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To explore the expression pattern of the TRIB1 gene in yak follicles and its effect on the steroidogenesis of granulosa cells (GCs). Here, 4-5 years old female yaks were treated as the subjects. Immunohistochemically assay found that TRIB1 protein was expressed in different developmental follicles. Among different cell types of follicles, including cumulus cells (CCs), granulosa cells (GCs) and theca cells (TCs), the TRIB1 protein was most abundant in GCs (P 0.0001). In addition, we cloned the coding sequence (CDS) of the yak TRIB1 gene, which is 1119 bp, encoding 372 amino acids (AA). The amino acid sequence homology of TRIB1 is80% to those of other species, except for zebrafish. To further explore the function of TRIB1 in steroidogenesis, the pcDNA3.1(+)-TRIB1 eukaryotic expression vector was constructed and then transfected into GCs. The data showed that overexpression of TRIB1 significantly reduced the progesterone (P4) secretion of granulosa cells measured by ELISA assay (P 0.05), but not Estradiol (E2) secretion. Consistently, TRIB1 gain-of-function downregulated the mRNA levels of steroidogenesis related genes steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (P 0.01), while cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) had no significant difference (P 0.05). Interestingly, mito-tracker staining showed that mitochondrial number significantly decreased in TRIB1 overexpressed GCs (P 0.01). Further, overexpression of TRIB1 inhibited mRNA levels of mitochondrial biogenesis related genes, including Mitochondrial transcription factor (TFAM) and Peroxisome proliferator-activated receptor alpha co-activator (PPARGC1A) (P 0.05). Conclusively, this work indicates that TRIB1 inhibited progesterone synthesis of GCs might be involved in the reduction of the mitochondria number.

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non-pregnant female yaks (4-year-old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (3.0 mm), medium (3.0-5.9 mm) and large (6.0-9.0 mm) follicles was detected by RT-qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si-PPP1R11 and si-NC (negative control) were transfected into GCs. RT-qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation- and apoptosis-related genes were detected by RT-qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si-PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway-related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation-related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis-related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including CDK4, HSD17B1 and MAP1LC3C, respectively. Both of these DE miRNAs and their target mRNAs exhibited dynamic expression profiles across the lifespan of yak CL. This study presents a general basis for understanding of the regulation of miRNA on yak CL and also provides a novel genetic resource for future analysis of the gene network during the estrous cycle in the yak.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10−6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10−6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10−6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3β-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Cattleyaks, a hybrid of (♂) and yak (♀), exhibit the marked productivity and adaptability of plateau, but suffer from male infertility. Small non-coding RNAs, especially miRNAs, play crucial roles in spermatogenesis and affect the growth of Sertoli cells (SCs). The objective of the present study was to explore the interaction between miR-34b-5p and protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11) and its effect on cattleyak SCs. RT-qPCR was used to determine the expression pattern of miR-34b-5p and PPP1R11, while the cellular and subcellular localization of PPP1R11 was determined by immunohistochemistry and immunocytochemistry. The interaction between MiR-34b-5p and PPP1R11 was evaluated by immunofluorescence, proliferation, apoptosis, and western blotting assays. The potential binding sites between miR-34b-5p and PPP1R11 were uncovered through targeted search of an online database, and verified using a dual luciferase reporter system. Our data show that miR-34b-5p is differentially expressed in the testes and SCs of cattleyaks compared to yaks. Overexpression of miR-34b-5p in SCs suppressed proliferation and induced apoptosis, while the effects of miR-34b-5p knockdown were the reverse. The 3'UTR of PPP1R11 was identified as a potential target site of miR-34b-5p, and this was validated by online database searches and our data from the dual-luciferase reporter assay, and it displayed an inverse expression pattern to miR-34b-5p in SCs. The effects of silencing PPP1R11 by siRNA were similar to the results of miR-34b-5p upregulation, but significantly different from miR-34b-5p downregulation in cattleyak SCs. The effects with PPP1R11 overexpression were opposite, suggesting a novel biofunctional role of PPP1R11 inactivation in depressing cattleyak SCs growth. Lastly, we confirmed that miR-34b-5p inhibited PPP1R11 expression and induced apoptosis by regulating proliferation- and apoptosis-related genes in SCs. Thus, miR-34b-5p regulates the apoptosis and proliferation of cattleyak SCs via targeting PPP1R11, which can provide an innovative direction for exploring the mechanism of cattleyak male sterility.

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR-342-3p during yak oocyte maturation. In this study, we investigated the role of miR-342-3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT-qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR-342-3p was verified by dual-luciferase reporter assay. Finally, miR-342-3p inhibitors were microinjected into yak cumulus-oocyte complex to evaluate the effects on oocyte maturation. MiR-342-3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV- and MI stages (p .05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3-year-old), DNMT1 showed an age-related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation-stage-related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR-342-3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR-342-3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'-untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process.

The adaptation and diversity of animals to the extreme environments of the Qinghai-Tibet Plateau (QTP) are typical materials to study adaptive evolution. The recently discovered Jinchuan yak population has many individuals with multiple ribs. However, little is known about this yak's origin, evolution, and the genetic mechanisms that formed its unique multirib trait. Here, we report a valuable population genome resource of the Jinchuan yak by resequencing the whole genome of 150 individuals. Population genetic polymorphism and structure analysis reveal that Jinchuan yak can be differentiated as a unique and original yak population among the domestic yak. Combined with geological change, the Jinchuan yak's evolutionary origin is speculated to be about 6290 years ago, which may be related to the unique geographical environment of the eastern edge of the QTP during this period. Compared with other domestic yaks, this new population has 280 positively selected genes. The genes related to skeletal function hold a considerable and remarkable proportion, suggesting that the specific skeletal characteristics have been enhanced in the adaptive evolution of Jinchuan yak in the extreme plateau environment. The genome-wide association study has revealed that TUBA8 and TUBA4A, the genes that regulate the cytoskeleton, are potential genes associated with the multirib trait. Our findings provide a basis to further understand the generation mechanism of the adaptive evolution of this new population in high-altitude extreme environments and the multivertebrate trait of domestic animals.

Cattleyak, which are interspecific hybrids between cattle and yak, display much higher growth performances than yak. However, F1 male cattleyak are infertile due to defective testicular development. Sirtuin 1 (SIRT1) is a histone deacetylase that is essential for various biological processes, while the roles of testicular SIRT1 in yak and cattleyak are still poorly understood. Here, we found that SIRT1 was localized in various kinds of yak testicular cells except elongated spermatids while it was deficient in cattleyak testis. Further studies indicated that cattleyak testis exhibited decreased histone acetylation levels on H3 and H4. One of SIRT1 co-factors, steroidogenic factor-1 (SF-1), was lost in cattleyak testis at protein level. Expressions of several SF-1 target genes responsible for Sertoli cell development and steroidogenesis, including STAR, CYP11A1, CYP26B1, FDX1 and HSD3B, decreased significantly in cattleyak testis. In addition, SIRT1-mediated P53 acetylation was not responsible for the cell apoptosis in cattleyak testis. Taken together, our results suggested the deficiency of SIRT1 in yak testis caused inactivation of SF-1 and the impairment of testicular development. This research provides theoretical bases for understanding the mechanism of cattleyak sterility and gives new insights in revealing the roles of SIRT1 in regulating yak testicular development.

Simple Summary Increasing milk fat rate has a good effect on the milk quality of cattleyak. SNPs can help us find potential molecular markers for the milk fat traits of cattleyak, and they can be screened according to molecular markers when they are young. It provides a reference for cultivating high milk fat cattle population in the future. The results of this study suggest that the SORBS1 gene polymorphism is closely related to the milk fat traits of cattleyak, which could be used as a candidate genetic marker for milk fat trait selection in cattleyak. This study provides a new molecular marker and theoretical basis for screening the milk fat traits of cattleyak. It has a certain reference value for the research and improvement of milk quality. Abstract This study aimed to find the SNPs in the SORBS1 gene of cattleyak, analyze the relationship between its polymorphisms and the milk fat traits, and find potential molecular markers for the milk fat traits of cattleyak. The polymorphism of the SORBS1 gene in 350 cattleyak from Hongyuan County (Sichuan, China) were detected by PCR and DNA sequencing, and the correlation between these SNPs and the milk production traits of cattleyak was analyzed. The results showed that there were nine SNPs in the CDS and their adjacent non-coding regions of the SORBS1 gene, and all SNPs have three genotypes. The correlation analysis found that the genotypes with superior milk fat traits in the other eight alleles were homozygous genotypes with a high genotype frequency except the g.96284 G > A (c.3090 G > A) (p < 0.05). However, at locus g.96284 G > A, the milk fat percentage, monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) of the GA genotype were significantly higher than that of GG and AA genotypes (p < 0.05). Among these SNPs, three SNPs (g.6256 C > T (c.298 C > T), g.24791 A > G (c.706 A > G) and g.29121 A > G (c.979 A > G)) caused the amino acids change. The genotypes of the three SNPs consist of three haplotypes and four diplotypes. The amino acid mutation degree of diplotype H1–H1 (CCAAAA) was the highest, and its milk fat percentage, MUFAs, PUFAs and SFAs were also the highest (p < 0.05). Taken together, we found nine SNPs in the SORBS1 gene that are closely related to the milk fat traits of cattleyak. Moreover, the mutation of amino acids caused by SNPs had positive effects on the milk fat traits of cattleyak. H1-H1 is the dominant diplotype which significantly related to the milk fat traits of cattleyak. This study provides a new molecular marker and theoretical basis for screening the milk fat traits of cattleyak.

Epigenetic signals and chromatin-modifying proteins play critical roles in adipogenesis, which determines the risk of obesity and which has recently attracted increasing interest. Histone demethylase 2A (KDM2A) is an important component of histone demethylase, however, its direct effect on fat deposition remains unclear. Here, a KDM2A loss of function was performed using two unbiased methods, small interfering RNA (siRNA) and Cre-Loxp recombinase systems, to reveal its function in adipogenesis. The results show that the knockdown of KDM2A by siRNAs inhibited the proliferation capacity of 3T3-L1 preadipocytes. Furthermore, the promotion of preadipocyte differentiation was observed in siRNA-treated cells, manifested by the increasing content of lipid droplets and the expression level of adipogenic-related genes. Consistently, the genetic deletion of KDM2A by Adipoq-Cre in primary adipocytes exhibited similar phenotypes to those of 3T3-L1 preadipocytes. Interestingly, the knockdown of KDM2A upregulates the expression level of Transportin 1(TNPO1), which in turn may induce the nuclear translocation of PPARγ and the accumulation of lipid droplets. In conclusion, the ablation of KDM2A inhibits preadipocyte proliferation and promotes its adipogenic differentiation. This work provides direct evidence of the exact role of KDM2A in fat deposition and provides theoretical support for obesity therapy that targets KDM2A.

The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17β-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including

MiR-155 regulates the development of germinal-center and the generation of immunoglobulin class-switched plasma cells. However, whether miR-155 is involved in immune response in fish is still unclear. Here, CIK cells transfected with miR-155 overexpressed plasmid inhibited mRNA expression of mIg and Rag2 (P

Functional reprogramming of a differentiated cell toward pluripotent cell may have long-term applications in numerous aspects, especially in regenerative medicine. Evidences accumulating from recent studies suggest that cellular extracts from stem cells or pluripotent cells can induce epigenetic reprogramming and facilitate pluripotency in otherwise highly differentiated cell types. Epigenetic reprogramming using cellular extracts has gained increasing attention and applied to recognize the functional factors, acquire the target cell types, and explain the mechanism of reprogramming. Now, more and more researches have proved that cellular extract treatment is an important strategy of cellular reprogramming. Thus, this review mainly focused on the progresses and potential mechanisms in epigenetic reprogramming using cellular extracts.

Yak milk, a high-quality milk, is one of the best raw materials for dairy products and economically important to pastoral herdsmen. To make a further understanding of the molecular differences in mammary tissues of the yaks with different milk production during lactation, in this study, we took the use of RNA-seq to perform high-throughput sequencing and analysis of the mammary gland transcriptomes of both high-yielding yak and low-yielding yaks during lactation. By the comparison and analysis of the transcriptome data for the mammary gland tissue of high-yielding yak and low-yield yak, 144 differential genes were screened out, of which 49 were upregulated and 95 were downregulated. Further functional analysis indicated that these differential genes involved in multiple classes based on Gene Ontology (GO) and multiple Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO analysis showed that the functions of the differential genes are closely related to the carbohydrate metabolism and other biological processes. KEGG pathway analysis revealed that these genes are mostly enriched in the pathway of antigen processing and presentation, phagosome pathway and type I diabetes pathway and enriched followed by extracellular matrix receptor interaction pathway. Moreover, several other pathways related to amino acid metabolism also showed significant enrichment. Here, the mammary gland transcriptomes of high-yielding yak and low-yielding yaks during lactation have for the first time been compared, and the related differential genes have been screened out and analyzed. Our study paves a way for the further elucidation of the basic molecular mechanism of yak mammary gland tissue, and at the same time provides new ideas for improving the milk production of yaks.

Sertoli cells (SCs) are important testicular somatic cells that carry out various functions in spermatogenesis. Understanding the biological mechanisms underlying SC development may facilitate the understanding of animal reproduction. Anti-Mullerian hormone (AMH) is a dimeric glycoprotein produced by SCs and plays essential roles in spermatogenesis. In this study, we cloned the coding sequence of the yak AMH, predicated the structure of AMH protein, analyzed AMH expression in the testis at different stages, and studied the functions of AMH in yak SCs. The open reading frame (ORF) of the yak AMH contained 1728 bp and encoded 575 amino acids. Structural analysis revealed that the yak AMH protein had a highly conserved transforming growth factor-β (TGF-β) domain. The mRNA expression level for the AMH gene in yak testis increased significantly from the fetal stage to calf stage, then decreased with the increase of age. The highest expression was found in calf stage. Cell proliferation was depressed in AMH-deficient SCs. Expression of several genes involved in SC proliferation and development, including PCNA, BCL-2, BAX, CASP3, AR and AMHR2 were altered after knockdown of AMH. Also, three SC-secreted factors essential for spermatogenesis, SCF, GDNF and ABP, were repressed at the transcription level after AMH knockdown in yak SCs. Moreover, supplementation with exogenous AMH protein partially rescued SC proliferation, and the expression of PCNA, BCL-2, AR and AMHR2 after AMH gene interference. This research provided theoretical basis for understanding the mechanism by which AMH regulates yak spermatogenesis and might give new insights in improving yak reproductive performance in the future.

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.

Microbial communities of human gut directly influence health and bear adaptive potential to different geography environment and lifestyles. However, knowledge about the influences of altitude and geography on the gut microbiota of Tibetans is currently limited. In this study, fecal microbiota from 208 Tibetans across six different locations were analyzed by MiSeq sequencing; these locations included Gannan, Gangcha, Tianzhu, Hongyuan, Lhasa and Nagqu, with altitudes above sea level ranging from 2800 m to 4500 m across the Tibetan plateau. Significant differences were observed in microbial diversity and richness in different locations. At the phylum level, gut populations of Tibetans comprised Bacteroidetes (60.00%), Firmicutes (29.04%), Proteobacteria (5.40%), and Actinobacteria (3.85%) and were marked by a low ratio (0.48) of Firmicutes to Bacteroidetes. Analysis based on operational taxonomic unit level revealed that core microbiotas included Prevotella, Faecalibacterium, and Blautia, whereas Prevotella predominated all locations, except Gangcha. Four community state types were detected in all samples, and they mainly belong to Prevotella, Bacteroides, and Ruminococcaceae. Principal component analysis and related correspondence analysis results revealed that bacterial profiles in Tibetan guts varied significantly with increasing altitude, BMI, and age, and facultative anaerobes were rich in Tibetan guts. Gut microbiota may play important roles in regulating high-altitude and geographical adaptations.

Sound development and maturation of oocytes serve as important indicators in evaluating female fertility. Development and maturation of oocytes are complex processes involving transcriptional regulation of a large number of genes. Transcriptome studies are crucial in understanding such processes. In this study, oocytes obtained from yak—a unique bovine found in high-altitude areas—were used as research subject. RNA-seq was applied in comparative transcriptome analysis of yak oocytes, which were arrested at the germinal vesicle (GV) stage and metaphase II (MII) stage after in vitro maturation (IVM). After deep sequencing, 51380686 clean reads totaling 4624261740 bp were obtained from a GV yak oocyte library, whereas 50303412 clean reads with a total of 4527307080 bp were obtained from a MII yak oocyte library. Gene coverage statistics showed that 16719 and 16339 yak genes were mapped in GV and MII libraries, respectively. Comparative transcriptome analysis of oocytes at MII and GV stages revealed 4767 differentially expressed genes (DEGs), including 1418 upregulated and 3349 downregulated genes. Gene Ontology (GO) functional classification analysis showed that these DEGs are closely related to “metabolism” and other biological processes; moreover, molecular events related to molecular binding function are active. The interaction analysis of Kyoto Encyclopedia of Genes and Genome (KEGG) pathways involved DEGs were conducted according to two parts of upregulation and downregulation respectively. Results of analysis of upregulated DEGs revealed that apoptosis, endocytosis, and metabolic pathways are key regulatory pathways. By contrast, downregulated genes are involved in metabolic pathways, pyruvate metabolism, focal adhesion, and oxidative phosphorylation. Therefore, metabolic pathways play critical regulatory roles in upregulation and downregulation of pathways. Further analysis showed that histidine, glycine, serine, and threonine metabolism; glyoxylic acid and dicarboxylic acid metabolism; and glutathione metabolism are key control nodes in entire metabolic pathways. Interestingly, immune-related interleukins and interferon family genes were also differentially expressed in IVM of yak oocytes. The present study provides basis for analyzing molecular mechanism of IVM of yak oocytes and for further understanding specificity of yak reproduction.

Toll-like receptors (TLRs) are innate pattern recognition receptors that play an important role in host resistance to pathogenic microbes. In this study, we cloned the coding region of the yak TLR family (1–10) genes and used bioinformatics to analyze gene characteristics. Real-time fluorescence quantitative polymerase chain reaction was used to detect TLR expression levels in different tissues. Yak TLR genes exhibited high homologies with other species. At the nucleotide level, yak shared more than 96 % homology with cattle and sheep and 75–87 % homology with human and mouse. At the amino acid level, yak shared 90–99 % homology with cattle and sheep and 64–86 % homology with human and mouse. Yak showed close evolutionary relationship with cattle and sheep, which formed a branch of mammals together with TLRs from human, horse, and mouse, among others, and formed a branch with a longer genetic distance with chicken. TLR1, 2, 6, and 10 and TLR7, 8, and 9 were clustered in 2 individual branches, respectively. Fluorescent quantitation results showed that TLRs were expressed in all yak tissues, but different members showed different expression patterns. TLR2, 4, and 6 showed the highest expression in the spleen, followed by ovary, small intestine, kidney, and liver. TLR1, 5, 7, 8, 9, and 10 were most highly expressed in the kidney and showed higher expression in the liver, kidney, spleen, and other tissues. Our results will be useful for studies on immune molecular mechanisms and disease resistance breeding of yak and other plateau animals.

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The objective of this study was to investigate the mechanism that regulates pre-implantation development of the yak (Bos grunniens). We determined the transcriptomes of in vitro-produced yak embryos at two-cell, four-cell, eight-cell stages, and morula and blastocyst using the Illumina RNA-seq for the first time. We obtained 47.36-50.86 million clean reads for each stage, of which, 85.65%-90.02% reads were covered in the reference genome. A total of 17,368 genes were expressed during the two-cell stage to blastocyst of the yak, of which 7,236 genes were co-expressed at all stages, whereas 10,132 genes were stage-specific expression. Transcripts from 9,827 to 14,893 different genes were detected in various developmental stages. When |log2 ratio| ≥ 1 and q-value

Contents To increase the current understanding of the gene expression in the pre-ovulatory ovary and identify the key genes involved in the regulation of ovulation rate, we compared the transcriptomes of ovaries from the prolific Jintang black goat (JTG) and the non-prolific Tibetan goat (TBG) during the follicular phase using the Illumina RNA-Seq method. Three ovarian libraries were constructed for each breed. On average, we obtained approximately 49.2 and 45.9 million reads for each individual ovary of TBGs and JTGs, respectively, of which 79.76% and 78.67% reads were covered in the genome database. A total of 407 differentially expressed genes (DEG) were detected between these two breeds, in which 316 were upregulated, and 91 were downregulated in the ovaries of JTGs versus TBGs. Based on the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, some of these DEGs potentially play an important role in controlling the development of ovarian follicles. SRD5A2, MSMB, STAR and 3BHSD, etc. were the most significantly differentially expressed between these two distinct breeds. In addition, each ovary expressed 1,066 versus 989 novel transcripts, and 171,829 versus 140,529 putative SNPs in TBGs versus JTBs, respectively. All data sets (GEO and dbSNP) were available via public repositories. Our study provides insight into the transcriptional regulation of the ovaries of two distinct breeds of goats that might serve as a key resource for understanding goat fecundity. SRD5A2, MSMB, STAR and 3BHSD may be associated with the high fecundity of JTGs.

Introduction We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing–thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. Material and methods H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen–thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. Results The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated ( P 0.05). Conclusions This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles.

Estrogen and its receptors are essential hormones for normal reproductive function in males and females during developmental stage. To better understand the effect of estrogen receptor (ER) gene in yak (Bos grunniens), reverse transcription-polymerase chain reaction (PCR) was carried out to clone ERα and ERβ genes. Bioinformatics methods were used to analyze the evolutionary relationship between yaks and other species, and real-time PCR was performed to identify the mRNA expression of ERα and ERβ. Sequence analysis showed that the ER open reading frames (ORFs) encoded 596 and 527 amino acid proteins. The yak ERα and ERβ shared 45.3% to 99.5% and 53.9% to 99.1% protein sequence identities with other species homologs, respectively. Real-time PCR analysis revealed that ERα and ERβ were expressed in a variety of tissues, but the expression level of ERα was higher than that of ERβ in all tissues, except testis. The mRNA expression of ERα was highest in the mammary gland, followed by uterus, oviduct, and ovary, and lowest in the liver, kidney, lung, testis, spleen, and heart. The ERβ mRNA level was highest in the ovary; intermediary in the uterus and oviduct; and lowest in the heart, liver, spleen, lung, kidney, mammary gland, and testis. The identification and tissue distribution of ER genes in yaks provides a foundation for the further study on their biological functions.

Contents With an aim to improve the efficiency of yak–bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak–bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P

SummaryInterspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear–cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine–yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and ‘corrected’ the gene expression patterns of yak iSCNT embryos.

The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos.

Secreted frizzled related protein 5 (SFRP5), an anti-inflammatory adipokine, is relevant to the adipocyte differentiation. In order to clarify its role in regulating intramuscular fat (IMF) deposition in Tibetan chicken, the full-length sequence of the Tibetan chicken SFRP5 gene was cloned. The relative expression of SFRP5 gene was detected using quantitative RT-PCR in various tissues of 154 days old Tibetan chicken, as well as in breast muscle, thigh muscle, and adipose tissue at different growth stages. The results showed that SFRP5 gene was expressed in all examined tissues but highly enriched in adipose tissue. Temporal expression profile showed that the expression of SFRP5 was gradually decreased in breast muscle, but was fluctuated in thigh muscle and adipose tissue with the growth of Tibetan chicken. Furthermore, correlation analysis demonstrated that the expression of SFRP5 in breast muscle, thigh muscle and adipose tissue was correlated with IMF content at different levels. The results indicated that Tibetan chicken SFRP5 is involved in IMF deposition.

Background Yaks (Bos grunniens) are endemic species that can adapt well to thin air, cold temperatures, and high altitude. These species can survive in harsh plateau environments and are major source of animal production for local residents, being an important breed in the Qinghai–Tibet Plateau. However, compared with ordinary cattle that live in the plains, yaks generally have lower fertility. Investigating the basic physiological molecular features of yak ovary and identifying the biological events underlying the differences between the ovaries of yak and plain cattle is necessary to understand the specificity of yak reproduction. Therefore, RNA-seq technology was applied to analyze transcriptome data comparatively between the yak and plain cattle estrous ovaries. Results After deep sequencing, 3,653,032 clean reads with a total of 4,828,772,880 base pairs were obtained from yak ovary library. Alignment analysis showed that 16992 yak genes mapped to the yak genome, among which, 12,731 and 14,631 genes were assigned to Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, comparison of yak and cattle ovary transcriptome data revealed that 1307 genes were significantly and differentially expressed between the two libraries, wherein 661 genes were upregulated and 646 genes were downregulated in yak ovary. Functional analysis showed that the differentially expressed genes were involved in various Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO annotations indicated that the genes related to “cell adhesion,” “hormonal” biological processes, and “calcium ion binding,” “cation transmembrane transport” molecular events were significantly active. KEGG pathway analysis showed that the “complement and coagulation cascade” pathway was the most enriched in yak ovary transcriptome data, followed by the “cytochrome P450” related and “ECM–receptor interaction” pathways. Moreover, several novel pathways, such as “circadian rhythm,” were significantly enriched despite having no evident associations with the reproductive function. Conclusion Our findings provide a molecular resource for further investigation of the general molecular mechanism of yak ovary and offer new insights to understand comprehensively the specificity of yak reproduction.

The present study was carried out to examine the effect of valproic acid (VPA), an important histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 lysine 9 (H3K9ac) in bovine somatic cell nuclear transfer (SCNT) embryos. We found that treatment with 4 mM VPA for 24 h could significantly improve the development of bovine SCNT embryos. Compared with the no-treatment group, the cleavage rate was higher (69.79 ± 0.99% vs. 65.11 ± 1.02%, p0.05), as was the blastocyst rate (39.99 ± 1.29% vs. 34.87 ± 1.74%, p0.05). Moreover, the rate of apoptosis (1.91 ± 0.48% vs. 5.67 ± 0.40%, p0.05) in blastocysts was greatly reduced after VPA treatment. Valproic acid treatment also increased the immunofluorescent signal for H3K9ac in SCNT embryos in a pattern similar to that of in vitro fertilized (IVF) embryos. In conclusion, we demonstrated that VPA can significantly improve the in vitro developmental competence and enhance the nuclear reprogramming of bovine SCNT embryos.

The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, P

We previously reported that treatment of both donor cells and early cloned embryos with a combination of 0.01 μM 5-aza-2(/)-Deoxycytidine (5-aza-dC) and 0.05 μM trichostatin A (TSA) significantly improved development of cloned bovine embryos in vitro. In the present study, we investigated the effect of this combination treatment on the in vivo development potency and postnatal survivability of cloned calves. Blastocysts (77 and 82 blastocysts derived from untreated (control) and treated groups, respectively) were individually transferred to recipient cows. Relative to the control group, the combination treatment of both donor cells and early embryos with 5-aza-dC and TSA dramatically increased the cleavage rate (49.2 vs 63.6%, P < 0.05) at 24 h of culture, and blastocyst development rate on Days 6 and 7 of culture (18.8 vs 33.9% and 27.1 vs 38.5% respectively, P < 0.05). Although pregnancy rate did not differ 40 d after transfer, it was lower in the treated than control group 90 d after transfer (7.8 vs 29.3%, P < 0.05). In the control group, there were three calves born to 77 recipients (only two survived beyond 60 d), whereas in the treated group, 17 calves were born to 82 recipients, and 11 survived beyond 60 d. In conclusion, a combination treatment of donor cells and early cloned embryos with 5-aza-dC and TSA significantly enhanced development of somatic cell cloned bovine embryos in vivo; cloning efficiency (number of surviving calves at 60 d of birth/number of recipient cows) was increased from 2.6 to 13.4%.

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.