Transcriptomic insight into the hybridization mechanism of the Tambacu, a hybrid from Colossoma macropomum (Tambaqui) and Piaractus mesopotamicus (Pacu).

Interspecific hybrids are highly complex organisms, especially considering aspects related to the organization of genetic material. The diversity of possibilities created by the genetic combination between different species makes it difficult to establish a large-scale analysis methodology. An example of this complexity is Tambacu, an interspecific hybrid of Colossoma macropomum (Tambaqui) and Piaractus mesopotamicus (Pacu). Either genotype represents an essential role in South American aquaculture. However, despite this importance, the genetic information for these genotypes is still highly scarce in specialized databases. Using RNA-Seq analysis, we characterized the transcriptome of white muscle from Pacu, Tambaqui, and their interspecific hybrid (Tambacu). The sequencing process allowed us to obtain a significant number of reads (approximately 53 billion short reads). A total of annotated contigs were 37,285, 96,738, and 158,709 for Pacu, Tambaqui, and Tambacu. After that, we performed a comparative analysis of the transcriptome of the three genotypes, where we evaluated the differential expression (Tambacu vs Pacu = 11,156, and Tambacu vs Tambaqui = 876) profile of the transcript and the degree of similarity between the nucleotide sequences between the genotypes. We assessed the intensity and pattern of expression across genotypes using differential expression information. Clusterization analysis showed a closer relationship between Tambaqui and Tambacu. Furthermore, digital differential expression analysis selected some target genes related to essential cellular processes to evaluate and validate the expression through the RT-qPCR. The RT-qPCR analysis demonstrated significantly (p < 0.05) elevated expression of the mafbx, foxo1a, and rgcc genes in the hybrid compared to the parents. Likewise, we can observe genes significantly more expressed in Pacu (mtco1 and mylpfa) and mtco2 in Tambaqui. Our results showed that the phenotype presented by Tambacu might be associated with changes in the gene expression profile and not necessarily with an increase in gene variability. Thus, the molecular mechanisms underlying these "hybrid effects" may be related to additive and, in some cases, dominant regulatory interactions between parental alleles that act directly on gene regulation in the hybrid transcripts.

Maternal protein restriction changes structural and metabolic gene expression in the skeletal muscle of aging offspring rats.

Maternal protein restriction affects postnatal skeletal muscle physiology with impacts that last through senility. To investigate the morphological and molecular characteristics of skeletal muscle in aging rats subjected to maternal protein restriction, we used aged male rats (540 days old) born of dams fed a protein restricted diet (6% protein) during pregnancy and lactation. Using morphological, immunohistochemical and molecular analyses, we evaluated the soleus (SOL) and extensor digitorum longus (EDL) muscles, muscle fiber cross-sectional area (CSA) (n=8), muscle fiber frequency (n=5) and the gene expression (n=8) of the oxidative markers (succinate dehydrogenase-Sdha and citrate synthase-CS) and the glycolytic marker (lactate dehydrogenase-Ldha). Global transcriptome analysis (n=3) was also performed to identify differentially regulated genes, followed by gene expression validation (n=8). The oxidative SOL muscle displayed a decrease in muscle fiber CSA (*p<0.05) and in the expression of oxidative metabolism marker Sdha (***p<0.001), upregulation of the anabolic Igf-1 (**p<0.01), structural Chad (**p<0.01), and Fmod (*p<0.05) genes, and downregulation of the Hspb7 (**p<0.01) gene. The glycolytic EDL muscle exhibited decreased IIA (*p<0.05) and increased IIB (*p<0.05) fiber frequency, and no changes in muscle fiber CSA or in the expression of oxidative metabolism genes. In contrast, the gene expression of Chad (**p<0.01) was upregulated and the Myog (**p<0.01) gene was downregulated. Collectively, our morphological, immunohistochemical and molecular analyses showed that maternal protein restriction induced changes in the expression of metabolic, anabolic, myogenic, and structural genes, mainly in the oxidative SOL muscle, in aged offspring rats.

The combination of resveratrol and exercise enhances muscle growth characteristics in pacu (Piaractus mesopotamicus).

Pacu is a tropical fish with important value to aquaculture. During cellular metabolism, reactive oxygen species (ROS) are produced, which can influence muscle growth. Resveratrol is an effective antioxidant that scavenges ROS and can modulate physical performance preventing oxidative stress. We investigated the effects of resveratrol and exercise on pacu muscle growth characteristics. Four groups were used: fish fed with control diet /without exercise (C); fish fed with control diet/subjected to exercise (CE); fish fed resveratrol-supplemented diet/without exercise (R); and fish fed resveratrol-supplemented diet/subjected to exercise (RE). At 30 days, the RE group presented a significant increase in body weight, fewer muscle fibers in the 20-40 µm and more fibers in the >60 µm diameter class compared to the C group. At day 7, catalase activity decreased in CE and RE groups. Superoxide dismutase activity decreased only in the CE group. Myod and mtor gene expression was higher in R and RE and igf-1 was up-regulated in the RE group. Murf1a level decreased in CE, R, and RE, while sdha expression was higher in the RE group. We suggest that resveratrol in combination with exercise was beneficial for muscle growth and metabolism, increasing the expression levels of genes related to muscle anabolism and oxidative metabolism, besides the decrease of catabolic gene expression. Notably, all of these changes occurred together with muscle hypertrophy and increased body weight. Our results show a positive application for resveratrol in association with exercise as a strategy to improve the growth performance of juvenile pacus.

Association of CAST2, HSP90AA1, DNAJA1 and HSPB1 genes with meat tenderness in Nellore cattle.

The objective of this study was to evaluate the association of expression of CAPN1, CAPN2, CAST, HSP90AA1, DNAJA1 and HSPB1 genes with meat tenderness in Nellore cattle. Three experimental groups were selected by shear force (SF): moderately tender (SF=34.3±5.8N), moderately tough (SF=56.8±7.8N), and very tough meat (SF=80.4±15N). Gene expression was evaluated by real-time PCR. Expression of the CAPN1, CAPN2, CAST and CAST1 genes did not differ between groups. Expression of the CAST2 was up-regulated (P<0.05) in the moderately tough and very tough meat groups. Down-regulation of the HSP90AA1, DNAJA1 and HSPB1 genes (P<0.05) was observed in the moderately tender meat group. The present results suggest that meat tenderness in Nellore cattle does not directly depend on the expression of the CAPN1 and CAPN2 genes, but is associated with the expression of other genes such as CAST2, HSP90AA1, DNAJA1 and HSPB1.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.