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.

Effect of second booster vaccination on clinical outcomes of Omicron-variant breakthrough infection: A propensity score matching cohort study.

Objective: To further explore the effect of vaccination regimen and frequency on clinical outcomes of breakthrough infections caused by the Omicron variant, as well as the durability of vaccine effectiveness. Methods: A retrospective, propensity score matching, real-world cohort study was conducted. Vaccination frequency was categorized into regular vaccination, first booster, and second booster. Results: A total of 7428 cases were included, with 3910 (53 %) being male. The median age was 39 years. BA.2 than BA.5/5.2 infection presented with more pulmonary symptoms and fewer influenza-like symptoms. Among the 3516 cases of BA.5/5.2 breakthrough infections, patients who received the second booster than the first booster or regular vaccination had higher first IgM and IgG titers and first cycle thredhold values for N gene on admission, a lower percentage of fever, lower peak body temperatures, and a higher percentage of asymptomatic cases. Patients who received the first booster vaccinated with homologous mRNA or heterologous inactivated plus mRNA vaccines than homologous inactivated vaccines had higher first IgM and IgG titers, a higher percentage of asymptomatic cases, and a lower percentage of fever. Moreover, significantly different first IgG titers were observed among patients receiving the second booster vaccinated with any of the three regimens. There was no statistical difference between booster regimens of homologous mRNA vaccines and heterologous inactivated plus mRNA vaccines. Patients in Month 7- than Month 0-6 after the first booster had lower first IgM and IgG titers and first cycle thredhold values, a lower percentage of asymptomatic cases, and a higher percentage of fever; and a higher percentage of pneumonia after the second booster. Conclusions: Repeated booster vaccinations every six months, with priority given to heterologous mRNA vaccine booster regimens in countries previously primarily using inactivated vaccines, may provide protection for adult patients with Omicron-variant breakthrough infections and improve clinical outcomes.

Do microbial-gut-muscle mediated by SCFAs, microbial-gut-brain axis mediated by insulin simultaneously regulate yak IMF deposition?

Ruminant rumen plays an important role in the digestibility of cellulose, hemicellulose, starch and fat. In this study, the yaks under graze and stall feeding were chosen as the models of different rumen bacteria and intramuscular fat (IMF). The characteristics of IMF deposition, serum indexes in yaks were detected; the bacteria, metabolites in rumen was explored by 16S rRNA sequencing technology, untargeted metabolomics based on liquid chromatography-mass spectrometer and gas chromatography, respectively; the transcriptome of longissimus thoracis was identified by RNA-Sequencing analysis. Based on above results, a hypothesis that yak IMF deposition is regulated by the combined action of microbiome-gut-brain and muscle axis was proposed. The short-chain fatty acids (SCFAs) and neurotransmitters precursors like acetylcholine produced in yak rumen promoted insulin secretion via central nervous system. These insulin resulted in the high expression of SREBF1 gene by gut-brain axis; SCFAs can directly arrive to muscular tissue via blood circulation system, then activated the expression of PPARγ gene by gut-muscle axis. The expression of lipogenesis gene SCD, FABP3, CPT1, FASN and ACC2 was accordingly up-regulated. This study firstly introduce the theory of microbiome-gut-brain/muscle axis into the study of ruminant, and comprehensively expounded the regulatory mechanism of yak IMF deposition.

Analysis of multiple organ function damage in patients with severe COVID-19 pneumonia.

Background: This study aims to analyze the changes and significance of organ function indices in patients with severe Coronavirus Disease 2019 (COVID-19) pneumonia for prediction of major organ damages and guiding treatment schemes. Methods: 63 patients with severe COVID-19 pneumonia were selected as the severe group and 73 patients with mild syndromes were selected as the mild group. SAS9.4 software was used for statistical analysis of the data. Results: Levels of ALT, AST, cTnI, Cr, PT, APTT and Ddimer of the severe group were significantly higher while PLT was lower than those of the mild group. The data of all quantitative variables were converted into categorical variables. Significantly higher levels of AST, ALB, D-dimer and higher proportion of bilateral lung involvement were observed from the severe group comparing to those in the mild group, while the difference in the other indices between the two groups was insignificant in statistical perspective. Conclusions: There are significant differences in the levels of multiple organ function indices between the severe group and the mild group of patients with COVID-19 pneumonia infection. Through examining the relevant indices, conditions of patients' multiple organ function damage could be predicted and used as guidance of treatment.

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.

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.

Efficacy and safety of SIM0417 (SSD8432) plus ritonavir for COVID-19 treatment: a randomised, double-blind, placebo-controlled, phase 1b trial.

Background: SIM0417 (SSD8432) is an orally administered coronavirus main proteinase (3CLpro) inhibitor with potential anti-SARS-CoV-2 activity. This study aimed to evaluate the efficacy and safety of SIM0417 plus ritonavir (a pharmacokinetic enhancer) in adults with COVID-19. Methods: This was a randomised, double-blind, placebo-controlled, phase 1b study in China. Adults with asymptomatic infection, mild or moderate COVID-19 were randomly assigned (3:3:2) to receive either 750 mg SIM0417 plus 100 mg ritonavir, 300 mg SIM0417 plus 100 mg ritonavir or placebo every 12 h for 10 doses. The main efficacy endpoints included SARS-CoV-2 viral load, proportion of participants with positive SARS-CoV-2 nucleic acid test and time to alleviation of COVID-19 symptoms. This trial is registered with ClinicalTrials.gov, NCT05369676. Findings: Between May 12 and August 29, 2022, 32 participants were enrolled and randomised to high dose group (n = 12), low dose group (n = 12) or placebo (n = 8). The viral load change from baseline in high dose group was statistically lower compared with placebo, with a maximum mean difference of -2.16 ± 0.761 log10 copies/mL (p = 0.0124) on Day 4. The proportion of positive SARS-CoV-2 in both active groups were lower than the placebo. The median time to sustained alleviation of COVID-19 symptoms was 2.0 days in high dose group versus 6.0 days in the placebo group (HR = 3.08, 95% CI 0.968-9.818). SIM0417 plus ritonavir were well tolerated with all adverse events in grade 1. Interpretation: SIM0417 plus ritonavir was generally well tolerated. The efficacy of SIM0417 showed a monotonic dose-response relationship, and the 750 mg SIM0417 plus 100 mg ritonavir was selected as the recommended clinical dose. Funding: The study was funded by Jiangsu Simcere Pharmaceutical Co., Ltd.

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.

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.

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.

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.

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.

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.

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.

Low-to-Moderate Dosage and Short-Term Use of Corticosteroids Benefit Patients With Severe COVID-19 Infections.

Although the FDA has given emergency use authorization (EUA) for some antiviral drugs for the treatment of COVID-19, no direct antiviral drugs have been identified for the treatment of critically ill patients, the most important treatment is suppression of the hyperinflammation. The purpose of this study was to evaluate the role of corticosteroids in hospitalized severe or critical patients positive for COVID-19. This is a retrospective single-center descriptive study. Patients classified as having severe or critical COVID-19 infections with acute respiratory dysfunction syndrome in Shenzhen Third People's Hospital were enrolled from January 11th to March 30th, 2020. Ninety patients were classified as having severe or critical COVID-19 infections. The patients were treated with methylprednisolone with a low-to-moderate dosage and short duration. The days from the symptom onset to methylprednisolone were about 8 days. Eighteen patients were treated with invasive ventilation and intensive care unit (ICU) care. All the patients in the severe group and ten in the critical group recovered and were discharged. Three critical cases with invasive ventilation died. Although cases were much more severe in the corticosteroid-treated group, the mortality was not significantly increased. Early use of low-to-moderate dosage and short duration of corticosteroid may be the more accurate immune-modulatory treatment and brings more benefits to severe patients with COVID-19.

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.

The transcriptome-wide N6-methyladenosine (m6A) map profiling reveals the regulatory role of m6A in the yak ovary.

BACKGROUND AND AIM: Yak estrus is a seasonal phenomenon, probably involving epigenetic regulation of synthesis and secretion of sex hormones as well as growth and development of follicles. N6-methyladenosine (m6A) is the most common internal modification of the eukaryotic mRNA. However, there are no detailed reports on the m6A transcriptome map of yak ovary. Therefore, this study aimed to collected the yak ovarian tissues at three different states of anestrus (YO-A), estrus (YO-F), and pregnancy (YO-P), and obtained the full transcriptome m6A map in yak by MeRIP-seq. RESULTS: The HE staining revealed that the number of growing follicles and mature follicles in the ovary during the estrus period was relatively higher than those in the anestrus period and the pregnancy period. The RT-qPCR showed that the expression of METTL3, METTL14, FTO, YTHDC1 were significantly different across different periods in the ovaries, which suggests that m6A may play a regulatory role in ovarian activity. Next, we identified 20,174, 19,747 and 13,523 m6A peaks in the three ovarian samples of YO-A, YO-F and YO-P using the methylated RNA immunoprecipitation sequencing (MeRIP-seq). The m6A peaks are highly enriched in the coding sequence (CDS) region and 3'untranslated region (3'UTR) as well as the conserved sequence of "RRACH." The GO, KEGG and GSEA analysis revealed the involvement of m6A in many physiological activities of the yak's ovary during reproductive cycle. The association analysis found that some genes such as BNC1, HOMER1, BMP15, BMP6, GPX3, and WNT11 were related to ovarian functions. CONCLUSIONS: The comparison of the distribution patterns of methylation peaks in the ovarian tissues across different periods further explored the m6A markers related to the regulation of ovarian ovulation and follicular development in the yak ovary. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the yak ovary.

Changes in Transcriptomic Profiles in Different Reproductive Periods in Yaks.

Yak reproductive characteristics have received extensive attention, though the molecular regulation mechanism of its ovarian activity remains to be explored. Therefore, this study initially conducted a comparative analysis of yak ovarian activities in anestrus, estrus, and pregnancy regarding their morphology and histology, followed by implementing RNA sequencing (RNA-seq) technology to detect the overall gene expression and biological mechanism in different reproductive stages. H&E staining showed that there were more growing follicles and mature follicles in ovarian tissue sections during estrus than ovarian tissues during non-estrus. The RNA-seq analysis of yak ovary tissues in three periods showed that DEGs related to follicular development and hormone metabolism were screened in the three comparison groups, such as COL1A2, NR4A1, THBS2, PTGS2, SCARB1, STAR, and WNT2B. Bioinformatics analysis showed that these DEGs are involved in ion binding, cell development, metabolic processes, enriched in ECM-receptor interactions, steroid biosynthesis, together with aldosterone generation/discharge and Wnt/PI3K-Akt signaling pathways. In addition, we speculate alternate splice development events to have important role/s in regulating ovarian functional genomic expression profiles. These results provide essential knowledge aimed at scrutinizing pivotal biomarkers for yak ovarian activity, together with paving the way for enhancing researchers' focus on improving yak reproductive performance.