O-GlcNAcylation is a gatekeeper of porcine myogenesis.

Although it has long been known that growth media withdrawal is a prerequisite for myoblast differentiation and fusion, the underpinning molecular mechanism remains somewhat elusive. Using isolated porcine muscle satellite cells (SCs) as the model, we show elevated O-GlcNAcylation by O-GlcNAcase (OGA) inhibition impaired SC differentiation (D5 P < 0.0001) but had unnoticeable impacts on SC proliferation. To explore the mechanism of this phenotype, we examined the expression of the transcription factor myogenin, a master switch of myogenesis, and found its expression was downregulated by elevated O-GlcNAcylation. Because insulin/IGF-1/Akt axis is a strong promoter of myoblast fusion, we measured the phosphorylated Akt and found that hyper O-GlcNAcylation inhibited Akt phosphorylation, implying OGA inhibition may also work through interfering with this critical differentiation-promoting pathway. In contrast, inhibition of O-GlcNAc transferase (OGT) by its specific inhibitor had little impact on either myoblast proliferation or differentiation (P > 0.05). To confirm these in vitro findings, we used chemical-induced muscle injury in the pig as a model to study muscle regenerative myogenesis and showed how O-GlcNAcylation functions in this process. We show a significant decrease in muscle fiber cross sectional area (CSA) when OGA is inhibited (P < 0.05), compared to nondamaged muscle, and a significant decrease compared to control and OGT inhibited muscle (P < 0.05), indicating a significant impairment in porcine muscle regeneration in vivo. Together, the in vitro and in vivo data suggest that O-GlcNAcylation may serve as a nutrient sensor during SC differentiation by gauging cellular nutrient availability and translating these signals into cellular responses. Given the importance of nutrition availability in lean muscle growth, our findings may have significant implications on how muscle growth is regulated in agriculturally important animals.

Cells use a variety of post translational modifications (PTMs) as a mechanism to transduce extracellular signals and adapt their behaviors in response to intracellular nutrient abundance. O-GlcNAcylation, the addition of single sugars to a protein's serine/threonine residues, has been established as a nutrient sensing PTM in a wide range of cell types. Here, we show the functional importance O-GlcNAcylation in porcine myogenesis. We used isolated porcine satellite cells as the model and pharmacological inhibitors to O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) as the tool to study the role of O-GlcNAcylation in porcine myogenesis. Our data show that although O-GlcNAcylation does not play a significant role in muscle cell proliferation, low level of O-GlcNAcylation is critical for muscle cell differentiation. We demonstrate that inhibition of OGA leads to higher level of O-GlcNAcylation and inhibition of myoblast fusion even though the growth medium (high nutrients) has been shifted to the differentiation medium (low nutrients). Together, these data show that porcine muscle cells use O-GlcNAcylation to sense the cellular nutrient levels and adjust their fate in accordance with the strength of the O-GlcNAcylation signals.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

BACKGROUND: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence. METHODS: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes. RESULTS: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex. CONCLUSIONS: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Driving an Oxidative Phenotype Protects Myh4 Null Mice From Myofiber Loss During Postnatal Growth.

Postnatal muscle growth is accompanied by increases in fast fiber type compositions and hypertrophy, raising the possibility that a slow to fast transition may be partially requisite for increases in muscle mass. To test this hypothesis, we ablated the Myh4 gene, and thus myosin heavy chain IIB protein and corresponding fibers in mice, and examined its consequences on postnatal muscle growth. Wild-type and Myh4 -/- mice had the same number of muscle fibers at 2 weeks postnatal. However, the gastrocnemius muscle lost up to 50% of its fibers between 2 and 4 weeks of age, though stabilizing thereafter. To compensate for the lack of functional IIB fibers, type I, IIA, and IIX(D) fibers increased in prevalence and size. To address whether slowing the slow-to-fast fiber transition process would rescue fiber loss in Myh4 -/- mice, we stimulated the oxidative program in muscle of Myh4 -/- mice either by overexpression of PGC-1α, a well-established model for fast-to-slow fiber transition, or by feeding mice AICAR, a potent AMP kinase agonist. Forcing an oxidative metabolism in muscle only partially protected the gastrocnemius muscle from loss of fibers in Myh4 -/- mice. To explore whether traditional means of stimulating muscle hypertrophy could overcome the muscling deficits in postnatal Myh4 -/- mice, myostatin null mice were bred with Myh4 -/- mice, or Myh4 -/- mice were fed the growth promotant clenbuterol. Interestingly, both genetic and pharmacological stimulations had little impact on mice lacking a functional Myh4 gene suggesting that the existing muscle fibers have maximized its capacity to enlarge to compensate for the lack of its neighboring IIB fibers. Curiously, however, cell signaling events responsible for IIB fiber formation remained intact in the tissue. These findings further show disrupting the slow-to-fast transition of muscle fibers compromises muscle growth postnatally and suggest that type IIB myosin heavy chain expression and its corresponding fiber type may be necessary for fiber maintenance, transition and hypertrophy in mice. The fact that forcing muscle metabolism toward a more oxidative phenotype can partially compensates for the lack of an intact Myh4 gene provides new avenues for attenuating the loss of fast-twitch fibers in aged or diseased muscles.

Dual effects of obesity on satellite cells and muscle regeneration.

Obesity is a complex metabolic disorder that often leads to a decrease in insulin sensitivity, chronic inflammation, and overall decline in human health and well-being. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury; however, the mechanism underlying these changes has yet to be determined. To test whether there is a negative impact of obesity on satellite cell (SC) decisions and behaviors, we fed C57BL/6 mice normal chow (NC, control) or a high-fat diet (HFD) for 10 weeks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller size (p < .001) compared to those from NC mice, and this decreased proliferation was confirmed (p < .05) by BrdU incorporation. Moreover, in vitro assays showed that HFD SCs exhibited diminished (p < .001) fusion capacity compared to NC SCs. In single fiber explants, a higher ratio of SCs experienced apoptotic events (p < .001) in HFD mice compared to that of NC-fed mice. In vivo lineage tracing using H2B-GFP mice showed that SCs from HFD treatment also cycled faster (p < .001) than their NC counterparts. In spite of all these autonomous cellular effects, obesity as triggered by high-fat feeding did not significantly impair muscle regeneration in vivo, as reflected by the comparable cross-sectional area (p > .05) of the regenerating fibers in HFD and NC muscles, suggesting that other factors may mitigate the negative impact of obesity on SCs properties.

Eye of the Finch: characterization of the ocular microbiome of house finches in relation to mycoplasmal conjunctivitis.

Vertebrate ocular microbiomes are poorly characterized and virtually unexplored in wildlife species. Pathogen defense is considered a key function of microbiomes, but determining microbiome stability during disease is critical for understanding the role of resident microbial communities in infectious disease dynamics. Here, we characterize the ocular bacterial microbiome of house finches (Haemorhous mexicanus), prior to and during experimental infection with an inflammatory ocular disease, Mycoplasmal conjunctivitis, caused by Mycoplasma gallisepticum. In ocular tissues of healthy house finches, we identified 526 total bacterial operational taxonomic units (OTUs, 97% similarity), primarily from Firmicutes (92.6%) and Proteobacteria (6.9%), via 16S rRNA gene amplicon sequencing. Resident ocular communities of healthy female finches were characterized by greater evenness and phylogenetic diversity compared to healthy male finches. Regardless of sex, ocular microbiome community structure significantly shifted 11 days after experimental inoculation with M. gallisepticum. A suite of OTUs, including taxa from the genera Methylobacterium, Acinetobacter and Mycoplasma, appear to drive these changes, indicating that the whole finch ocular microbiome responds to infection. Further study is needed to quantify changes in absolute abundance of resident taxa and to elucidate potential functional roles of the resident ocular microbiome in mediating individual responses to this common songbird bacterial pathogen.

Incubation temperature causes skewed sex ratios in a precocial bird.

Many animals with genetic sex determination are nonetheless capable of manipulating sex ratios via behavioral and physiological means, which can sometimes result in fitness benefits to the parent. Sex ratio manipulation in birds is not widely documented, and revealing the mechanisms for altered sex ratios in vertebrates remains a compelling area of research. Incubation temperature is a key component of the developmental environment for birds, but despite its well-documented effects on offspring phenotype it has rarely been considered as a factor in avian sex ratios. Using ecologically relevant manipulations of incubation temperature within the range 35.0-37.0°C, we found greater mortality of female embryos during incubation than males regardless of incubation temperature, and evidence that more female than male embryos die at the lowest incubation temperature (35.0°C). Our findings in conjunction with previous work in brush turkeys suggest incubation temperature is an important determinant of avian secondary sex ratios that requires additional study, and should be considered when estimating the impact of climate change on avian populations.

Experimental support for food limitation of a short-distance migratory bird wintering in the temperate zone.

The Winter Food Limitation Hypothesis (WFLH) states that winter food abundance is a dominant source of population limitation of migratory birds. Evidence is accumulating that long-distance migratory birds wintering in tropical climates have high overwinter survival probabilities and that winter food limitation mainly affects their fitness nonlethally by limiting energetic reserves necessary for successful reproduction. In contrast, the relative roles of direct mortality vs. indirect effects caused by food limitation have not been investigated thoroughly on short-distance migratory birds wintering in temperate zones, where they face thermal challenges. We performed the first test of the WFLH for a temperate migratory bird in the wild on the Swamp Sparrow (Melospiza georgiana), with a replicated plot-wide food supplementation experiment. In contrast to tropical, but consistent with other temperate-wintering migrants, Swamp Sparrows on unmanipulated plots showed relatively low apparent survival across the winter. Following food addition, birds (1) immigrated to experimental plots, which subsequently supported approximately 50% higher abundances, (2) experienced increases of within-season apparent survival of 8-10%, depending on age/sex class, and (3) had higher-scaled mass index values, all supporting winter food limitation. The last two findings are interrelated because birds with higher scaled mass had higher survival probabilities, further supporting direct effects of winter food limitation. Food limitation of fat reserves might also have indirect effects on reproductive success by limiting migration timing and survival during migration. Increases in scaled mass were higher in females, suggesting that they are disproportionately affected by food limitation, possibly through competition. Based on Robust Design Modeling, we found no support for emigration prior to food addition, indicating that our estimates of mortality are unbiased.