, early-spermatocytes, late-spermatocytes, and spermatids in yak testis. Pseudo-timing analysis showed that Leydig and myoid cells originated from common progenitor cells in yaks. Moreover, functional enrichment analysis demonstrated that the top expressed genes in yak testicular somatic cells were significantly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and ECM receptor interactions. Throughout the spermatogenesis process, genes related to spermatogenesis, cell differentiation, DNA binding, and ATP binding were expressed. Using immunohistochemical techniques, we identified candidate marker genes for spermatogonial stem cells and Sertoli cells. Our research provides new insights into yak spermatogenesis and the development of various types of cells in the testis, and presents more reliable marker proteins for in vitro culture and identification of yak spermatogonial stem cells in the later stage.

Analysis of Chromatin Openness in Testicle Tissue of Yak and Cattle-Yak.

Cattle-yak, a crossbreed of yak and cattle, which can exhibit obvious heterosis and can adapt to the harsh environmental conditions of the Qinghai Tibet Plateau (QTP). However, F1 cattle-yak were found to be sterile because they were unable to produce sperm, which adversely restricted the fixation of heterosis. Many prior attempts have been made to decipher the mechanism underlying the spermatogenesis stagnation of cattle-yak. However, the open chromatin region (OCR) map of yak and cattle-yak testes has not been generated yet. Here, we have analyzed the OCRs landscape of testicular tissues of cattle-yak and yaks by performing ATAC-seq technology. The OCRs of cattle-yak and yak testes displayed similar genome distribution and showed priority in intergenic regions, introns and promoters. The pathway enrichment analysis indicated that the differential OCRs-related genes were involved in spermatogenesis, involving the cell cycle, as well as Hippo, mTOR, MAPK, Notch, and Wnt signaling pathways. The integration of ATAC-seq and mRNA-seq indicated that the majority of the gene expression levels were positively correlated with chromatin openness. At the same time, we have identified a number of transcription factors (TFs) related to spermatogenesis and the differential expression of these TFs may contribute to the spermatogenesis stagnation of the cattle-yak. Overall, the findings of this study provide valuable information for advancing the research related to yak crossbreeding improvement and sperm production stagnation of cattle-yak.

Complete characterization of the yak testicular development using accurate full-length transcriptome sequencing.

Alternative splicing is a prevalent phenomenon in testicular tissues. Due to the low assembly accuracy of short-read RNA sequencing technology in analyzing post-transcriptional regulatory events, full-length (FL) transcript sequencing is highly demanded to accurately determine FL splicing variants. In this study, we performed FL transcriptome sequencing of testicular tissues from 0.5, 1.5, 2.5, and 4-year-old yaks and 4-year-old cattle-yaks using Oxford Nanopore Technologies. The obtained sequencing data were predicted to have 47,185 open reading frames (ORFs), including 26,630 complete ORFs, detected 7645 fusion transcripts, 15,355 alternative splicing events, 25,798 simple sequence repeats, 7628 transcription factors, and 35,503 long non-coding RNAs. A total of 40,038 novel transcripts were obtained from the sequencing data, and the proportion was almost close to the number of known transcripts identified. Structural analysis and functional annotation of these novel transcripts resulted in the successful annotation of 9568 transcripts, with the highest and lowest annotation numbers in the Nr and KOG databases, respectively. Weighted gene co-expression network analysis revealed the key regulatory pathways and hub genes at various stages of yak testicular development. Our findings enhance our comprehension of transcriptome complexity, contribute to genome annotation refinement, and provide foundational data for further investigations into male sterility in cattle-yaks.

Single-cell RNA sequencing and UPHLC-MS/MS targeted metabolomics offer new insights into the etiological basis for male cattle-yak sterility.

The variety of species can be efficiently increased by interspecific hybridization. However, because the males in the hybrid progeny are usually sterile, this heterosis cannot be employed when other cattle and yaks are hybridized. While some system-level studies have sought to explore the etiological basis for male cattle-yak sterility, no systematic cellular analyses of this phenomenon have yet been performed. Here, single-cell RNA sequencing and UPHLC-MS/MS targeted metabolomics methods were used to study the differences in testicular tissue between 4-year-old male yak and 4-year-old male cattle-yak, providing new and comprehensive insights into the causes of male cattle-yak sterility. Cattle-yak testes samples detected 6 somatic cell types and one mixed germ cell type. Comparisons of these cell types revealed the more significant differences in Sertoli cells (SCs) and [Leydig cells and myoid cells (LCs_MCs)] between yak and cattle-yak samples compared to other somatic cell clusters. Even though the LCs and MCs from yaks and cattle-yaks were derived from the differentiation of the same progenitor cells, a high degree of overlap between LCs and MCs was observed in yak samples. Still, only a small overlap between LCs and MCs was observed in cattle-yak samples. Functional enrichment analyses revealed that genes down-regulated in cattle-yak SCs were primarily enriched in biological activity, whereas up-regulated genes in these cells were enriched for apoptotic activity. Furthermore, the genes of up-regulated in LCs_MCs of cattle-yak were significantly enriched in enzyme inhibitor and molecular function inhibitor activity. On the other hand, the genes of down-regulated in these cells were enriched for signal receptor binding, molecular function regulation, positive regulation of biological processes, and regulation of cell communication activity. The most significant annotated differences between yak and cattle-yak LCs_MCs were associated with cell-to-cell communication. While yak LCs_MCs regulated spermatogenic cells at spermatogonia, spermatocyte, and spermatid levels, no such relationships were found between cattle-yak LCs_MCs and germ cells. This may suggest that the somatic niche in male cattle-yak testes is a microenvironment that is ultimately not favorable for spermatogenesis.

Comparative Analysis of Epididymis Cauda of Yak before and after Sexual Maturity.

Epididymis development is the basis of male reproduction and is a crucial site where sperm maturation occurs. In order to further understand the epididymal development of yak and how to regulate sperm maturation, we conducted a multi-omics analysis. We detected 2274 differential genes, 222 differential proteins and 117 co-expression genes in the cauda epididymis of yak before and after sexual maturity by RNA-seq and proteomics techniques, which included TGFBI, COL1A1, COL1A2, COL3A1, COL12A1, SULT2B1, KRT19, and NPC2. These high abundance genes are mainly related to cell growth, differentiation, adhesion and sperm maturation, and are mainly enriched via extracellular matrix receptor interaction, protein differentiation and absorption, and lysosome and estrogen signaling pathways. The abnormal expression of these genes may lead to the retardation of epididymal cauda development and abnormal sperm function in yak. In conclusion, through single and combined analysis, we provided a theoretical basis for the development of the yak epididymal cauda, sperm maturation, and screening of key genes involved in the regulation of male yak reproduction.

The Study of Yak Colostrum Nutritional Content Based on Foodomics.

The utilization of yak milk is still in a primary stage, and the nutrition composition of yak colostrum is not systematically characterized at present. In this study, the lipids, fatty acids, amino acids and their derivatives, metabolites in yak colostrum, and mature milk were detected by the non-targeted lipidomics based on (ultra high performance liquid chromatography tandem quadrupole mass spectrometer) UHPLC-MS, the targeted metabolome based on gas chromatography-mass spectrometer (GC-MS), the targeted metabolome analysis based on UHPLC-MS, and the non-targeted metabolome based on ultra high performance liquid chromatography tandem quadrupole time of flight mass spectrometer (UHPLC-TOF-MS), respectively. Meanwhile, the nutrition composition of yak colostrum was compared with the data of cow mature milk in the literatures. The results showed that the nutritive value of yak colostrum was higher by contrast with yak and cow mature milk from the perspective of the fatty acid composition and the content of Σpolyunsaturated fatty acids (PUFAs), Σn-3PUFAs; the content of essential amino acid (EAA) and the ratio of EAA/total amino acid (TAA) in yak colostrum were higher than the value in yak mature milk; and the content of functional active lipids including phosphatidylcholines (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), lyso-phosphatidylcholine (LPC), lyso-phosphatidylglycerol (LPG), lyso-phosphatidylinositol (LPI), sphingomyelin (SM), ganglioside M3 (GM3), ganglioside T3 (GT3), and hexaglycosylceramide (Hex1Cer) in yak colostrum, was higher than the value of yak mature milk. Moreover, the differences of nutritive value between yak colostrum and mature milk were generated by the fat, amino acids and carbohydrate metabolism that were regulated by the ovarian hormone and referencesrenin-angiotensin-aldosterone system in yaks. These research results can provide a theoretical basis for the commercial product development of yak colostrum.

Transcriptome Studies Reveal the N6-Methyladenosine Differences in Testis of Yaks at Juvenile and Sexual Maturity Stages.

Studying the mechanism of spermatogenesis is key to exploring the reproductive characteristics of male yaks. Although N6-methyladenosine (m6A) RNA modification has been reported to regulate spermatogenesis and reproductive function in mammals, the molecular mechanism of m6A in yak testis development and spermatogenesis remains largely unknown. Therefore, we collected testicular tissue from juvenile and adult yaks and found that the m6A level significantly increased after sexual maturity in yaks. In MeRIP-seq, 1702 hypermethylated peaks and 724 hypomethylated peaks were identified. The hypermethylated differentially methylated RNAs (DMRs) (CIB2, AK1, FOXJ2, PKDREJ, SLC9A3, and TOPAZ1) mainly regulated spermatogenesis. Functional enrichment analysis showed that DMRs were significantly enriched in the adherens junction, gap junction, and Wnt, PI3K, and mTOR signaling pathways, regulating cell development, spermatogenesis, and testicular endocrine function. The functional analysis of differentially expressed genes showed that they were involved in the biological processes of mitosis, meiosis, and flagellated sperm motility during the sexual maturity of yak testis. We also screened the key regulatory factors of testis development and spermatogenesis by combined analysis, which included BRCA1, CREBBP, STAT3, and SMAD4. This study indexed the m6A characteristics of yak testicles at different developmental stages, providing basic data for further research of m6A modification regulating yak testicular development.

F1 Male Sterility in Cattle-Yak Examined through Changes in Testis Tissue and Transcriptome Profiles.

Male-derived sterility in cattle-yaks, a hybrid deriving from yak and cattle, is a challenging problem. This study compared and analyzed the histomorphological differences in testis between sexually mature yak and cattle-yak, and examined the transcriptome differences employing RNA-seq. The study found that yak seminiferous tubules contained spermatogenic cells at all levels, while cattle-yak seminiferous tubules had reduced spermatogonia (SPG) and primary spermatocyte (Pri-SPC), fewer secondary spermatocytes (Sec-SPC), an absence of round spermatids (R-ST) and sperms (S), and possessed large vacuoles. All of these conditions could have significantly reduced the volume and weight of cattle-yak testis compared to that of yak. RNA-seq analysis identified 8473 differentially expressed genes (DEGs; 3580 upregulated and 4893 downregulated). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment evaluations for DEGs found their relation mostly to spermatogenesis and apoptosis. Among the DEGs, spermatogonia stem cell (SSCs) marker genes (Gfra1, CD9, SOHLH1, SALL4, ID4, and FOXO1) and genes involved in apoptosis (Fas, caspase3, caspase6, caspase7, caspase8, CTSK, CTSB and CTSC) were significantly upregulated, while differentiation spermatogenic cell marker genes (Ccna1, PIWIL1, TNP1, and TXNDC2) and meiosis-related genes (TEX14, TEX15, MEIOB, STAG3 and M1AP) were significantly downregulated in cattle-yak. Furthermore, the alternative splicing events in cattle-yak were substantially decreased than in yak, suggesting that the lack of protein subtypes could be another reason for spermatogenic arrest in cattle-yak testis.

Huang Bai Jian Pi decoction alleviates diarrhea and represses inflammatory injury via PI3K/Akt/NF-κB pathway: In vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang Bai Jian Pi (HBJP) decoction, a Chinese herbal formula based on the Pulsatilla decoction (PD) and Si Junzi decoction, is efficacy to treat clinical diarrhea in calves. AIM OF THE STUDY: The mechanism of HBJP decoction to treat calf diarrhea remains unclear. This study was to investigate the therapeutic effect and anti-inflammatory mechanism of HBJP decoction on diarrhea in rats. MATERIALS AND METHODS: Thirty-six Sprague Dawley rats were randomly divided into control group, model group, PD group and three treated groups with HBJP decoction. The diarrheal model in rats was established by multiple factors including high-sugar and fat diet, high temperature and dampness environment, biological pathogenic factors. The diarrheal animals were treated with HBJP decoction or PD for 5 days. The inflammatory model of the intestinal epithelioid cell line 6 (IEC-6) was induced by TNF-α. The clinical symptoms, blood routine and biochemistry parameters, histopathology of main organs were detected. The proteins associated with PI3K/Akt/NF-κB pathway and the expression levels of cytokines associated with inflammation were detected in vivo and in vitro by Western blot and ELISA. RESULTS: The model rats showed obvious diarrheal symptoms, and the obvious systemic inflammatory response accompanied with abnormal change in blood routine, biochemistry parameters and histopathology. HBJP decoction alleviated obviously the clinical symptoms, and pathological changes of the liver, colon and lung, and abnormal blood routine and biochemistry indexes in rats. The expression of P-PI3K, P-Akt, P-NF-κB, IL-1ß, IL-6 was significantly increased, and the expression of IL-10 was markedly decreased in diarrheal rats and IEC-6 with inflammation. HBJP decoction significantly inhibited the PI3K/AKT/NF-κB signal pathway and adjusted the expression of these inflammatory cytokines. CONCLUSIONS: The finding suggested that HBJP decoction alleviate the inflammation in diarrhea through inhibiting the PI3K/Akt/NF-κB signal pathway, which provides scientific evidences for the clinical application of HBJP decoction in diarrhea.

Characterization of N6-methyladenosine in cattle-yak testis tissue.

N6-methyladenosine (m6A) is the most common form of eukaryotic mRNA modification, and it has been shown to exhibit broad regulatory activity in yeast, plants, and mammals. The specific role of m6A methylation as a regulator of spermatogenesis, however, has yet to be established. In this experiment, through a series of preliminary studies and methylated RNA immunoprecipitation sequencing, the m6A map of cattle-yak testicular tissue was established as a means of exploring how m6A modification affects cattle-yak male infertility. Cattle-yak testis tissues used in this study were found to contain sertoli cells and spermatogonia. Relative to sexually mature yak samples, those isolated from cattle-yak testis exhibited slightly reduced levels of overall methylation, although these levels were significantly higher than those in samples from pre-sexually mature yaks. Annotation analyses revealed that differentially methylated peaks were most concentrated in exonic regions, with progressively lower levels of concentration in the 3'-untranslated region (UTR) and 5'-UTR regions. To further explore the role of such m6A modification, enrichment analyses were performed on differentially methylated and differentially expressed genes in these samples. For the cattle-yaks vs. 18-months-old yaks group comparisons, differentially methylated genes were found to be associated with spermatogenesis-related GO terms related to the cytoskeleton and actin-binding, as well as with KEGG terms related to the regulation of the actin cytoskeleton and the MAPK signaling pathway. Similarly, enrichment analyses performed for the cattle-yaks vs. 5-years-old yaks comparison revealed differentially methylated genes to be associated with GO terms related to protein ubiquitination, ubiquitin ligase complexes, ubiquitin-dependent protein catabolism, and endocytotic activity, as well as with KEGG terms related to apoptosis and the Fanconi anemia pathway. Overall, enrichment analyses for the cattle-yaks vs. 18-months-old yaks comparison were primarily associated with spermatogenesis, whereas those for the cattle-yaks vs. 5-years-old yaks comparison were primarily associated with apoptosis.

Identification of the TSSK4 Alternative Spliceosomes and Analysis of the Function of the TSSK4 Protein in Yak (Bos grunniens).

In mammals, the testis-specific serine/threonine kinase (TSSK) is essential for spermatogenesis and male fertility. TSSK4 belongs to the family of the testis-specific serine/threonine-protein kinase (TSSK), with a crucial role in spermatogenesis. This study aimed to analyze the variable spliceosome of the TSSK4 gene in the yak for understanding the regulatory function of the TSSK4 spliceosome in yak testis development using PCR amplification and cloning techniques. The GST pull-down was used for pulling down the protein interacting with TSSK4, and then the protein interacting with TSSK4 was identified using LC-MS/MS. The results of the PCR amplification demonstrated multiple bands of the TSSK4 gene in the yak. The cloning and sequencing yielded a total of six alternative spliceosomes, which included only two alternative spliceosomes before sexual maturity and four alternative spliceosomes after sexual maturity. The sub-cells of the alternative spliceosomes were found to localize in the nucleus before sexual maturity and in the cytoplasm after sexual maturity. The LC-MS/MS analysis of the alternative spliceosome with the highest expression after sexual maturity yielded a total of 223 interacting proteins. The enrichment analysis of the 223 interacting proteins revealed these proteins participate in biological processes, cell composition, and molecular functions. The KEGG analysis indicated that the TSSK4-interacting protein participates in the estrogen signaling pathways, tight junctions, endoplasmic reticulum protein processing, and other signaling pathways. This study cloned the six alternative spliceosomes of the TSSK4 gene laying the foundation for studying the function of each spliceosome in the future.

Whole-Genome Resequencing Highlights the Unique Characteristics of Kecai Yaks.

Kecai yaks are regarded as an important genetic resource in China owing to their high fecundity and flavorful meat. However, the genetic characteristics of Kecai yaks have not been effectively characterized to date, and the relationship between Kecai yaks and other yak breeds remains to be fully characterized. In this paper, the resequencing of the Kecai yak genome is performed leading to the identification of 11,491,383 high-quality single nucleotide polymorphisms (SNPs). Through principal component, phylogenetic, and population genetic structure analyses based on these SNPs, Kecai yaks were confirmed to represent an independent population of yaks within China. In this study, marker and functional enrichment analysis of genes related to positive selection in Kecai yak was carried out, and the results show that such selection in Kecai yaks is associated with the adaptation to alpine environments and the deposition of muscle fat. Overall, these results offer a theoretical foundation for the future utilization of Kecai yak genetic resources.