RESUMEN
The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.
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Senescencia Celular , Miostatina , Animales , Miostatina/genética , Miostatina/metabolismo , Bovinos , Senescencia Celular/genética , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Transducción de Señal , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células CultivadasRESUMEN
Myostatin (MSTN), a growth and differentiation factor, plays an important role in regulating skeletal muscle growth and development. MSTN knockout (MSTN-KO) leads to skeletal muscle hypertrophy and regulates metabolic homeostasis. Moreover, MSTN is also detected in smooth muscle. However, the effect of MSTN-KO on smooth muscle has not yet been reported. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in esophageal smooth muscles of MSTN-KO Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 608.5 ± 17.62 kg) and wild-type (WT) Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 528.25 ± 11.03 kg). The transcriptome was sequenced using the Illumina Novaseq™ 6000 sequence platform. In total, 337 significantly up- and 129 significantly down-regulated genes were detected in the MSTN-KO cattle compared with the WT Chinese Luxi Yellow cattle. Functional enrichment analysis indicated that the DEGs were mainly enriched in 67 signaling pathways, including cell adhesion molecules, tight junction, and the cGMP-PKG signaling pathway. Metabolomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 130 differential metabolites between the groups, with 56 up-regulated and 74 down-regulated in MSTN knockout cattle compared with WT cattle. Differential metabolites were significantly enriched in 31 pathways, including glycerophospholipid metabolism, histidine metabolism, glutathione metabolism, and purine metabolism. Transcriptome and metabolome were combined to analyze the significant enrichment pathways, and there were three metabolically related pathways, including histidine metabolism, purine metabolism, and arginine and proline metabolism. These results provide important references for in-depth research on the effect of MSTN knockout on smooth muscle.
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Miostatina , Transcriptoma , Animales , Bovinos , Miostatina/genética , Miostatina/metabolismo , Cromatografía Liquida , Histidina/metabolismo , Espectrometría de Masas en Tándem , Músculo Liso/metabolismo , Metaboloma , Purinas/metabolismo , Músculo Esquelético/metabolismoRESUMEN
Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine-bovine (B-B), ovine-ovine (O-O) SCNT, and ovine-bovine (O-B) iSCNT embryos and found that a developmental block existed in the 8-cell stage in O-B iSCNT embryos. RNA sequencing and q-PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O-B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O-B iSCNT embryos. In addition, the nucleolin exhibited a clear circular-ring structure in B-B 8-cell stage embryos, whereas this was shell-like or dot-like in the O-B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring-like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell-like nucleolin-structure in B-B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos.
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Desarrollo Embrionario , Fibroblastos/citología , Técnicas de Transferencia Nuclear , Fosfoproteínas/fisiología , Proteínas de Unión al ARN/fisiología , Animales , Bovinos , Células Cultivadas , Embrión de Mamíferos , Oocitos , Ovinos , NucleolinaRESUMEN
Cloned animals generated by somatic cell nuclear transfer (SCNT) have been reported for many years; however, SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT-mediated somatic cell reprogramming. To identify candidate factors that facilitate ZGA in SCNT-mediated reprogramming, we performed siRNA-repressor and mRNA-inducer screenings, which reveal Dux, Dppa2, and Dppa4 as key factors enhancing ZGA in SCNT. We show that direct injection of ZGA inducers has no significant effect on SCNT blastocyst formation; however, following the establishment of an inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improves SCNT efficiency but also increases that of chemically induced pluripotent stem cell reprogramming. Moreover, transcriptome profiling reveals that Dux-treated SCNT embryos are similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promotes the full embryonic development of SCNT-derived animals. These findings enhance our understanding of ZGA-regulator function in somatic reprogramming.
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Células Madre Pluripotentes Inducidas , Animales , Blastocisto , Reprogramación Celular/genética , Embrión de Mamíferos , Desarrollo Embrionario/genética , Genoma , Ratones , Proteínas Nucleares , Técnicas de Transferencia Nuclear , Factores de Transcripción/genética , CigotoRESUMEN
Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.
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Proteínas Quinasas Activadas por AMP , Miostatina , Animales , Ratones , Aminoimidazol Carboxamida/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Miostatina/genética , Miostatina/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Myostatin (MSTN) is an important negative regulator of skeletal muscle growth in animals. A lack of MSTN promotes lipolysis and glucose metabolism but inhibits oxidative phosphorylation (OXPHOS). Here, we aimed to investigate the possible mechanism of MSTN regulating the mitochondrial energy homeostasis of skeletal muscle. To this end, MSTN knockout mice were generated by the CRISPR/Cas9 technique. Expectedly, the MSTN null (Mstn-/-) mouse has a hypermuscular phenotype. The muscle metabolism of the Mstn-/- mice was detected by an enzyme-linked immunosorbent assay, indirect calorimetry, ChIP-qPCR, and RT-qPCR. The resting metabolic rate and body temperature of the Mstn-/- mice were significantly reduced. The loss of MSTN not only significantly inhibited the production of ATP by OXPHOS and decreased the activity of respiratory chain complexes, but also inhibited key rate-limiting enzymes related to the TCA cycle and significantly reduced the ratio of NADH/NAD+ in the Mstn-/- mice, which then greatly reduced the total amount of ATP. Further ChIP-qPCR results confirmed that the lack of MSTN inhibited both the TCA cycle and OXPHOS, resulting in decreased ATP production. The reason may be that Smad2/3 is not sufficiently bound to the promoter region of the rate-limiting enzymes Idh2 and Idh3a of the TCA cycle, thus affecting their transcription.
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Mitocondrias , Músculo Esquelético , Miostatina , Fosforilación Oxidativa , Animales , Ratones , Adenosina Trifosfato/metabolismo , Ratones Noqueados , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Miostatina/genética , Miostatina/metabolismoRESUMEN
Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.
Asunto(s)
Núcleo Celular , Embrión de Mamíferos , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Núcleo Celular/metabolismo , Clonación de Organismos , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Femenino , Mitocondrias/metabolismo , Técnicas de Transferencia Nuclear , Oocitos/metabolismo , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Ovinos/genéticaRESUMEN
Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator-activated receptor-γ (PPARγ) site-directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse-transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit-8 cell proliferation analysis, 5-ethynyl-2'-deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high-quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Técnicas de Sustitución del Gen , Mutagénesis Sitio-Dirigida , Mutación , Mioblastos/metabolismo , Miostatina/genética , PPAR gamma/genética , Adipogénesis , Animales , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Bovinos , Línea Celular , Proliferación Celular , Transdiferenciación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Estudios de Factibilidad , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos , Miostatina/metabolismo , Técnicas de Transferencia Nuclear , PPAR gamma/metabolismoRESUMEN
Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.
Asunto(s)
Integrina alfa6/genética , Factor de Transcripción PAX7/genética , Espermatogénesis/genética , Testículo/crecimiento & desarrollo , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Autorrenovación de las Células/genética , Regulación del Desarrollo de la Expresión Génica/genética , Cabras/genética , Cabras/crecimiento & desarrollo , Masculino , MicroARNs/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Espermatogonias/crecimiento & desarrollo , Células Madre/citología , Células Madre/metabolismo , Testículo/metabolismoRESUMEN
Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied.
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Células Madre Germinales Adultas/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inflamación/patología , Melatonina/farmacología , Células Madre Germinales Adultas/efectos de los fármacos , Células Madre Germinales Adultas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Lipopolisacáridos , Masculino , Ratones , Modelos Biológicos , Receptores de Melatonina/metabolismo , Testículo/efectos de los fármacos , Testículo/patologíaRESUMEN
Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1ß (IL-1ß) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1ß expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.
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Virus de la Encefalitis Japonesa (Especie)/fisiología , Regulación de la Expresión Génica , Interleucina-1beta/biosíntesis , MicroARNs/metabolismo , Estabilidad del ARN , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Línea Celular Tumoral , Cricetinae , Mesocricetus , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
The ten-eleven translocation (TET) proteins play a crucial role in promoting locus-specific reversal of DNA methylation, a type of chromatin modification. Considerable evidences have demonstrated that the sequence motifs with specific codes are important to determine the functions of domains and active sites. Here, we surveyed major studies and reviews regarding the multiple functions of the TET proteins and established the patterns of the motif arrangements that determine the functions of TET proteins. First, we summarized the functional sequence basis of TET proteins and identified the new functional motifs based on the phylogenetic relationship. Next, we described the sequence characteristics of the functional motifs in detail and provided an overview of the relationship between the sequence motifs and the functions of TET proteins, including known functions and potential functions. Finally, we highlighted that sequence motifs with diverse post-translational modifications perform unique functions, and different selection pressures lead to different arrangements of sequence motifs, resulting in different paralogs and isoforms.
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Secuencia de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/químicaRESUMEN
Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.
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Autorrenovación de las Células , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Dominios y Motivos de Interacción de Proteínas , Espermatogénesis , Células Madre/citología , Testículo/citología , Factores de Transcripción/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Cabras , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Animales , Células Madre/metabolismo , Testículo/metabolismoRESUMEN
Fat deposition in sheep tails is as a result of a complicated mechanism. Mongolian sheep (MG) and Small Tail Han sheep (STH) are two fat-tailed Chinese indigenous sheep breeds while DairyMeade and East Friesian (DS) are two thin-tailed dairy sheep breeds recently introduced to China. In this study, population genomics analysis was applied to identify candidate genes associated with sheep tails based on an in-depth whole-genome sequencing of MG, STH and DS. The selective signature analysis demonstrated that GLIS1, LOC101117953, PDGFD and T were in the significant divergent regions between DS and STH-MG. A nonsynonymous point mutation (g.27807636G>T) was found within GLIS1 in STH-MG and resulted in a Pro to Thr substitution. As a pro-adipogenic factor, GLIS1 may play critical roles in the mesodermal cell differentiation during fetal development affecting fat deposition in sheep tails. This study gives a new insight into the genetic basis of species-specific traits of sheep tails.
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Metabolismo de los Lípidos/genética , Oveja Doméstica , Cola (estructura animal)/metabolismo , Dedos de Zinc/genética , Animales , China , Genómica , Metagenómica , Mutación , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Polimorfismo de Nucleótido Simple , Oveja Doméstica/genética , Oveja Doméstica/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuenciación Completa del GenomaRESUMEN
We have previously bred Chinese local dairy sheep through grading up with local Small-Tailed Han (STH) sheep as female parent and DairyMeade (DM) sheep as male parent. In this research communication we characterize the whey protein profile of STH sheep and their offspring (F1, F2) to reveal physiological differences and variation in milk traits. A total of 1032 whey proteins were identified through tandem mass tag labeling (TMT) proteome profiling. Three proteins were significantly differentially abundant between F1 and STH milk, six between F2 and STH milk and five between F1 and F2 milk. In terms of differential changes between generations, WASHC4 and CUTA of F1 and Ig-like domain-containing protein of F2 milk were dominant whey proteins. Overall, the results showed that the whey protein profiles of different generations varied little. The crossbreeds of STH and DM sheep would be suitable for the development of the Chinese local sheep milk industry, and the F2 may be a better population for sheep milk production.
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Leche/química , Proteómica , Ovinos , Proteína de Suero de Leche/análisis , Animales , Cruzamiento , China , Cruzamientos Genéticos , Industria Lechera , Femenino , Masculino , Proteómica/métodosRESUMEN
We characterized the proteome profile of mid-lactation small-tailed Han (STH) and DairyMeade (DM) ovine milk in order to explore physiological variation and differences in milk traits between the two breeds. Methodology combined a tandem mass tag (TMT) proteomic approach with LC-MS/MS technology. A total of 656 proteins were identified in STH and DM ovine milk, of which 17and 29 proteins were significantly upregulated (P < 0.05) in STH and DM, respectively. Immune-related proteins and disease-related proteins were highly expressed in STH milk, whereas S100A2 and AEBP1 were highly expressed in DM milk, which had beneficial effects on mammary gland development and milk yield. Our results provide a theoretical basis for future breeding of dairy sheep.
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Leche , Proteoma , Animales , Cromatografía Liquida/veterinaria , Femenino , Lactancia , Proteómica , Ovinos , Espectrometría de Masas en Tándem/veterinaria , Suero LácteoRESUMEN
Hydraulic piston pump is the heart of hydraulic transmission system. On account of the limitations of traditional fault diagnosis in the dependence on expert experience knowledge and the extraction of fault features, it is of great meaning to explore the intelligent diagnosis methods of hydraulic piston pump. Motivated by deep learning theory, a novel intelligent fault diagnosis method for hydraulic piston pump is proposed via combining wavelet analysis with improved convolutional neural network (CNN). Compared with the classic AlexNet, the proposed method decreases the number of parameters and computational complexity by means of modifying the structure of network. The constructed model fully integrates the ability of wavelet analysis in feature extraction and the ability of CNN in deep learning. The proposed method is employed to extract the fault features from the measured vibration signals of the piston pump and realize the fault classification. The fault data are mainly from five different health states: central spring failure, sliding slipper wear, swash plate wear, loose slipper, and normal state, respectively. The results show that the proposed method can extract the characteristics of the vibration signals of the piston pump in multiple states, and effectively realize intelligent fault recognition. To further demonstrate the recognition property of the proposed model, different CNN models are used for comparisons, involving standard LeNet-5, improved 2D LeNet-5, and standard AlexNet. Compared with the models for contrastive analysis, the proposed method has the highest recognition accuracy, and the proposed model is more robust.
RESUMEN
The efficiency of somatic cell nuclear transfer (SCNT) reprogramming is extremely low in terms of production of cloned animals. Here, we found that telomere rejuvenation is a critical event for SCNT reprogramming. Through small-molecule screening, we identified that melatonin significantly improved the in vitro and in vivo developmental competence of SCNT-derived embryos. Through use of embryonic biopsy, single-cell RNA sequencing, and quantitative FISH experiments, we revealed that melatonin not only attenuated the zygotic genome activation defect but also facilitated telomere elongation in the SCNT embryos. Further investigation indicated that melatonin inhibited heterochromatic epigenetic modification related to gene silencing including DNA methylation and histone H3 lysine 9 trimethylation. In addition, melatonin could increase the level of activation markers such as acetylated histone H3. This is the first study to characterize melatonin-treatment and telomere rejuvenation in SCNT-mediated reprogramming. Moreover, combinational use of melatonin-treated donor embryos and pseudopregnant recipients achieved synergistic enhancement of the production of cloned animals.-Yang, L., Liu, X., Song, L., Su, G., Di, A., Bai, C., Wei, Z., Li, G. Inhibiting repressive epigenetic modification promotes telomere rejuvenation in somatic cell reprogramming.
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Reprogramación Celular , Embrión de Mamíferos/efectos de los fármacos , Represión Epigenética/efectos de los fármacos , Telómero/fisiología , Animales , Clonación de Organismos , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/efectos de los fármacos , Melatonina/farmacología , Ratones , Estrés Oxidativo/efectos de los fármacosRESUMEN
Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus-L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3-specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT-derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.
Asunto(s)
Embrión de Mamíferos/metabolismo , Retrovirus Endógenos/genética , Genes Reporteros , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Técnicas de Transferencia Nuclear , Animales , Blastocisto/citología , Blastocisto/metabolismo , Reprogramación Celular , Desarrollo Embrionario , Femenino , Fertilización , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones Endogámicos C57BL , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transcriptoma/genética , Cigoto/metabolismoRESUMEN
N6-methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long-term-cultured ESCs. Pluripotency studies indicated that 10 µmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c-Myc, by preventing m6A-dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1-JAK2/STAT3-Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.