RESUMO
Microchromosomes are prevalent in nonmammalian vertebrates [P. D. Waters et al., Proc. Natl. Acad. Sci. U.S.A. 118 (2021)], but a few of them are missing in bird genome assemblies. Here, we present a new chicken reference genome containing all autosomes, a Z and a W chromosome, with all gaps closed except for the W. We identified ten small microchromosomes (termed dot chromosomes) with distinct sequence and epigenetic features, among which six were newly assembled. Those dot chromosomes exhibit extremely high GC content and a high level of DNA methylation and are enriched for housekeeping genes. The pericentromeric heterochromatin of dot chromosomes is disproportionately large and continues to expand with the proliferation of satellite DNA and testis-expressed genes. Our analyses revealed that the 41-bp CNM repeat frequently forms higher-order repeats (HORs) at the centromeres of acrocentric chromosomes. The centromere core regions where the kinetochore attaches often encompass telomeric sequence (TTAGGG)n, and in a one of the dot chromosomes, the centromere core recruits an endogenous retrovirus (ERV). We further demonstrate that the W chromosome shares some common features with dot chromosomes, having large arrays of hypermethylated tandem repeats. Finally, using the complete chicken chromosome models, we reconstructed a fine picture of chordate karyotype evolution, revealing frequent chromosomal fusions before and after vertebrate whole-genome duplications. Our sequence and epigenetic characterization of chicken chromosomes shed insights into the understanding of vertebrate genome evolution and chromosome biology.
Assuntos
Centrômero , Galinhas , Animais , Masculino , Galinhas/genética , Centrômero/genética , Telômero , Heterocromatina , Sequências de Repetição em TandemRESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ferro , Oxirredução , Ribonucleoproteínas , Edição de Genes/métodos , Ferro/química , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Lipossomos/química , Técnicas de Transferência de Genes , Proteína 9 Associada à CRISPR/metabolismoRESUMO
Liver lipid metabolism disruption significantly contributes to excessive fat buildup in waterfowl. Research suggests that the supplementation of Threonine (Thr) in the diet can improve liver lipid metabolism disorder, while Thr deficiency can lead to such metabolic disorders in the liver. The mechanisms through which Thr regulates lipid metabolism remain unclear. STAT3 (signal transducer and activator of transcription 3), a crucial transcription factor in the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway, participates in various biological processes, including lipid and energy metabolism. This research investigates the potential involvement of STAT3 in the increased lipid storage seen in primary duck hepatocytes as a result of a lack of Thr. Using small interfering RNA and Stattic, a specific STAT3 phosphorylation inhibitor, we explored the impact of STAT3 expression patterns on Thr-regulated lipid synthesis metabolism in hepatocytes. Through transcriptome sequencing, we uncovered pathways related to lipid synthesis and metabolism jointly regulated by Thr and STAT3. The results showed that Thr deficiency increases lipid deposition in primary duck hepatocytes (p < 0.01). The decrease in protein and phosphorylation levels of STAT3 directly caused this deposition (p < 0.01). Transcriptomic analysis revealed that Thr deficiency and STAT3 knockdown jointly altered the mRNA expression levels of pathways related to long-chain fatty acid synthesis and energy metabolism (p < 0.05). Thr deficiency, through mediating STAT3 inactivation, upregulated ELOVL7, PPARG, MMP1, MMP13, and TIMP4 mRNA levels, and downregulated PTGS2 mRNA levels (p < 0.01). In summary, these results suggest that Thr deficiency promotes lipid synthesis, reduces lipid breakdown, and leads to lipid metabolism disorders and triglyceride deposition by downregulating STAT3 activity in primary duck hepatocytes.
Assuntos
Patos , Hepatócitos , Fator de Transcrição STAT3 , Treonina , Triglicerídeos , Animais , Fator de Transcrição STAT3/metabolismo , Hepatócitos/metabolismo , Fosforilação , Treonina/metabolismo , Triglicerídeos/metabolismo , Metabolismo dos Lipídeos , Células CultivadasRESUMO
Duck meat is known for its taste and high nutritive value. To preserve local genetic diversity while maintaining commercial viability, we obtained a crossbreed (CB) between high-performing Cherry Valley (CV) and traditional Chinese crested (CC) ducks. We compared carcass traits and meat quality characteristics of CB and parental breeds. Meat from the above ducks at their respective marketable ages was evaluated for proximate composition, amino acid and fatty acid profiles, and selected mineral content. The live weights, carcass weights, and breast muscle percentage of CB were higher than CC but lower than CV; the leg muscle of CB was lower than CV and CC. CB had higher intramuscular fat content than CV; its collagen content was lower than CC but higher than CV in breast and thigh muscles. Additionally, the saturated fatty acid content of CB muscle was lower than CV and higher than CC. CB contained more monounsaturated fatty acids than CV and CC. Zn content was higher in CB breast than CV and CC. CB, obtained by crossing CV and CC, has partial advantages over both the breeds suggesting that these characteristics aligned with standards to breed ducks with high-quality meat.
Assuntos
Patos , Carne , Animais , Aminoácidos/análise , Patos/genética , Ácidos Graxos/análise , Carne/análise , Minerais/análise , Composição Corporal/genética , ChinaRESUMO
With the development of high-throughput sequencing, circular RNA has come into people's vision and attracted more and more attention. Many studies have found that circular RNA plays an important role in a variety of biological processes and the occurrence and development of diseases. According to the previous sequencing results, circRNA_3238 was differentially expressed in ALV-J infected group and the non-infected group was selected for subsequent verification and analysis. We found that circRNA_3238 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, lymph, and bursa of fabricius. Bioinformatics results show that circRNA_3238 and the predicted target genes enriched MAPK signaling pathway, Notch signaling pathway, and other pathways related to disease or immune, revealing circRNA_3238 may indirectly regulate the process of ALV-J infection by regulating target genes.
Assuntos
MicroRNAs , RNA Circular , Animais , RNA Circular/genética , RNA Circular/metabolismo , RNA/genética , Galinhas/genética , Galinhas/metabolismo , Transdução de Sinais/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/genéticaRESUMO
The growth and development of duck skeletal muscle is an important economic trait that is genetically regulated. The internal mechanism underlying the regulation of skeletal muscle growth and development in ducks remains unclear. The purpose of this study was to identify candidate genes related to the growth of duck skeletal muscle. RNA-sequencing technology was used to compare the transcriptome of duck breast muscles in an F2 population with the high breast muscle rate (HB) and the low breast muscle rate (LB). A total of 14,522 genes were confirmed to be expressed in the breast muscle, and 173 differentially expressed genes (DEGs) were identified between the HB and LB groups. Functional analysis showed that these DEGs were mainly involved in biological processes and pathways of fat metabolism and muscle growth, especially the FABP3 and MYL4 involved in the PPAR signaling pathway and cardiac muscle contraction pathway. These findings deepened our understanding of the molecular mechanisms involved in muscle growth in ducks and provided a theoretical basis for improving duck production and breeding of ducks.
Assuntos
Fenômenos Biológicos , Transcriptoma , Animais , Transcriptoma/genética , Patos/genética , Perfilação da Expressão Gênica , Músculo Esquelético/metabolismoRESUMO
Circular RNA (circRNA) is a new non-coding RNA with a highly conserved and stable covalently closed loop structure, and it plays an important role in a variety of biological processes and the occurrence of diseases. Based on the sequencing results, circRNA_3079 had the most significant difference between the infected group and normal group, up to about 8 times. It has attracted our attention and was selected for further verification and analysis. Though the characteristics analysis of circRNA_3079 in chicken, we found circRNA_3079 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, spleen, lymph and bursa of fabricius. Bioinformatics analysis results showed that circRNA_3079 and the predicted target genes are enriched in many pathways related to immunity or tumors, such as p53 signaling pathway, Jak-STAT signaling pathway and NOD-like receptor signaling pathway, which revealed that circRNA_3079 may indirectly regulate the ALV-J infection process through the regulation of target genes.HIGHLIGHTSCircRNA_3079 is an abundant and stable circular RNA expressed in different tissues and cells in chicken.The circularization of circRNA_3079 does not depend on the reverse complementary repetitive sequence of the flanking sequence.CircRNA_3079 may indirectly regulate the ALV-J infection process.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Animais , Leucose Aviária/genética , Vírus da Leucose Aviária/genética , Galinhas/genética , Proteínas NLR , RNA Circular/genética , RNA não Traduzido , Proteína Supressora de Tumor p53RESUMO
Muscle growth rate and muscle mass are important economic traits in animal production. Musculoskeletal embryonic nuclear protein 1 (MUSTN1) gene has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and expression of MUSTN1 in different duck breeds are not fully understood. To gain insights into the biological functions of MUSTN1 in skeletal muscle development, the MUSTN1 coding sequence of Pekin ducks (BD) and Cherry Valley ducks (CD) was compared to various other animals using the Editseq in DNAstar and MEGA software. The results showed that the duck had the highest homology with chicken. The RT-qPCR and western blot were performed to estimate the mRNA and protein expression pattern of MUSTN1 in leg muscles of BD and CD at 3 and 6-weeks of age. At 3 weeks of age, the mRNA and protein expression levels of MUSTN1 were significantly higher in BD than in CD (p < 0.05). At 6 weeks, the expression level was higher in BD than in CD. In conclusion, MUSTN1 might play a key role in positive regulation of muscle growth and development of ducks.
Assuntos
Patos , Desenvolvimento Muscular , Animais , Patos/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Totally, 315 42-day-old male Xueshan chickens were allocated into 3 caging densities, 14, 21 and 28 birds/m2. Each treatment was represented by 5 replicates. The body weight (BW), slaughter performance, meat quality, behavioral assessment, and the cecal microorganisms were detected at the market age. The results showed that the BW of broilers in the low- and medium-density groups was significantly higher (p < 0.05) than that of the high-density group from the age of 10 weeks. Only the feather quality of the broilers in the low-density group improved significantly (p < 0.05) compared with those of the other two groups. And, the abdominal fat percentage and the fat content of thigh muscle of broilers in the low- and medium-density groups were higher (p < 0.05) than those in the high-density group. No significant difference (p > 0.05) was noted in the other traits. The abundance of some microbial like Akkermansiaceae, Lactobacillaceae and Faecalibacterium may be correlated with the BW and fat content of broilers. The findings of this study suggest that increasing the stocking density decreased the final BW, fat content and the feather quality, whereas no evidence was found that stocking density caused changes in other parameters.
Assuntos
Criação de Animais Domésticos , Peso Corporal , Galinhas , Microbioma Gastrointestinal , Carne , Animais , Masculino , Criação de Animais Domésticos/métodos , Ceco/microbiologia , Carne/análise , Carne/normas , Densidade DemográficaRESUMO
The imbalance in polyunsaturated fatty acid (PUFA) composition in human food is ubiquitous and closely related to obesity and cardiovascular diseases. The development of n-3 PUFA-enriched poultry products is of great significance for optimizing fatty acid composition. This study aimed to improve our understanding of the effects of dietary linseed oil on hepatic metabolism using untargeted metabolomics and 4D label-free proteome analysis. A total of 91 metabolites and 63 proteins showed differences in abundance in duck livers between the high linseed oil and control groups. Pathway analysis revealed that the biosynthesis of unsaturated fatty acids, linoleic acid, glycerophospholipid, and pyrimidine metabolisms were significantly enriched in ducks fed with linseed oil. Meanwhile, dietary linseed oil changed liver fatty acid composition, which was reflected in the increase in the abundance of downstream metabolites, such as α-linolenic acid (ALA; 18:3n-3) as a substrate, including n-3 PUFA and its related glycerophospholipids, and a decrease in downstream n-6 PUFA synthesis using linoleic acid (LA; 18:2n-6) as a substrate. Moreover, the anabolism of PUFA in duck livers showed substrate-dependent effects, and the expression of related proteins in the process of fatty acid anabolism, such as FADS2, LPIN2, and PLA2G4A, were significantly regulated by linseed oil. Collectively, our work highlights the ALA substrate dependence during n-3 PUFA synthesis in duck livers. The present study expands our knowledge of the process products of PUFA metabolism and provides some potential biomarkers for liver health.
Assuntos
Gorduras Insaturadas na Dieta , Ácidos Graxos Ômega-3 , Linho , Animais , Humanos , Óleo de Semente do Linho/metabolismo , Gorduras Insaturadas na Dieta/metabolismo , Patos , Linho/metabolismo , Proteômica , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Ácido Linoleico/metabolismoRESUMO
The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.
Assuntos
Proteína Morfogenética Óssea 4/genética , Epigênese Genética/genética , Histona Metiltransferases/genética , Testículo/crescimento & desenvolvimento , Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/metabolismo , Animais , Blastoderma/crescimento & desenvolvimento , Diferenciação Celular/genética , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Genitália/crescimento & desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Masculino , Transdução de Sinais/genética , Testículo/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Proteína Wnt-5a/genéticaRESUMO
Fatty acids (FAs) are essential for the vital movement of humans and animals. Their metabolism is, in part, regulated by FABP3. In our previous study, a novel lncRNA (ENSGALG00000021686, L21686) was identified, and FABP3 was predicted as its target gene. Here, using chicken myocytes, lymphocytes, and different tissues, L21686 target on the FABP3 gene, FABP3 mRNA expression, and their effect on FA metabolism are explored. The results show that the highest expression of L21686 is in muscle tissue, a significant energy-consuming tissue. L21686 expression is consistent with FABP3 mRNA expression. We also show that under the different treatments, the levels of FABP3 mRNA and protein in myocytes and lymphocytes change in tandem with L21686 expression. Moreover, the dual-luciferase reporter assay provided direct evidence that L21686 targets the FABP3 gene. Finally, it was found that the content of free FAs increases along with the up-regulation of L21686 and the FABP3 gene. Malonyl CoA content does not change under the different treatments, suggesting that L21686 regulates the intake of extracellular FAs in chicken. Further, the changes in lipoprotein lipase (LPL), sterol-regulatory element binding protein 1 (SREBP-1), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) mRNA levels support this view. In summary, our data show that the new lncRNA (L21686) regulates the intake of extracellular FAs in chicken cells in vitro by targeting the expression of the FABP3 gene. Our findings will help to establish the groundwork and provide a new clue for deciphering the regulation of FAs metabolism in chicken.
Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Proteína 3 Ligante de Ácido Graxo/genética , Ácidos Graxos/metabolismo , RNA Longo não Codificante/genética , Animais , Proteínas Aviárias/metabolismo , Transporte Biológico , Células Cultivadas , Galinhas/metabolismo , Proteína 3 Ligante de Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Linfócitos/metabolismo , Células Musculares/metabolismoRESUMO
Skin and feather follicle morphogenesis are important processes for duck development; however, the mechanisms underlying morphogenesis at the embryonic stage remain unclear. To improve the understanding of these processes, we used transcriptome and weighted gene co-expression network analyses to identify the critical genes and pathways involved in duck skin development. Five modules were found to be the most related to five key stages in skin development that span from embryonic day 8 (E8) to postnatal day 7 (D7). Using STEM software, 6519 genes from five modules were clustered into 10 profiles to reveal key genes. Above all, we obtained several key module genes including WNT3A, NOTCH1, SHH, BMP2, NOG, SMAD3, and TGFß2. Furthermore, we revealed that several pathways play critical roles throughout the skin development process, including the Wnt pathway and cytoskeletal rearrangement-related pathways, whereas others are involved in specific stages of skin development, such as the Notch, Hedgehog, and TGF-beta signaling pathways. Overall, this study identified the pathways and genes that play critical roles in skin development, which may provide a basis for high-quality down-type meat duck breeding.
Assuntos
Patos/embriologia , Patos/genética , Desenvolvimento Embrionário/genética , Pele/embriologia , Animais , Patos/crescimento & desenvolvimento , Plumas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Anotação de Sequência Molecular , Morfogênese/genética , Organogênese , Pele/metabolismo , TranscriptomaRESUMO
MicroRNA (miRNA)-1 and miRNA-133 are derived from the same bicistronic pairs with roles in skeletal muscle development. Many investigations have focused on the role of miRNA-1 and miRNA-133 in the regulation of skeletal muscle development in mammals and fish. However, the mechanisms of miRNA-1 and miRNA-133 underlying the differences in skeletal muscle development between different breeds are not well known. Our study found that the weights of body and breast at 42 days of age were greater in Cherry Valley ducks than in Putian ducks and the areas of breast muscle fibers increased with age; the areas of muscle fibers of Cherry Valley ducks were always greater than those of Putian ducks. Besides, quantitative reverse-transcriptase polymerase chain reaction analysis revealed that relatively high levels of miRNA-1 and miRNA-133 were detected in heart, breast, and leg muscles compared with the liver, spleen, lung, kidney, and the expression levels of miRNA-1 and miRNA-133 remained stable in the embryo stage, and in the growth period, the fluctuation in miRNA expression levels in Putian ducks was considerably higher than that in Cherry Valley ducks, especially from 7 to 28 days. However, in the late growth period, the expression of miRNA-1 and miRNA-133 of Cherry Valley duck was higher than that of Putian duck, which may indicate that miRNA-1 and miRNA-133 play a more important role during the growth period. To determine the function of miRNA-1 and miRNA-133 in skeletal muscle development, we found that the overexpression of miRNA-1, but not miRNA-133, promoted fusion of adjacent myoblasts. By contrast, a repressor of miRNA-1 promoted, whereas a miRNA-133 inhibitor inhibited, myoblast proliferation. Accordingly, the expression levels of myocyte enhancer factor 2D (MEF2D) and myogenic differentiation ( MYOD) were significantly increased by an miRNA-1 mimic and the miRNA-133 inhibitor. In addition, we found that the expression levels of miRNA-1 significantly affected the expression of histone deacetylase 4 ( HDAC4), and miRNA-133 affected serum response factor ( SRF) and transforming growth factor ß receptor 1 ( TGFBR1) levels. However, dual-luciferase reporter assays revealed that only miRNA-1 directly inhibited pGL- HDAC4 luciferase reporter activity, whereas miRNA-133 did not affect pGL- SRF or pGL- TGFBR1 fluorescence activity. Taken together, these results suggest that miRNA-1 targets HDAC4 to promote the differentiation of duck myoblasts and miRNA-133 may affect SRF and TGFBR1 expression to promote proliferation, which indicates that miRNA-1 and miRNA-133 play different important roles in skeletal muscle development.
Assuntos
Diferenciação Celular , Proliferação de Células , MicroRNAs/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Animais , Células Cultivadas , Patos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , MicroRNAs/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de SinaisRESUMO
Spermatogenesis is a complex process. Some studies have shown that Piwi-interacting RNAs (piRNAs) play an important role in spermatogenesis. To verify the evaluate between piRNAs and PIWI proteins in chicken and its possible role in spermatogenesis and reproductive stem cell proliferation and differentiation, we performed immunoprecipitation and deep sequencing analyses and determined the expression profiles of small RNAs in primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia (Sa) cells, and spermatozoa. Length analysis showed that piRNAs bound to PIWIL1 mainly contained 23-30 nt. Base preference analysis showed "1U-10A"; moreover, base preference of piRNAs was obvious in all of germline cells. Here we reported the TE family of gallus gallus, and targeted by piRNA. Target gene of piRNA annotation enrichment analysis identified candidate genes KIT, SRC, WNT4, and HMGB2. Kyoto Encyclopedia of Genes and Genomes analysis showed that these genes were associated with steroid hormone biosynthesis, Notch signaling pathway, and melanogenesis. These results indicate that chicken piRNAs perform important regulatory roles during spermatogenesis similar to mice piRNAs. Chicken piRNAs interacted with PIWI proteins and regulated spermatogenesis and germ cell proliferation and differentiation. Further, we observed a negative correlation between piRNA-19128 and KIT expression. Results of dual-luciferase reporter assay confirmed that piRNA-19128 directly interacted with KIT, suggesting that it plays a key role in the regulation spermatogenesis by inhibiting KIT expression. Thus, the present study provides information on the length and base preference of chicken piRNAs and suggests that piRNA-19128 regulates spermatogenesis in chicken by silencing KIT.
Assuntos
Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Interferente Pequeno/metabolismo , Espermatogênese/fisiologia , Animais , Galinhas , Biologia Computacional , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-kit/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermatogênese/genética , Proteína Wnt4/genética , Proteína Wnt4/metabolismoRESUMO
Male germ cell differentiation is a subtle and complex regulatory process. Currently, its regulatory mechanism is still not fully understood. In our experiment, we performed the first comprehensive genome and transcriptome-wide analyses of the crucial genes and signaling pathways in three kinds of crucial cells (embryonic stem cells, primordial germ cell, and spermatogonial stem cells) that are associated with the male germ cell differentiation. We identified thousands of differentially expressed genes in this process, and from these we chose 173 candidate genes, of which 98 genes were involved in cell differentiation, 19 were involved in the metabolic process, and 56 were involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, and PSMD7 and so on. In addition, we found that 18 key signaling pathways were involved mainly in cell proliferation, differentiation, and signal transduction processes like TGF-ß, Notch, and Jak-STAT. Further exploration found that the candidate gene expression patterns were the same between in vitro induction experiments and transcriptome results. Our results yield clues to the mechanistic basis of male germ cell differentiation and provide an important reference for further studies.
Assuntos
Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Células Germinativas/metabolismo , Transdução de Sinais , Espermatogônias/metabolismo , Células-Tronco Adultas/citologia , Animais , Proliferação de Células , Células Cultivadas , Galinhas , Células-Tronco Embrionárias/citologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Células Germinativas/citologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogônias/citologiaRESUMO
BACKGROUND: Dopamine ß-hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines. This enzyme's role in neuroendocrine regulation is well known, but there are some indications that it may also modulate reproduction and endocrine in mammals and birds. We selected goose (Anas cygnoides) as an ideal model species for investigating the role of DBH in avian reproduction. RESULTS: Full-length cDNA encoding DBH was cloned from Zhedong goose using reverse transcription PCR and rapid amplification of cDNA ends. The cDNA consisted of a 126-base pair (bp) 5'-untranslated region (UTR), a 379-bp 3'-UTR, and an 1896-bp open reading frame encoding a polypeptide of 631 amino acids. The deduced amino acid sequence of gDBH shared high homology with an analogue from other birds and contained three conserved domains from a mono-oxygenase family including a DOMON domain and two Cu2_mono-oxygen domains. Real-time quantitative PCR analysis showed that gDBH mRNA was expressed in both reproductive and endocrine tissues of Zhedong goose, specifically in the hypothalamus, pituitary, ovary, and oviduct. More DBH mRNA of reproductive and endocrine tissues was detected at ovulation than at oviposition in Zhedong goose. Evidence of opposite trend of gDBH expression was found between the hypothalamus-pituitary and oviduct during the ovulation phase and the broody phase. In addition, we assessed DBH mRNA expression during ovulation in two breeds of geese that differ in egg production. The reproductive and endocrine tissues of Yangzhou geese with higher egg production had more gDBH expression than Zhedong geese. Finally, the five non-synonymous SNP(c.1739 C > T, c.1760G > T, c.1765A > G, c.1792 T > C and c.1861G > C) were identified in the coding region of DBH gene between Zhedong goose and Yangzhou goose. CONCLUSIONS: We conclude that goose DBH mRNA show obvious periodically variation in reproductive and endocrine tissues during the reproductive cycle in geese.
Assuntos
Dopamina beta-Hidroxilase/genética , Gansos/genética , Reprodução/genética , Sequência de Aminoácidos , Animais , Catecolaminas/biossíntese , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Regulação da Expressão Gênica , Dados de Sequência Molecular , Ovulação , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
The P-element induced wimpy testis (Piwi) protein family is responsible for initiating spermatogenesis and maintaining the integrity of germ cells and stem cells, but little is known regarding its transcriptional regulation in poultry. Here, we characterized the methylation status of the Piwil1 promoter in five different spermatogenic cell lines using direct bisulfite pyrosequencing and determined that methylation correlates negatively with germ cell type-specific expression patterns of piwil1. We demonstrated that methylation of the -148 CpG site, which is the predicted binding site for the transcription factors TCF3 and NRF1, was differentially methylated in different spermatogenic cells. This site was completely methylated in PGCs (primordial germ cells), but was unmethylated in round spermatids. A similar result was obtained in the region from +121 to +139 CpG sites of the Piwil1 promoter CpG island, which was predicted to contain SOX2 binding sites. In addition, demethylation assays further demonstrated that DNA methylation indeed regulates Piwil1 expression during chicken spermatogenesis. Combined with transcription factor binding site prediction, we speculate that methylation influences the recruitment of corresponding transcription factors. Collectively, we show the negative correlation between promoter methylation and piwil1 expression and that the spatiotemporal expression of chicken Piwil1 from the PGC stage to the round spermatid stage is influenced by methylation-mediated transcription factor regulation.
Assuntos
Proteínas Argonautas/genética , Regulação da Expressão Gênica , Fatores de Transcrição SOXB1/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Animais , Proteínas Argonautas/metabolismo , Sítios de Ligação , Linhagem Celular , Galinhas , Ilhas de CpG , Metilação de DNA , Masculino , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/metabolismoRESUMO
The immune performance, SNPs and expression levels of candidate genes (IL1-ß, Nramp1, TLR4, MyD88, NF-κB and NLRC5) were analyzed in carrier chickens of a Chinese indigenous breed infected with Salmonella enterica Serovar Pullorum at different persistence periods (12, 19 and 24 weeks of age). Carrier birds at 19 weeks of age presented significant difference in most immune parameters, as compared to carriers at 12 and 24 weeks of age, while no significant difference in most immune parameters was observed between carriers at 12 and 24 weeks of age. The genotype distributions of IL1-ß and TLR4 presented significant differences between carriers and healthy birds. The expression levels of most candidate genes in carriers at 19 weeks of age were significantly higher than that in carriers at 12, 24 weeks of age and healthy birds and reached 1% level of significance between carriers at 19 weeks of age and healthy birds. The expression patterns of all genes, but IL-1fl and NLRC5 between carriers at 12 and 24 weeks of age in all tissues were similar. Compared with carriers at 12 weeks of age, IL1-ß was significantly down-regulated, but NLRC5 was significantly up-regulated in carriers at 24 weeks of age. Our study demonstrated that immune performance of carrier birds was severely impaired at age of sexual maturation and NLRC5 might play as a negative mediator of NF-κB pathway involved in immune response to asymptomatic infection by S. Pullorum. The TLR4/MyD88/NF-κB pathway might be suitable for study on S. Pullorum infection in Chinese indigenous breeds.
Assuntos
Galinhas/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Salmonella enterica/patogenicidade , Transdução de Sinais , Regulação para Cima , Animais , Sequência de Bases , Relação CD4-CD8 , Galinhas/genética , Primers do DNA , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Copy number variation (CNV) has been recently examined in many species and is recognized as being a source of genetic variability, especially for disease-related phenotypes. In this study, the PennCNV software, a genome-wide CNV detection system based on the 60 K SNP BeadChip was used on a total sample size of 1,310 Beijing-You chickens (a Chinese local breed). After quality control, 137 high confidence CNVRs covering 27.31 Mb of the chicken genome and corresponding to 2.61 % of the whole chicken genome. Within these regions, 131 known genes or coding sequences were involved. Q-PCR was applied to verify some of the genes related to disease development. Results showed that copy number of genes such as, phosphatidylinositol-5-phosphate 4-kinase II alpha, PHD finger protein 14, RHACD8 (a CD8α- like messenger RNA), MHC B-G, zinc finger protein, sarcosine dehydrogenase and ficolin 2 varied between individual chickens, which also supports the reliability of chip-detection of the CNVs. As one source of genomic variation, CNVs may provide new insight into the relationship between the genome and phenotypic characteristics.