Comprehensive cognition of yak (Bos grunniens) AIFM2 gene and its anti-ferroptosis role in bisphenol A-induced fetal fibroblast model.

Apoptosis-inducing factor mitochondrion-associated 2 (AIFM2) has been identified as a gene with anti-ferroptosis properties. To explore whether AIFM2 exerts anti-ferroptosis role in yaks (Bos grunniens), we cloned yak AIFM2 gene and analyzed its biological characteristics. The coding region of AIFM2 had 1122 bp and encoded 373 amino acids, which was conserved in mammals. Next, RT-qPCR results showed an extensive expression of AIMF2 in yak tissues. Furthermore, we isolated yak skin fibroblasts (YSFs) and established a bisphenol A (BPA)-induced ferroptosis model to further investigate the role of AIFM2. BPA elevated oxidative stress (reactive oxygen species, ROS) and lipid peroxidation (malondialdehyde, MDA and BODIPY), and reduced cell viability and antioxidant capacity (glutathione, GSH), with the severity depending on the dosage. Of note, a supplement of Ferrostatin-1 (Fer), an inhibitor of ferroptosis, restored the previously mentioned indicators. Subsequently, we constructed an AIFM2 overexpression vector and designed AIFM2 specific interfering siRNAs, which were transfected into YSFs. The results showed that overexpressing AIFM2 alleviated ferroptosis, characterizing by significant changes of cell viability, ROS, BODIPY, MDA and GSH. Meanwhile, interfering AIFM2 aggravated ferroptosis, demonstrating the critical anti-ferroptosis role of the yak AIFM2 gene. This study shed light on further exploring the molecular mechanism of AIFM2 in plateau adaptability.

Pangenome and multi-tissue gene atlas provide new insights into the domestication and highland adaptation of yaks.

BACKGROUND: The genetic diversity of yak, a key domestic animal on the Qinghai-Tibetan Plateau (QTP), is a vital resource for domestication and breeding efforts. This study presents the first yak pangenome obtained through the de novo assembly of 16 yak genomes. RESULTS: We discovered 290 Mb of nonreference sequences and 504 new genes. Our pangenome-wide presence and absence variation (PAV) analysis revealed 5,120 PAV-related genes, highlighting a wide range of variety-specific genes and genes with varying frequencies across yak populations. Principal component analysis (PCA) based on binary gene PAV data classified yaks into three new groups: wild, domestic, and Jinchuan. Moreover, we proposed a 'two-haplotype genomic hybridization model' for understanding the hybridization patterns among breeds by integrating gene frequency, heterozygosity, and gene PAV data. A gene PAV-GWAS identified a novel gene (BosGru3G009179) that may be associated with the multirib trait in Jinchuan yaks. Furthermore, an integrated transcriptome and pangenome analysis highlighted the significant differences in the expression of core genes and the mutational burden of differentially expressed genes between yaks from high and low altitudes. Transcriptome analysis across multiple species revealed that yaks have the most unique differentially expressed mRNAs and lncRNAs (between high- and low-altitude regions), especially in the heart and lungs, when comparing high- and low-altitude adaptations. CONCLUSIONS: The yak pangenome offers a comprehensive resource and new insights for functional genomic studies, supporting future biological research and breeding strategies.

Enhanced glutathione production protects against zearalenone-induced oxidative stress and ferroptosis in female reproductive system.

Zearalenone (ZEN, a widespread fusarium mycotoxin) causes evoked oxidative stress in reproductive system, but little is known about whether this is involved in ferroptosis. Melatonin, a well-known antioxidant, has demonstrated unique anti-antioxidant properties in several studies. Here, this study was aimed to investigate whether ZEN-induced oxidative stress in female pig's reproductive system was involved in ferroptosis, and melatonin was then supplemented to protect against ZEN-induced abnormalities in vitro cell models [human granulosa cell (KGN) and mouse endometrial stromal cell (mEC)] and in vivo mouse model. According to the results from female pig's reproductive organs, ZEN-induced abnormalities in vulvar swelling, inflammatory invasion and pathological mitochondria, were closely linked with evoked oxidative stress. Using RNA-seq analysis, we further revealed that ZEN-induced reproductive toxicity was due to activated ferroptosis. Mechanistically, by using in vitro cell models (KGN and mEC) and in vivo mouse model, we observed that ZEN exposure resulted in oxidative stress and ferroptosis in a glutathione-dependent manner. Notably, these ZEN-induced abnormalities above were alleviated by melatonin supplementation through enhanced productions of glutathione peroxidase 4 and glutathione. Herein, the present results suggest that potential strategies to improve glutathione production protect against ZEN-induced reproductive toxicity, including oxidative stress and ferroptosis.

Cloning and Characterization of Yak DHODH Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts.

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of de novo biosynthesis of pyrimidine. Although the involvement of DHODH in resisting ferroptosis has been successively reported in recent years, which greatly advanced the understanding of the mechanism of programmed cell death (PCD), the genetic sequence of the yak DHODH gene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence of DHODH (1188 bp) from yak liver and conducted a characterization analysis of its predictive protein that consists of 395 amino acids. We found that the coding region of the yak DHODH gene presented high conservation among species. Second, the expression profile of the DHODH gene in various yak tissues was investigated using RT-qPCR. The results demonstrated that DHODH was widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement of DHODH in regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce the in vitro ferroptosis model of YSFs. We observed that BPS decreased the cell viability (CCK8) and membrane potential (JC-1) of YSFs in a dose-dependent manner and induced oxidative stress by elevating reactive oxygen species (ROS). Simultaneously, it was evident that BPS effectively augmented the indicators associated with ferroptosis (MDA and BODIPY staining) and reduced GSH levels. Importantly, the co-administration of Ferrostatin-1 (Fer), a potent inhibitor of ferroptosis, significantly alleviated the aforementioned markers, thereby confirming the successful induction of ferroptosis in YSFs by BPS. Finally, overexpression plasmids and siRNAs of the yak DHODH gene were designed and transfected respectively into BPS-cultured YSFs to modulate DHODH expression. The findings revealed that DHODH overexpression alleviated the occurrence of BPS-induced ferroptosis, while interference of DHODH intensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yak DHODH gene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of the DHODH gene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yak DHODH gene and the underlying mechanisms of ferroptosis in this species.

ß-Nicotinamide Mononucleotide Alleviates Hydrogen Peroxide-Induced Cell Cycle Arrest and Death in Ovarian Granulosa Cells.

Maintaining normal functions of ovarian granulosa cells (GCs) is essential for oocyte development and maturation. The dysfunction of GCs impairs nutrition supply and estrogen secretion by follicles, thus negatively affecting the breeding capacity of farm animals. Impaired GCs is generally associated with declines in Nicotinamide adenine dinucleotide (NAD+) levels, which triggers un-controlled oxidative stress, and the oxidative stress, thus, attack the subcellular structures and cause cell damage. ß-nicotinamide mononucleotide (NMN), a NAD+ precursor, has demonstrated well-known antioxidant properties in several studies. In this study, using two types of ovarian GCs (mouse GCs (mGCs) and human granulosa cell line (KGN)) as cell models, we aimed to investigate the potential effects of NMN on gene expression patterns and antioxidant capacity of both mGCs and KGN that were exposed to hydrogen peroxide (H2O2). As shown in results of the study, mGCs that were exposed to H2O2 significantly altered the gene expression patterns, with 428 differentially expressed genes (DEGs) when compared with those of the control group. Furthermore, adding NMN to H2O2-cultured mGCs displayed 621 DEGs. The functional enrichment analysis revealed that DEGs were mainly enriched in key pathways like cell cycle, senescence, and cell death. Using RT-qPCR, CCK8, and ß-galactosidase staining, we found that H2O2 exposure on mGCs obviously reduced cell activity/mRNA expressions of antioxidant genes, inhibited cell proliferation, and induced cellular senescence. Notably, NMN supplementation partially prevented these H2O2-induced abnormalities. Moreover, these similar beneficial effects of NMN on antioxidant capacity were confirmed in the KGN cell models that were exposed to H2O2. Taken together, the present results demonstrate that NMN supplementation protects against H2O2-induced impairments in gene expression pattern, cell cycle arrest, and cell death in ovarian GCs through boosting NAD+ levels and provide potential strategies to ameliorate uncontrolled oxidative stress in ovarian GCs.

Factors Influencing the Maturation and Developmental Competence of Yak (Bos grunniens) Oocytes In Vitro.

The yak (Bos grunniens) is a unique breed living on the Qinghai-Tibet Plateau and its surrounding areas, providing locals with a variety of vital means of living and production. However, the yak has poor sexual maturity and low fertility. High-quality mature oocytes are the basis of animal breeding technology. Recently, in vitro culturing of oocytes and embryo engineering technology have been applied to yak breeding. However, compared to those observed in vivo, the maturation rate and developmental capacity of in vitro oocytes are still low, which severely limits the application of in vitro fertilization and embryo production in yaks. This review summarizes the endogenous and exogenous factors affecting the in vitro maturation (IVM) and developmental ability of yak oocytes reported in recent years and provides a theoretical basis for obtaining high-quality oocytes for in vitro fertilization and embryo production in yaks.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Testis-specific knockout of Kdm2a reveals nonessential roles in male fertility but partially compromises spermatogenesis.

Lysine-specific histone demethylase 2 (Kdm2a) is a regulatory factor of histone modifications that participates in gametogenesis and embryonic development. The mis-regulation of Kdm2a can lead to aberrant gene expression, thereby contributing to abnormal cell proliferation, differentiation, apoptosis, and tumorigenesis. However, due to the potential confounding effects that are secondary to the loss of Kdm2a function from the soma in existing whole-animal mutants, the in vivo function of Kdm2a in spermatogenesis for male fertility remains unknown. Herein, we focus on exploring the spatiotemporal expression profile and biological functions of Kdm2a in the spermatogenesis and fertility of male mice. A testis-specific knockout Kdm2a model (Kdm2a cKO) was established by using the Stra8-Cre/loxP recombinase system to explore the roles of Kdm2a in male fertility. Our results showed that Kdm2a was ubiquitously expressed and dynamically distributed in multiple tissues and cell types in the testis of mice. Surprisingly, Kdm2a-deficient adult males were completely fertile and comparable with their control (Kdm2aflox/flox) counterparts. Despite the significantly reduced total number of sperm and density of seminiferous tubules in Kdm2a cKO testis accompanied by the degeneration of spermatogenesis, the fertilization ability and embryonic developmental competence of the Kdm2a cKO were comparable with those of their control littermates, suggesting that Kdm2a disruption did not markedly affect male fertility, at least during younger ages. Furthermore, Kdm2a homozygous mutants exhibited a lower total number and motility of sperm than the control group and showed notably affected serum 17ß-estradiol concentration. Interestingly, the transcriptome sequencing revealed that the loss of Kdm2a remarkably upregulated the expression level of Kdm2b. This effect, in turn, may induce compensative effects in the case of Kdm2a deficiency to maintain normal male reproduction. Together, our results reveal that Kdm2a shows spatiotemporal expression during testicular development and that its loss is insufficient to compromise the production of spermatozoa completely. The homologous Kdm2b gene might compensate for the loss of Kdm2a. Our work provides a novel Kdm2a cKO mouse allowing for the efficient deletion of Kdm2a in a testis-specific manner, and further investigated the biological function of Kdm2a and the compensatory effects of Kdm2b. Our study will advance our understanding of underlying mechanisms in spermatogenesis and male fertility.

Molecular cloning and characterization of STC1 gene and its functional analyses in yak (Bos grunniens) cumulus granulosa cells.

Cumulus granulosa cells (CGCs), an important type of ovarian somatic cells, carries out various functions related to oogenesis, follicular development, and steroidogenesis. Studying the biological mechanisms involved in the development and function of CGCs makes a great contribution to understanding the reproductive regulation in female animals. Stanniocalcin-1 (STC1) is an important Ca2+-regulated glycoprotein hormone that exhibits high expression levels in ovaries. In this study, we cloned the coding sequence of the yak STC1, predicted the structure of STC1 protein, detected the expression and localization of STC1 in yak ovaries, and analyzed the functions of STC1 in yak CGCs. The CDS (coding sequence) region of yak STC1 gene was found to be 744 bp and encoded 247 amino acids. Homology comparison revealed that STC1 protein was highly conserved among mammals. The STC1 mRNA displayed dynamic expression profiles in different stages of yak ovaries, and the highest expression was found in the follicular phase. Regarding localization, STC1 protein was widely distributed in various kinds of yak ovarian cells, including oocytes, mural granulosa cells, CGCs, and thecal cells. Repressing the expression of STC1 resulted in defective proliferation and survival of yak CGCs. In addition, knockdown the expression of STC1 repressed the secretion of progesterone and promoted the secretion of estrogen. Overexpression of STC1 partially rescued the proliferation of CGCs and resulted in opposite effects on the secretion of progesterone and estrogen. Several apoptosis and steroidogenesis-related genes, including BAX, BCL2, HSD3B1, HSD17B1, CYP11A1 and CYP17A1 showed altered expressions after repressing or increasing the expression of STC1 in yak CGCs. To the best of our knowledge, this study is the first to focus on the role of STC1 in yak CGCs, and the outcomes offer fresh insights into the mechanism governing yak reproduction.

Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development.

Histone methylation plays an essential role in oocyte growth and preimplantation embryonic development. The modification relies on histone methyl-transferases and demethylases, and one of these, lysine-specific demethylase 2a (Kdm2a), is responsible for modulating histone methylation during oocyte and early embryonic development. The mechanism of how Kdm2a deficiency disrupts early embryonic development and fertility remains elusive. To determine if maternally deposited Kdm2a is required for preimplantation embryonic development, the expression profile of Kdm2a during early embryos was detected via immunofluorescence staining and RT-qPCR. The Kdm2a gene in oocytes was specifically deleted with the Zp3-Cre/LoxP system and the effects of maternal Kdm2a loss were studied through a comprehensive range of female reproductive parameters including fertilization, embryo development, and the number of births. RNA transcriptome sequencing was performed to determine differential mRNA expression, and the interaction between Kdm2a and the PI3K/Akt pathway was studied with a specific inhibitor and activator. Our results revealed that Kdm2a was continuously expressed in preimplantation embryos and loss of maternal Kdm2a suppressed the morula-to-blastocyst transition, which may have been responsible for female subfertility. After the deletion of Kdm2a, the global H3K36me2 methylation in mutant embryos was markedly increased, but the expression of E-cadherin decreased significantly in morula embryos compared to controls. Mechanistically, RNA-seq analysis revealed that deficiency of maternal Kdm2a altered the mRNA expression profile, especially in the PI3K/Akt signaling pathway. Interestingly, the addition of a PI3K/Akt inhibitor (LY294002) to the culture medium blocked embryo development at the stage of morula; however, the developmental block caused by maternal Kdm2a loss was partially rescued with a PI3K/Akt activator (SC79). In summary, our results indicate that loss of Kdm2a influences the transcriptome profile and disrupts the PI3K/Akt signaling pathway during the development of preimplantation embryo. This can result in embryo block at the morula stage and female subfertility, which suggests that maternal Kdm2a is a potential partial redundancy with other genes encoding enzymes in the dynamics of early embryonic development. Our results provide further insight into the role of histone modification, especially on Kdm2a, in preimplantation embryonic development in mice.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

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, 3ß-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.

Dynamic transcriptome analysis of Maiwa yak corpus luteum during the estrous cycle.

Maiwa yak is a special breed of animal living on the Qinghai-Tibet Plateau, which has great economic value, but its fertility rate is low. The corpus luteum (CL) is a temporary tissue that plays a crucial role in maintaining the physiological cycle. However, little is known about the transcriptome profile in Maiwa yak CL. In the present study, the transcriptome of Maiwa yak CL at early (EYCL), middle (MYCL) and late-stages (LYCL) was studied employing high-throughput sequencing. A total of 25,922 transcripts were identified, including 22,277 known as well as 3,645 novel ones. Furthermore, 690 and 212 differentially expressed (DE) mRNAs were detected in the EYCL vs. MYCL and MYCL vs. LYCL groups, respectively. KEGG pathway enrichment analysis of DEGs illustrated that the most enriched pathway was PI3K-Akt pathway. Furthermore, twenty-six DEGs were totally found to be associated with different biological processes of CL development. One of these genes, PGRMC1, displayed a dynamical expression trend during the lifespan of yak CL. The knockdown of PGRMC1 in luteinized yak granulosa cells resulted in defective steroidogenesis. In conclusion, this study analyzed the transcriptome profiles in yak CL of different stages, and provided a novel database for analyzing the gene network in yak CL.HIGHLIGHTSThe manuscript analyzed the transcriptome profiles in yak CL during the estrous cycle.Twenty-six DEGs were found to be associated with the development or function of CL.One of the DEGs, PGRMC1, was found to be responsible for steroidogenesis in luteinized yak granulosa cells.

Identification of microRNA transcriptome throughout the lifespan of yak (Bos grunniens) corpus luteum.

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.

hnRNP K Degrades Viral Nucleocapsid Protein and Induces Type I IFN Production to Inhibit Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteric coronavirus currently spreading in several nations and inflicting substantial financial damages on the swine industry. The currently available coronavirus vaccines do not provide adequate protection against the newly emerging viral strains. It is essential to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. This study shows that heterogeneous nuclear ribonucleoprotein K (hnRNP K), the host protein determined by the transcription factor KLF15, inhibits the replication of PEDV by degrading the nucleocapsid (N) protein of PEDV in accordance with selective autophagy. hnRNP K was found to be capable of recruiting the E3 ubiquitin ligase, MARCH8, aiming to ubiquitinate N protein. Then, it was found that the ubiquitinated N protein could be delivered into autolysosomes for degradation by the cargo receptor NDP52, thereby inhibiting PEDV proliferation. Moreover, based on the enhanced MyD88 expression, we found that hnRNP K activated the interferon 1 (IFN-1) signaling pathway. Overall, the data obtained revealed a new mechanism of hnRNP K-mediated virus restriction wherein hnRNP K suppressed PEDV replication by degradation of viral N protein using the autophagic degradation pathway and by induction of IFN-1 production based on upregulation of MyD88 expression. IMPORTANCE The spread of the highly virulent PEDV in many countries is still leading to several epidemic and endemic outbreaks. To elucidate effective antiviral mechanisms, it is important to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. In the work, we detected hnRNP K as a new host restriction factor which can hinder PEDV replication through degrading the nucleocapsid protein based on E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. In addition, via the upregulation of MyD88 expression, hnRNP K could also activate the interferon (IFN) signaling pathway. This study describes a previously unknown antiviral function of hnRNP K and offers a new vision toward host antiviral factors that regulate innate immune response as well as a protein degradation pathway against PEDV infection.

Oocyte-Specific Knockout of Histone Lysine Demethylase KDM2a Compromises Fertility by Blocking the Development of Follicles and Oocytes.

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 17ß-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.

Nuclear ribonucleoprotein RALY targets virus nucleocapsid protein and induces autophagy to restrict porcine epidemic diarrhea virus replication.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and dehydration in pigs and leads to great economic losses in the commercial swine industry. However, the underlying molecular mechanisms of host response to viral infection remain unclear. In the present study, we investigated a novel mechanism by which RALY, a member of the heterogeneous nuclear ribonucleoprotein family, significantly promotes the degradation of the PEDV nucleocapsid (N) protein to inhibit viral replication. Furthermore, we identified an interaction between RALY and the E3 ubiquitin ligase MARCH8 (membrane-associated RING-CH 8), as well as the cargo receptor NDP52 (nuclear dot protein 52 kDa), suggesting that RALY could suppress PEDV replication by degrading the viral N protein through a RALY-MARCH8-NDP52-autophagosome pathway. Collectively, these results suggest a preventive role of RALY against PEDV infection via the autophagy pathway and open up the possibility of inducing RALY in vivo as an effective prophylactic and preventive treatment for PEDV infection.

GPR50 participates in and promotes yak oocyte maturation: A new potential oocyte regulatory molecule.

Numerous molecular regulatory mechanisms are involved in the formation of mammalian oocytes, but many of their key proteins and molecules remain unknown. GPR50, the lone G protein-coupled receptor located in the X chromosome, is one of the superfamily members of the G protein-coupled receptor. GPR50 has been recently proved to play an important role in various physiological activities. However, the role of GPR50 in reproduction is currently unclear. In our previous research, we proved that the GPR50 receptor is present in yak oocytes. In the present study, the expression level and subcellular localization of GPR50 in the in vitro maturation process of yak oocytes were investigated to explore further its role in the maturation of yak oocytes. In the germinal vesicle (GV) stage, GPR50 was expressed and positioned in the cell membrane. By comparison, in the metaphase II (MII) stage, GPR50 had the highest expression level and was highly diffused in the cytoplasm. On the basis of these observations, the knockdown and overexpression of GPR50 yak oocyte models were constructed. Results showed that the expression level of GPR50 knockdown significantly reduced the excretion rate and maturity level of the yak oocyte PB1 (P < 0.01). However, the expression level of GPR50 overexpression exerted no significant influence on the excretion rate and maturity level of the yak oocytes (P > 0.05). This study verified that GPR50 plays an important role in the in vitro maturation process of yak oocytes.

Transcriptomic study of yak mammary gland tissue during lactation.

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.

Population genome of the newly discovered Jinchuan yak to understand its adaptive evolution in extreme environments and generation mechanism of the multirib trait.

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.