Silver Nanoparticles as Modulators of Myogenesis-Related Gene Expression in Chicken Embryos

The present study was conducted to investigate the effects of colloidal nanoparticles of silver (Nano-Ag) on the expression of myogenesis-related genes in chicken embryos. The investigated genes included the members of the myogenic regulatory factors family (MRFs) and myocyte enhancer factor 2A (MEF2A) genes. A total of 200 fertilized broiler eggs (Indian River) were randomly distributed into four groups; non-injected control, injected control with placebo, treatment I in ovo injected with 20 ppm Nano-Ag, and treatment II in ovo injected with 40 ppm Nano-Ag. The eggs were then incubated for 21 days at the optimum temperature and humidity conditions. Breast muscle tissues were collected at the 5th, 8th, and 18th days of the incubation period. The mRNA expression of myogenic determination factor 1 (MYOD1), myogenic factor 5 (MYF5), myogenic factor 6 (MYF6), myogenin (MYOG), and MEF2A was measured at the three sampling points using real-time quantitative PCR, while MYOD1 protein expression was evaluated on day 18 using western blot. Breast muscle tissues were histologically examined on day 18 to detect the changes at the cellular level. Our results indicate that myogenesis was enhanced with the low concentration (20 ppm) of Nano-Ag due to the higher expression of MYOD1, MYF5, and MYF6 at the transcriptional level and MYOD1 at the translational level. Moreover, histological analysis revealed the presence of hyperplasia (31.4% more muscle fibers) in treatment I (injected with 20 ppm). Our findings indicate that in ovo injection of 20 ppm Nano-Ag enhances the development of muscles in chicken embryos compared with the 40-ppm dosage and provide crucial information for the use of silver nanoparticles in poultry production.

Epigenetic effects induced by the ectopic expression of Pax7 in 3T3-L1

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

The Impact of Polyamine Precursors, Polyamines, and Steroid Hormones on Temporal Messenger RNA Abundance in Bovine Satellite Cells Induced to Differentiate

Emerging research suggests that hormones found in anabolic implants interact with polyamine biosynthesis. The objective of this study was to determine the effects of steroidal hormones, polyamines and polyamine precursors on bovine satellite cell (BSC) differentiation and polyamine biosynthesis temporally. Primary BSCs were induced to differentiate in 3% horse serum (CON) and treated with 10 nM trenbolone acetate (TBA), 10 nM estradiol (E2), 10 nM TBA and 10 nM E2, 10 mM methionine, 8 mM ornithine, 2 mM putrescine, 1.5 mM spermidine, or 0.5 mM spermine. Total mRNA was isolated 0, 2, 4, 8, 12, 24, and 48 h post-treatment. Abundance of mRNA for genes associated with induction of BSC differentiation: paired box transcription factor 7, myogenic factor 5, and myogenic differentiation factor 1 and genes in the polyamine biosynthesis pathway: ornithine decarboxylase and S-adenosylmethionine—were analyzed. Overall, steroidal hormones did not impact (p > 0.05) mRNA abundance of genes involved in BSC differentiation, but did alter (p = 0.04) abundance of genes involved in polyamine biosynthesis. Polyamine precursors influenced (p < 0.05) mRNA of genes involved in BSC differentiation. These results indicate that polyamine precursors and polyamines impact BSC differentiation and abundance of mRNA involved in polyamine biosynthesis, while steroidal hormones altered the mRNA involved in polyamine biosynthesis.

POLYMORPHISM OF GROWTH HORMONE RECEPTOR GENE AND MYOGENIC FACTOR 5 GENE IN DAIRY CATTLE POPULATIONS

Investigations of the genetic structure features of the populations of Ukrainian Black-and-White and Red-and-White dairy breeds by polymorphism of growth hormone receptor (GHR) and myogenic factor 5 (MYF5) genes were carried out. AluI-polymorphism in the promoter fragment of the GHR gene and TaqI-polymorphism in the second intron of the MYF5 gene was detected using polymerase chain reaction and restriction analysis (PCR-RFLP). According to the research results, it was shown that the growth hormone receptor and myogenic factor 5 genes in the populations of Ukrainian Black-and-White and Red-and-White dairy breeds are polymorphic. In the population of the Ukrainian Black-and-White dairy breed at the GHR locus, the frequency of the AluI+ allele was 0.61; allele AluI- – 0.39; at the MYF5 locus, the TaqI+ allele frequency was 0.65; allele Taq- – 0.35, respectively. In the population of the Ukrainian Red-and-White dairy breed at the GHR locus, the frequency of the AluI+ allele was 0.54; allele AluI- – 0.46; at the MYF5 locus, the TaqI+ allele frequency was 0.64; allele Taq- – 0.36, respectively. Both experimental populations for GHR and MYF5 loci are in a genetic equilibrium state according to Hardy-Weinberg, which indicates the absence of microevolutionary changes in the process of their reproduction. There were no significant fluctuations in the genetic structure according to the revealed features of the genotype and allele frequencies distribution. The breeding work that is carried out on both populations does not affect marker alleles (which are described in the work), which is reflected in the features of the genetic-population structure of the experimental groups and their equilibrium state. The features of the distribution of individuals with different genotypes by GHR and MYF5 loci in the populations of both breeds make it possible to carry out further studies to determine the relationship between the identified allelic variants of polymorphic loci with parameters of animal productivity.

Fructose 1,6-bisphosphate sensing by pyruvate kinase isozymes M2 (PKM2) controls MyoD stability and myogenic differentiation

Glucose exerts beneficial effects on myogenesis and muscle physiology. However, the mechanisms by which glucose regulates myogenesis remain ill-defined or incompletely understood. Here, we show that low glycolysis destabilizes MyoD protein, a master myogenic transcription factor. Intriguingly, MyoD is not controlled by the cellular energy status per se, but by the level of fructose 1,6-bisphosphate, an intermediate metabolite of glycolysis. Fructose 1,6-bisphosphate is sensed by pyruvate kinase M2 (PKM2). In the presence of fructose 1,6-bisphosphate, PKM2 form tetramers that sequester the Huwe1 E3 ubiquitin ligase to the cytoplasm. Reduced fructose 1,6-bisphosphate levels dissociate the tetramer, releasing Huwe1 into the nucleus where it targets MyoD for degradation. Genetic or pharmacological modulation of PKM2-Huwe1 axis restores myogenic differentiation in glucose restricted conditions. Our results show that glucose metabolism directly regulates protein stability of a key myogenic factor and provide a rationale for enhancing myogenesis.

200 Reduced growth performance of pigs fed methionine deficient diet may be associated with their reduced muscle cell differentiation

Methionine (Met) is the second or third limiting AA in typical swine diets and plays important roles in promoting the growth, especially, the muscle growth of pigs. This research studied the effects of dietary Met restriction on growth performance and myogenic gene expression in growing pigs. Eight genes in two families, including myogenic regulatory factor family comprising myogenic differentiation 1 (MyoD1), myogenic factor 5 (Myf5), myogenin (MyoG) and myogenic factor 6 (Myf6), and myocyte enhancer factor 2 (Mef2) family comprising Mef2A, Mef2B, Mef2C and Mef2D, were selected for analysis. Individually penned barrows (crossbred, 23.6±2.4 kg) were randomly assigned to two dietary treatments. A basal diet (Diet 1; Met-deficient) was formulated to contain 0.22% standardized ileal digestible (SID) Met and 0.52% SID Met+Cys but to meet the NRC (2012) recommendations for other nutrients. Crystalline DL-Met was added to the basal diet to generate Diet 2 adequate in Met (0.37% SID Met, 0.67% SID Met+Cys). During the four-week ad libitum feeding trial, ADG, ADFI and G:F were measured. Immediately before and after the trial, muscle samples were collected from the longissimus dorsi using a standard biopsy protocol. Total RNA was extracted from the muscle samples (TRIzol Reagent; Invitrogen) and subjected to cDNA reverse-transcription (QuantiTect Reverse Transcription Kit; Qiagen). Transcribed cDNA were used for qPCR analysis (Rotor-Gene Q System; Qiagen). Comparative ΔΔCT method was used for quantitation. Data were analyzed with Student’s T-test. Pigs fed Diet 1 (vs. Diet 2) had a lower ADG and G:F (P&lt; 0.01). Before the feeding trial, all the tested genes had comparable mRNA levels between the two treatments (P &gt;0.17). After the trial, Diet 1 pigs showed tendency for lower levels of Myf6 and Mef2D mRNA (P&lt; 0.09). These results suggest that the reduced ADG and G:F is associated with the possibly-reduced muscle cell differentiation in pigs fed Met-deficient diet.

Poly(ADP-ribose) Polymerase 1 (PARP1) restrains MyoD-dependent gene expression during muscle differentiation

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Effect of vitamin A restriction on carcass traits and blood metabolites in Korean native steers

The objective of the present study was to determine the effect of vitamin A restriction on serum metabolites and carcass performance in Korean native steers during a fattening period. In Study 1, 61 steers were divided into three groups and supplied diets with concentrate and roughage containing 890 IU/kg of provitamin A and then supplemented with either 8000 IU (control), 3000 IU (T1) or 0 IU (T2) of premix vitamin A per kilogram of dry matter. In Study 2, 19 steers were divided into two groups and provided with the same basic diets then supplemented with 8000 IU (control) or 0 IU (T) of premix vitamin A per kilogram of dry matter. In Study 1, we found that serum vitamin A concentrations were significantly (P &lt; 0.05) lower in the T2 group, along with significant (P &lt; 0.05) increases in blood urea nitrogen, albumin, creatinine and non-esterified fatty acid concentrations. In Study 2, the T group had a higher (P &lt; 0.05) skeletal muscle mRNA expression levels of myogenic factor 6 and a trend for the greater yield grade (P=0.095). However, marbling scores in the study showed no significant. Therefore, vitamin A restriction with vitamin premix during the fattening period was associated with a trend for a higher yield grade, but marbling scores that were not significantly higher. Metabolic parameters in this stage could be used as indicators in future metabolic studies and as early health status markers in Korean native steers.