ABSTRACT
Green eggs are mainly caused by inserting an avian endogenous retrovirus (EVA-HP) fragment into the SLCO1B3 gene. Although the genotypes for this insertion allele are consistent, eggshell color (ESC) may vary after a peak laying period; light-colored eggs are undesired by consumers and farmers and result in financial loss, so it is necessary to resolve this problem. miRNAs are small non-coding RNAs that exert essential functions in animal development and diseases. However, the regulatory miRNAs and detailed molecular mechanisms regulating eggshell greenness remain unclear. In the present study, we determined the genotype of green-eggshell hens through the detection of a homozygous allele insertion in the SLCO1B3 gene. The shell gland epithelium was obtained from green-eggshell hens that produced white and green shell eggs to perform transcriptome sequencing and investigate the important regulatory mechanisms that influence the ESC. Approximately 921 miRNAs were expressed in these two groups, which included 587 known miRNAs and 334 novel miRNAs, among which 44 were differentially expressed. There were 22 miRNAs that were significantly upregulated in the green and white groups, respectively, which targeted hundreds of genes, including KIT, HMOX2, and several solute carrier family genes. A Gene Ontology enrichment analysis of the target genes showed that the differentially expressed miRNA-targeted genes mainly belonged to the functional categories of homophilic cell adhesion, gland development, the Wnt signaling pathway, and epithelial tube morphogenesis. A KEGG enrichment analysis showed that the Hedgehog signaling pathway was significantly transformed in this study. The current study provides an overview of the miRNA expression profiles and the interaction between the miRNAs and their target genes. It provides valuable insights into the molecular mechanisms underlying green eggshell pigmentation, screening more effective hens to produce stable green eggs and obtaining higher economic benefits.
Subject(s)
Chickens , Egg Shell , MicroRNAs , Pigmentation , Transcriptome , Animals , Chickens/genetics , MicroRNAs/genetics , Egg Shell/metabolism , Pigmentation/genetics , Transcriptome/genetics , FemaleABSTRACT
The Jinling White duck represents a newly developed breed characterized by a rapid growth rate and a superior meat quality, offering significant economic value and research potential; however, the genetic basis underlying their body weight traits remains less understood. Here, we performed whole-genome resequencing for 201 diverse Jinling White male ducks and conducted population genomic analyses, suggesting a rich genetic diversity within the Jinling White duck population. Equipped with our genomic resources, we applied genome-wide association analysis for body weight on birth (BWB), body weight on 1 wk (BW1), body weight on 3 wk (BW3), body weight on 5 wk (BW5) and body weight on 7 wk (BW7) using 4 statistical models. Comparative studies indicated that factored spectrally transformed linear mixed models (FaST-LMM) demonstrated the most superior efficiency, yielding more results with the minimal false positives. We discovered that PUS7, FBXO11, FOXN2, MSH6, and SLC4A4 were associated with BWB. RAG2, and TMEFF2 were candidate genes for BW1, and STARD13, Klotho, ZAR1L are likely candidates for BW3 and BW5. PLXNC1, ATP1A1, CD58, FRYL, OCIAD1, and OCIAD2 were linked to BW7. These findings provide a genetic reference for the selection and breeding of Jinling White ducks, while also deepened our understanding of Growth and development phenotypic in ducks.
Subject(s)
Body Weight , Ducks , Genome-Wide Association Study , Animals , Ducks/genetics , Ducks/physiology , Ducks/growth & development , Genome-Wide Association Study/veterinary , Body Weight/genetics , Male , China , East Asian PeopleABSTRACT
OBJECTIVE: The objective of this study was to identify candidate genes that play important roles in skeletal muscle development in ducks. METHODS: In this study, we investigated the transcriptional sequencing of embryonic pectoral muscles from two specialized lines: Liancheng white ducks (female) and Cherry valley ducks (male) hybrid Line A (LCA) and Line C (LCC) ducks. In addition, prediction of target genes for the differentially expressed mRNAs was conducted and the enriched gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes signaling pathways were further analyzed. Finally, a protein-to-protein interaction network was analyzed by using the target genes to gain insights into their potential functional association. RESULTS: A total of 1,428 differentially expressed genes (DEGs) with 762 being up-regulated genes and 666 being down-regulated genes in pectoral muscle of LCA and LCC ducks identified by RNA-seq (p<0.05). Meanwhile, 23 GO terms in the down-regulated genes and 75 GO terms in up-regulated genes were significantly enriched (p<0.05). Furthermore, the top 5 most enriched pathways were ECM-receptor interaction, fatty acid degradation, pyruvate degradation, PPAR signaling pathway, and glycolysis/gluconeogenesis. Finally, the candidate genes including integrin b3 (Itgb3), pyruvate kinase M1/2 (Pkm), insulinlike growth factor 1 (Igf1), glucose-6-phosphate isomerase (Gpi), GABA type A receptorassociated protein-like 1 (Gabarapl1), and thyroid hormone receptor beta (Thrb) showed the most expression difference, and then were selected to verification by quantitative realtime polymerase chain reaction (qRT-PCR). The result of qRT-PCR was consistent with that of transcriptome sequencing. CONCLUSION: This study provided information of molecular mechanisms underlying the developmental differences in skeletal muscles between specialized duck lines.
ABSTRACT
To investigate the effect of genetic selection on meat quality in ducks, twenty of each fast growth ducks (LCA) and slow growth ducks (LCC) selected from F6 generation of Cherry Valley ducks (â) x Liancheng white ducks (â) were analyzed for carcass characteristics, meat quality (physicochemical and textural characteristics), amino acid and fatty acid profiles at 7 wk. Results showed that live body weight, slaughter weight, eviscerated yield and abdominal fat percentage of LCA were significantly higher than those in LCC ducks (P < 0.01). Moreover, the average area and diameter of myofiber were larger in LCA than LCC ducks (P < 0.01). The breast and thigh muscles of LCA exhibited significantly lower water holding capacity and thermal loss compared with LCC ducks (P < 0.01). In addition, the content of nonessential amino acids (Glu, Asp, and Arg) in breast muscles and Asp, Ser, Thr, and Met in thigh muscles was higher in LCC than LCA ducks (P < 0.05). The proportion of polyunsaturated fatty acids (PUFA) in breast muscles of LCC was higher than LCA ducks (P < 0.05). However, the content of saturated fatty acids (SFA) in breast and thigh muscles of LCA was higher compared with LCC ducks (P < 0.05). The proportion of monounsaturated fatty acids (MUFA) in thigh muscles was significantly higher in LCC compared with LCA ducks (P < 0.01). Finally, multiple traits were evaluated by applying principal component analysis (PCA) and the results indicated that PUFA and SFA in breast muscles of LCA played important roles in meat quality, followed by Warner-Bratzler shear force (WBSF) and MUFA. However, water holding capacity (WHC) had a dominant effect in meat quality of thigh muscles in both LCA and LCC ducks.
Subject(s)
Amino Acids , Ducks , Fatty Acids , Meat , Muscle, Skeletal , Animals , Ducks/physiology , Ducks/genetics , Ducks/growth & development , Meat/analysis , Amino Acids/metabolism , Amino Acids/analysis , Fatty Acids/analysis , Fatty Acids/metabolism , Muscle, Skeletal/chemistry , Male , Female , Body CompositionABSTRACT
OBJECTIVE: The objective of this study was to investigate the regulation relationship of Teneleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. METHODS: siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/ß-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. RESULTS: Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/ß-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/ß-catenin signaling pathway. CONCLUSION: These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/ß-catenin signaling pathway.
ABSTRACT
DNA methylation is a pivotal epigenetic regulatory mechanism in the development of skeletal muscles. Nonetheless, the regulators responsible for DNA methylation in the development of embryonic duck skeletal muscles remain unknown. In the present study, whole genome bisulfite sequencing (WGBS) and transcriptome sequencing were conducted on the skeletal muscles of embryonic day 21 (E21) and day 28 (E28) ducks. The DNA methylation pattern was found to fall mainly within the cytosine-guanine (CG) context, with high methylation levels in the intron, exon, and promoter regions. Overall, 7902 differentially methylated regions (DMRs) were identified, which corresponded to 3174 differentially methylated genes (DMGs). By using integrative analysis of both WGBS with transcriptomics, we identified 1072 genes that are DMGs that are negatively associated with differentially expressed genes (DEGs). The gene ontology (GO) analysis revealed significant enrichment in phosphorylation, kinase activity, phosphotransferase activity, alcohol-based receptors, and binding to cytoskeletal proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGGs) analysis showed significant enrichment in MAPK signaling, Wnt signaling, apelin signaling, insulin signaling, and FoxO signaling. The screening of enriched genes showed that hyper-methylation inhibited the expression of Idh3a, Got1, Bcl2, Mylk2, Klf2, Erbin, and Klhl38, and hypo-methylation stimulated the expression of Col22a1, Dnmt3b, Fn1, E2f1, Rprm, and Wfikkn1. Further predictions showed that the CpG islands in the promoters of Klhl38, Klf2, Erbin, Mylk2, and Got1 may play a crucial role in regulating the development of skeletal muscles. This study provides new insights into the epigenetic regulation of the development of duck skeletal muscles.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Animals , Ducks/genetics , Transcriptome , Muscle, Skeletal/metabolismABSTRACT
China boasts a rich diversity of indigenous duck species, some of which exhibit desirable economic traits. Here, we generated transcriptome sequencing datasets of breast muscle tissue samples from 1D of four groups: Pekin duck pure breeding group (P), Jinling White duck breeding group (J), P â × J â orthogonal group (PJ) and J â × P â reciprocal-cross group (JP) (n = 3), chosen based on the distinctive characteristics of duck muscle development during the embryonic period. We identified 5053 differentially expressed genes (DEGs) among the four groups. Network prediction analysis showed that ribosome and oxidative phosphorylation-related genes were the most enriched, and muscular protein-related genes were found in the 14-day-old embryonic group. We found that previously characterized functional genes, such as FN1, AGRN, ADNAMST3, APOB and FGF9, were potentially involved in muscle development in 14-day-old embryos. Functional enrichment analysis suggested that genes that participated in molecular function and cell component and key signaling pathways (e.g., hippo, ribosome, oxidative phosphorylation) were significantly enriched in the development of skeletal muscle at 14 days of embryonic age. These results indicate a possible role of muscle metabolism and myoglobin synthesis in skeletal muscle development in both duck parents and hybrids.
Subject(s)
Ducks , Gene Expression Profiling , Animals , Ducks/genetics , Gene Expression , Muscle Development/genetics , Muscle, SkeletalABSTRACT
Ascorbic acid (also called Vitamin C, VC) strengthens the function of Tets families and directly increases DNA demethylation level to affect myogenic differentiation. However, the precise regulatory mechanism of DNA methylation in chicken myogenesis remains unclear. Results of present study showed that the mRNA expression of MyoD significantly decreased and MyoG and MyHC increased in myoblasts treated with 5 µM 5-azacytidine (5-AZA) and 5 µM VC (p < 0.05). Results also indicated the formation of myotubes was induced by 5-AZA or VC, but this effect was attenuated after knockdown of Tet2. In addition, the protein expression of TET2, DESMIN and MyHC was remarkable increased by the addition of 5-AZA or VC, and the upregulation was inhibited after knockdown of Tet2 (p < 0.05). DNA dot blot and immunofluorescence staining results suggested that the level of 5hmC was significantly increased when treated with 5-AZA or VC, even by Tet2 knockdown (p < 0.05). Moreover, 5-AZA and VC reduced the level of dimethylation of lysine 9 (H3K9me2) and trimethylation of lysine 27 of histone 3 (H3K27me3), and this inhibitory effect was eliminated after Tet2 knockdown (p < 0.05). These data indicated that Tet2 knockdown antagonized the increased levels of 5hmC and H3K27me3 induced by 5-AZA and VC, and eventually reduced myotube formation by modulating the expression of genes involved in myogenic differentiation. This study provides insights that epigenetic regulators play essential roles in mediating the myogenic program of chicken myoblasts.
Subject(s)
Ascorbic Acid , Chickens , Animals , Ascorbic Acid/pharmacology , Ascorbic Acid/metabolism , Histones/metabolism , Lysine/metabolism , Myoblasts/metabolism , Muscle Development/genetics , AzacitidineABSTRACT
The Guangxi Partridge chicken is a well-known chicken breed in southern China with good meat quality, which has been bred as a meat breed to satisfy the increased demand of consumers. Compared with line D whose body weight is maintained at the average of the unselected group, the growth rate and weight of the selected chicken group (line S) increased significantly after breeding for four generations. Herein, transcriptome analysis was performed to identify pivotal genes and signal pathways of selective breeding that contributed to potential mechanisms of growth and development under artificial selection pressure. The average body weight of line S chickens was 1.724 kg at 90 d of age, which showed a significant increase at 90 d of age than line D chickens (1.509 kg), although only the internal organ ratios of lung and kidney changed after standardizing by body weight. The myofiber area and myofiber density of thigh muscles were affected by selection to a greater extent than that of breast muscle. We identified 51, 210, 31, 388, and 100 differentially expressed genes (DEGs) in the hypothalamus, pituitary, breast muscle, thigh muscle, and liver between the two lines, respectively. Several key genes were identified in the hypothalamus-pituitary-muscle axis, such as FST, THSB, PTPRJ, CD36, PITX1, PITX2, AMPD1, PRKAB1, PRKAB2, and related genes for muscle development, which were attached to the cytokine-cytokine receptor interaction signaling pathway, the PPAR signaling pathway, and lipid metabolism. However, signaling molecular pathways and the cell community showed that elevated activity in the liver of line S fowl was mainly involved in focal adhesion, ECM-receptor interaction, cell adhesion molecules, and signal transduction. Collectively, muscle development, lipid metabolism, and several signaling pathways played crucial roles in the improving growth performance of Guangxi Partridge chickens under artificial selection for growth rate. These results support further study of the adaptation of birds under selective pressure.
Subject(s)
Chickens , Gene Expression Profiling , Animals , Body Weight , Chickens/metabolism , China , Meat/analysis , Pectoralis Muscles/metabolismABSTRACT
Chicken blastoderm cells (cBCs) obtained from stage X (EG&K) embryos are easily available materials for the study of cell development. However, cBCs are not widely used because they are hard to maintain in long-term culture in vitro. To solve this problem, ascorbic acid (AA; also known as vitamin C (VC)) and all-trans retinoic acid (ATRA) were added into basic culture medium to promote cell growth. Results suggested that cultured cBCs possessed strongly proliferative activity and maintained their pluripotency on the support of chicken embryonic fibroblast (CEF) feeder. Moreover, when VC or/and ATRA was added, the number and area of cBC colonies increased significantly compared with the control group. The expression of pluripotency genes (Sox2 and Nanog) and cell cycle-regulated genes (CCND1 and CDK6) was upregulated obviously. Furthermore, results showed that 5hmC levels in VC and RA groups increased significantly by DNA dot blot and immunofluorescence staining. These results provide strong evidence that VC and ATRA induced DNA demethylation and enhanced 5hmC level. The level of H3K27me3 was raised, while the level of H3K9me2 was reduced by addition of VC and ATRA. Finally, the expression of Tet1 and Dnmt3b was upregulated remarkably. Therefore, these results indicated that VC and ATRA enhanced DNA demethylation and then promoted cBC survival and proliferation in vitro.
Subject(s)
Blastoderm , Chickens , Animals , Ascorbic Acid/pharmacology , Cell Proliferation , Chick Embryo , DNA Demethylation , Tretinoin/pharmacologyABSTRACT
The objective of this study was to investigate the effects of rearing systems on the bone quality parameters in chickens using a metabolomics strategy. A total of 419 male one-day-old chicks were randomly allocated to two groups, a floor rearing group (FRG, n = 173) and a cage rearing group (CRG, n = 246). At 6, 8, 10, and 12 weeks of age, all chickens were radiographed by a digital X-ray machine, and body weight was recorded. At 12 weeks of age, 12 birds were selected from each group to obtain tibia and femur, and bone quality parameters of bone mineral density (BMD), mineral content (BMC), breaking strength (BBS), stiffness, Young's modulus (YM), ash content, calcium content, and phosphorus content were determined. An untargeted metabolomics assay was performed to identify changes in the serum metabolic profile (n = 8 birds/group). The results showed that cage-reared chickens had wider tibiae and greater body weight compared with floor-reared chickens. There were no significant differences in BMC or BBS between the two groups (p > 0.05), but BMD, ash content, calcium content, and phosphorus content of the tibia and femur of FRG were significantly higher than those of CRG (p < 0.05). Greater stiffness and YM of the femur were also observed in birds raised in the FRG compared with those raised in the CRG (p < 0.05). Taken together, the results suggest that rearing systems affected bone quality parameters. Furthermore, 148 and 149 differential metabolites were identified in positive and negative ion modes by LC-MS/MS analysis, among which 257 metabolites were significantly correlated with 16 bone quality parameters, including leucine, myristoleic acid, glycocholic acid, and N-phenylacetamide. KEGG analysis indicated that 15 metabolic pathways, including six pathways of amino acid metabolism, two pathways of lipid metabolism, and two pathways of carbohydrate metabolism, were responsible for bone quality. Overall, the present study demonstrated the effect of rearing systems on bone quality parameters, and identified several metabolites and metabolic pathways associated with bone quality parameters.
ABSTRACT
Skeletal muscle development is an orchestrated progress that is primarily regulated by temporospatial expression of myogenic regulatory factors (MRFs). Recent studies demonstrated that DNA demethylation also exerted a critical role in myogenesis. However, the function of Tet2 in the regulation of chicken myogenesis still remains unknown. In the present study, the role of Tet2 in regulating myogenic differentiation was determined by using a model of primary myoblasts from chickens. The expression of Tet2 was significantly elevated during myoblast differentiation. Meanwhile, the level of 5hmC in genomic DNA was increased, but H3K9me2 and H3K27me3 were markedly reduced following differentiation. Knockdown of Tet2 significantly inhibited the formation of multinucleated myotubes, which was accompanied by a reduction of relevant pivotal MRFs. Moreover, the level of 5hmC decreased sharply in Tet2 knockdown myoblasts. Attenuated differentiated myoblasts that resulted from reduced Tet2 also demonstrated an increased level of H3K9me2 and H3K27me3. Collectively, these results indicated that Tet2 played an essential role during myogenesis, which affected demethylation of genomic DNA and histone to regulate expression of MRFs and therefore, contributed to myoblast differentiation.
Subject(s)
Cell Differentiation , DNA Methylation , DNA-Binding Proteins/metabolism , Muscle Development/physiology , Muscle Fibers, Skeletal/cytology , Myoblasts/cytology , Animals , Cells, Cultured , Chickens , DNA-Binding Proteins/genetics , Gene Knockdown Techniques , Models, Animal , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolismABSTRACT
This study was conducted to evaluate the effect of curcumin on laying performance, egg quality, biochemical indicators, hormone levels, and immune activity in hens under heat stress. Hy-Line brown hens (280-day-old) were fed with 0, 100, 150, and 200 mg/kg of curcumin during a 42-D experiment. Compared with the control treatment, supplementation with 150 mg/kg of curcumin improved laying performance and egg quality by significantly increasing egg production, eggshell thickness, eggshell strength (P < 0.01), and albumen height (P < 0.05) while decreasing the feed-to-egg ratio. Antioxidant activity was improved by significantly increasing the activity of superoxide dismutase and glutathione peroxidase but decreasing malondialdehyde levels in serum (P < 0.05) and significantly increasing the levels of follicle-stimulating hormone, luteinizing hormone, estradiol, IgG, IgA, and complement C3 activity in serum (P < 0.05). These results indicated that supplemental 150 mg/kg curcumin can improve productive performance, antioxidant enzyme activity, and immune function in laying hens under the heat stress conditions applied in the present study.
Subject(s)
Chickens/physiology , Curcumin/metabolism , Heat-Shock Response , Hormones/metabolism , Immunity/drug effects , Animal Feed/analysis , Animals , Chickens/blood , Chickens/immunology , Curcumin/administration & dosage , Diet/veterinary , Dietary Supplements/analysis , Dose-Response Relationship, Drug , Female , Random AllocationABSTRACT
Antibiotics stimulate the growth of animals but result in drug residues and bacterial resistance. In this study, the negative effect of antibiotics on abdominal fat deposition was evaluated in broilers. The results showed that adding both chlortetracycline (50 g/1,000 kg) and tylosin (50 g/1,000 kg) significantly increased abdominal fat weight, abdominal fat percentage (p < .05), and triglyceride and cholesterol levels (p < .05) in blood. Also, both products synchronously stimulated intestinal absorption and synthesis of liver fat. The expression levels of the peroxisome proliferator-activated receptor γ (PPARγ), diacylgycerol acyltransferase 2 (DGAT2), lipoprotein lipase (LPL), and fatty acid-binding protein (FABP4) genes in abdominal fat tissue significantly increased (p < .05 or 0.01) when antibiotics were added to the feed. However, no significant difference was found in expression of the fatty acid synthesis (FAS) or acetyl CoA carboxylase (ACC) genes. Further in vitro study results revealed that antibiotics had no effect on fat content or the related gene expression levels in preadipocytes. In summary, the antibiotics induced fat deposition in adipose tissues by activating extracellular absorption of fatty acids from intestinal absorption and synthesis of liver fat. However, it shows no direct regulation by adipose tissue.
Subject(s)
Abdominal Fat/metabolism , Anti-Bacterial Agents/pharmacology , Chickens/growth & development , Chickens/metabolism , Chlortetracycline/pharmacology , Tylosin/pharmacology , Adipose Tissue/metabolism , Animals , Anti-Bacterial Agents/adverse effects , Chlortetracycline/adverse effects , Cholesterol/blood , Diacylglycerol O-Acyltransferase/metabolism , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Gene Expression , Intestinal Absorption , Lipoprotein Lipase/genetics , Lipoprotein Lipase/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Triglycerides/blood , Tylosin/adverse effectsABSTRACT
High-quality chicken meat is an important source of animal protein for humans. Gene expression profiles in breast muscle tissue were determined, aiming to explore the common regulatory genes relevant to muscle and intramuscular fat (IMF) during the developmental stage in chickens. Results show that breast muscle weight (BMW), breast meat percentage (BMP, %), and IMF (%) continuously increased with development. A total of 256 common differentially expressed genes (DEGs) during the developmental stage were screened. Among them, some genes related to muscle fiber hypertrophy were upregulated (e.g., CSRP3, LMOD2, MUSTN1, MYBPC1), but others (e.g., ACTC1, MYL1, MYL4) were downregulated from Week 3 to Week 18. During this period, expression of some DEGs related to the cells cycle (e.g., CCNB3, CCNE2, CDC20, MCM2) changed in a way that genetically suggests possible inhibitory regulation on cells number. In addition, DEGs associated with energy metabolism (e.g., ACOT9, CETP, LPIN1, DGAT2, RBP7, FBP1, PHKA1) were found to regulate IMF deposition. Our data identified and provide new insights into the common regulatory genes related to muscle growth, cell proliferation, and energy metabolism at the developmental stage in chickens.
Subject(s)
Adipose Tissue/metabolism , Chickens/genetics , Lipid Metabolism/genetics , Meat , Adipose Tissue/growth & development , Animals , Chickens/growth & development , Chickens/metabolism , Gene Expression Regulation, Developmental , Muscle Proteins/genetics , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolismABSTRACT
Testosterone (T) is essential for muscle fiber formation and growth. However, the specific mechanism by which T regulates skeletal muscle development in chicken embryos remains unclear. In this study, the role of T in myoblast proliferation both in vivo and in vitro was investigated. Results showed that the T administration significantly increased the ratio of breast muscle and leg muscle. T induced a significant increase in the cross-sectional area (CSA) and density of myofiber and the ratio of PAX7-positive cells in the skeletal muscle. Exogenous T also induced the upregulation of myogenic regulatory factors (MRFs) and cyclin-dependent kinases (CDK2)/Cyclin D1 (CCND1) and protein levels of androgen receptor (AR), p-Akt and PAX7. Furthermore, T treatment significantly promoted myoblasts cultured in vitro entering a new cell cycle and increased PAX7-positive cells. The mRNA and protein expression of AR and PAX7 were upregulated when treated with T compared to that of the control. The addition of T induced proliferation accompanied by increasing AR level as well as PI3K (Phosphoinositide 3-kinase)/Akt activation. However, T-induced proliferation was attenuated by AR, PI3K, and Akt-specific inhibitors. These data indicated that the pro-proliferative effect of T was regulated though AR in response to the activation of PI3K/Akt signalling pathway.
Subject(s)
Avian Proteins/metabolism , Chick Embryo/cytology , Myoblasts/cytology , Receptors, Androgen/metabolism , Signal Transduction , Testosterone/metabolism , Animals , Cell Proliferation , Cells, Cultured , Chick Embryo/metabolism , Chickens/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myoblasts/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Ten-eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5-E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.
Subject(s)
DNA Methylation , Germ Cells/metabolism , Mixed Function Oxygenases/metabolism , Proto-Oncogene Proteins/metabolism , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Amino Acid Transport System y+L/genetics , Amino Acid Transport System y+L/metabolism , Animals , Cell Proliferation/genetics , Cell Survival/genetics , Chickens , Deleted in Azoospermia 1 Protein/genetics , Deleted in Azoospermia 1 Protein/metabolism , Gene Knockdown Techniques , Genomic Imprinting , Mixed Function Oxygenases/genetics , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Proto-Oncogene Proteins/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Bisphenol A (BPA) as an endocrine-disrupting chemical with weak estrogenic activity affects formation of primordial follicles. This study aimed to identify the potential effects and molecular mechanisms of BPA on meiosis and primordial follicle formation in chickens. The results suggest that the cortical layer was thickened and the number of germ cells that entered into meiosis was increased in BPA-treated ovaries. The percentage of γH2AX-positive cells increased significantly. In addition, up-regulated mRNA expression of meiotic genes, including stimulated by retinoic acid gene 8 (Stra8), disrupted meiotic cDNA 1 homologue (Dmc1) and synaptonemal complex protein 3 (Scp3) were observed in BPA-treated ovaries. Therefore, progression to meiosis prophase I was accelerated by exposure to BPA. Furthermore, the results demonstrated that injection of BPA resulted in hypomethylation of Dazl (Deleted in A Zoospermia-Like gene) and Stra8 and up-regulation mRNA expression of Dazl and Stra8 during meiotic onset. Finally, the relationship between estrogen receptor (ER) expression and BPA-induced meiosis was revealed using an in vitro ovarian culture system. BPA enhanced ERß expression at the levels of mRNA and protein, while BPA exerted no significant effect on ERα and membrane-bound estrogen receptor (GPR30) expression. The inducing effects of BPA on meiosis were blocked by ER inhibitor. Collectively, these results demonstrate the dynamic ovarian response to BPA exposure, which indicate that BPA affects the formation of primordial follicles by promoting meiotic progression of oocytes via hypomethylation of Dazl and Stra8 and ERß signaling pathways.
Subject(s)
Benzhydryl Compounds/toxicity , Chickens/metabolism , Estrogen Receptor beta/metabolism , Meiosis/drug effects , Phenols/toxicity , Signal Transduction/drug effects , Animals , Chick Embryo , DNA/metabolism , DNA Methylation/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , Female , Gene Expression Regulation/drug effects , Germ Cells/cytology , Germ Cells/drug effects , Germ Cells/metabolism , Ovarian Follicle/cytology , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Regulation of skeletal muscle development requires many of the regulatory networks that are fundamental to developmental myogenesis. ErbB3 binding protein-1 (Ebp1) is involved in the control of myoblasts development in chicken. However, the expression and biological functions of Ebp1 in the progress of myogenesis are unclear. This study focused on determining the effect of Ebp1 on myogenic proliferation and differentiation using a primary myoblasts culture model. Ebp1 was found to upregulate in proliferating myoblasts and decrease at the early stage of myogenic differentiation. The level of endogenous Ebp1 increased from E9 to E20 chicken leg muscles. Knockdown of Ebp1 had no effect on myoblasts proliferation. However, myogenic differentiation into multinucleated myotubes was significantly reduced. The mRNA and protein expression of MRFs was decreased when Ebp1 was knocked down. Downregulation of Ebp1, accompanied by elevated levels of pSMAD2/3, suggests that Ebp1 is involved in regulating myogenic differentiation via SMAD2/3 inhibition. The phosphorylation of SMAD2/3 was activated and the expression of MYOD and MYOG was reduced in Ebp1 knockdown myoblasts, but addition of LY2109761 (an inhibitor specified to SMAD2/3) blocked these effects. Collectively, these results indicate that Ebp1 promotes myoblast differentiation by inhibition of SMAD2/3 signaling pathway during chicken myogenesis. These data provide new insights into the biological role of Ebp1 in embryonic chicken skeletal muscle development.
Subject(s)
Cell Differentiation/physiology , Muscle Development/physiology , Myoblasts/cytology , Myoblasts/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Blotting, Western , Cell Differentiation/genetics , Chick Embryo , Muscle Development/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
BACKGROUND: Producing transgenic chickens with chicken blastodermal cells (cBCs) is inefficient due to the extremely low germline transmission capacity of cBCs. As chicken primordial germ cells (PGCs) have been reported as an efficient method for producing transgenic chickens, the inefficiency of cBCs could potentially be resolved by inducing them to differentiate into germ cells. However, whether chemical inducers are able to enhance cBCs germline competence in vitro is unknown and the molecular mechanisms of differentiation of chicken pluripotent cells into germ cells are poorly understood. RESULTS: We cultured cBCs with a monolayer morphology in E8 medium, a xeno- and feeder-free medium. We showed that retinoic acid (RA) treatment increased expression of germ cell-specific genes in cBCs. Using western blot, we determined that RA stimulated Smad1/5 phosphorylation. Moreover, Smad1/5 activation regulates the expression of germ cell-specific genes, as co-treatment with a Smad1/5 phosphorylation inhibitor or activator alters expression of these genes. We also demonstrate that Smad1/5 is required for RA-induced differentiation by RNA interference knockdown. CONCLUSION: Our results demonstrated that E8 medium is able to maintain cBC growth for weeks and RA treatment induced germ cell differentiation of cBCs through the BMP-Smad1/5 signaling pathway.