Francesco Mascarello

DIFFERENTIATION AND GROWTH OF MUSCLE IN THE FISH SPARUS AURATA (L). II: HYPERPLASTIC AND HYPERTROPHIC GROWTH OF LATERAL MUSCLE FROM HATCHING TO ADULT

SummaryPost-hatching growth of lateral muscle in a teleost fish, Sparus aurata (L) was studied morphometrically to identify and quantify muscle fibre hyperplasia and hypertrophy, and by in vivo nuclear labelling with 5-bromo-deoxyuridine to identify areas of myoblast proliferation. Muscle fibre types were identified principally by myosin ATPase histochemistry and immunostaining, and labelled nuclei were identified at light and electronmicroscope level by immunostaining with a specific monoclonal antibody. Hyperplastic growth was slow at hatching, but then increased to a maximum at the mid-point of larval life. Larval hyperplastic growth occured by apposition of new fibres along proliferation zones, principally just under the lateral line and in the apical regions of the myotome, but also just under the superficial monolayer at intermediate positions. The first of these zones gave rise to slow and pink muscle fibres, in a process which continued through into postlarval life. The other zones added new fibres to the fast-white muscle layer in a process which was exhausted by the end of larval life. Post-larvally, between 60 and 90 days posthatching, a new hyperplastic process started in the fast-white muscle as nuclei proliferated and new muscle fibres were formed throughout the whole layer. This process resulted in a several-fold increase in the number of fast-white fibres over a few weeks, and then waned to very low levels in juveniles. Hyperplasia by apposition continued for some time postlarvally on the deep surface of the superficial monolayer, but at this stage gave rise to slow fibres only. Hypertrophic growth occurred at all ages, but was the dominant mechanism of muscle growth only in the juvenile and adult stages. Mechanisms giving rise to these different growth processes in fish muscle are discussed, and compared with muscle development in higher vertebrates.

Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern.

Abstract: We report on the sequence and expression analysis of the myostatin gene (MSTN) in the gilthead seabream Sparus aurata. A 2189-bp transcript was isolated, encoding an open reading frame (385 amino acids) that showed 74% to 60% protein similarity with other vertebrate myostatins. Phylogenetic analysis of MSTN and other related genes confirmed the evolutionary relationships of the isolated sequence. The complete sequences of two introns were also determined. Intron-exon boundaries were conserved when compared with those of mammalian MSTN genes, whereas intron size was smaller. Reverse transcriptase polymerase chain reaction on total RNA extracted from different tissues and developmental stages revealed MSTN expression in the skeletal muscle, but also in other tissues. The observed expression profile differed from that in mammals, suggesting possible additional functions of myostatin in the teleost fish.

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation.

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Fast fibres in a large animal: fibre types, contractile properties and myosin expression in pig skeletal muscles

SUMMARY Little is known about the influence of Myosin Heavy Chain (MHC) isoforms on the contractile properties of single muscle fibres in large animals. We have studied MHC isoform composition and contractile properties of single muscle fibres from the pig. Masseter, diaphragm, longissimus, semitendinosus, rectractor bulbi and rectus lateralis were sampled in female pigs (aged 6 months, mass 160 kg). RT-PCR, histochemistry, immunohistochemistry and gel electrophoresis were combined to identify and separate four MHC isoforms: MHC-slow and three fast MHC (2A, 2X, 2B). Maximum shortening velocity (Vo) and isometric tension (Po) were measured in single muscle fibres with known MHC isoform composition. Six groups of fibres (pure: slow, 2A, 2X and 2B, and hybrid: 2A-2X and 2X-2B) with large differences in Vo and Po were identified. Slow fibres had mean Vo=0.17±0.01 length s-1 and Po=25.1±3.3 mN mm-2. For fast fibres 2A, 2X and 2B, mean Vo values were 1.86±0.18, 2.55±0.19 and 4.06±0.33 length s-1 and mean Po values 74.93±8.36, 66.85±7.58 and 32.96±7.47 mN mm-2, respectively. An in vitro motility assay confirmed that Vo strictly reflected the functional properties of the myosin isoforms. We conclude that pig muscles express high proportions of fast MHC isoforms, including MHC-2B, and that Vo values are higher than expected on the basis of the scaling relationship between contractile parameters and body size.

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

Abstract. In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata (sea bream), Solea solea (sole) and Brachydanio rerio (zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).

Differentiation and growth of muscle in the fish Sparus aurata (L): I. Myosin expression and organization of fibre types in lateral muscle from hatching to adult

SummaryPost-hatching development of lateral muscle in a teleost fish, Sparus aurata (L) was examined. At hatching only two fibre types were present; several layers of mitochondria-poor, myofibril-rich deep muscle fibres surrounded the notochord and were covered by a superficial monolayer of mitochondria-rich, myofibril-poor fibres. A third ultrastructurally distinct fibre type first appeared as one or two fibres located just under the lateral line at 6 days post-hatching. This type, which gradually increased in number during larval life, contained a slow isoform of myosin, identified by mATPase staining and immunostaining with myosin isoform-specific antibodies. Deep muscle fibres — the presumptive fast-white type — contained a fast myosin, and superficial monolayer fibres an isoform similar but not identical to that in adult pink muscle fibres. The only fibres present during larval life which showed a clear change in myosin expression were the superficial monolayer fibres, which gradually transformed into the slow type post-larvally. Pink muscle fibres first appeared near the end of larval life. Both slow and pink muscle fibres remained concentrated around the horizontal septum under the lateral line during larval life, expanding outwards towards the apices of the myotomes only after metamorphosis. Between 60 and 90 days very small diameter fibres with a distinct mATPase profile appeared scattered throughout the deep, fast-white muscle layer, giving it a ‘mosaic’ appearance, which persisted into adult life. A marked expansion in the slow muscle layer began at the same time, partly by transformation of superficial monolayer fibres, but mainly by addition of new fibres both on the deep surface of the superficial monolayer and close to the lateral line. The order of appearance of these fibre types, their myosin composition, and the significance of the superificial monolayer layer are discussed and compared to muscle fibre type development in higher vertebrates.

Regeneration of skeletal muscle in two teleost fish: Sparus aurata and Brachydanio rerio.

Abstract.Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5′-bromo-2′-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.

Effect of swimming on myostatin expression in white and red gastrocnemius muscle and in cardiac muscle of rats.

The aim of this study was to test the hypothesis that swimming training might impact differentially myostatin expression in skeletal muscles, depending on fibre type composition, and in cardiac muscle of rats. Myostatin expression was analysed by real time reverse transcriptase‐polymerase chain reaction, Western blot and immunohistochemistry of the red deep portion (mainly composed of slow and type II A fibres) and in the superficial, white portion (composed of fast type II X and II B fibres) of the gastrocnemius muscle in adult male Wistar rats: (i) subjected to two consecutive swimming bouts for 3 h; (ii) subjected to intensive swimming training for 4 weeks; and (iii) sedentary control rats. Myostatin mRNA content was in all cases higher in white than in red muscles. Two bouts of swimming did not alter myostatin expression, whereas swimming training for 4 weeks resulted in a significant reduction of myostatin mRNA contents, significant both in white and red muscles but more pronounced in white muscles. Western blot did not detect any change in the amount of myostatin protein. Immunohistochemistry showed that, in control rats, myostatin was localized in presumptive satellite cells of a few muscle fibres. After training, the number of myostatin‐positive spots decreased significantly. Myostatin mRNA content in cardiac muscle was lower than in skeletal muscle and was significantly increased by swimming training. In conclusion, the results obtained showed that intense training caused a decreased expression of myostatin mRNA in white and red skeletal muscles but an increase in cardiac muscle.

Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell‐based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB‐MSCs), platelet‐rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood‐derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

MUSCLE GROWTH IN RESPONSE TO CHANGING DEMANDS OF FUNCTIONS IN THE TELEOST SPARUS AURATA (L.) DURING DEVELOPMENT FROM HATCHING TO JUVENILE

Abstract Growth of laterarl muscle in the teleost fish Sparus aurata (L.) was examined from hatching to juvenile by a basic morphofunctional approach that takes into account structural and ecophysiological aspects and combines in vivo observations and LM and TEM microscopic analysis. As shown in most teleost fishes, muscle growth proceeds by a double mechanism of hyperplasia and hypertrophy that contribute differentially to the overall development of the lateral muscle, giving rise in each myomere to a typical pattern of structurally and functionally different fibre types (slow-red and fast-white fibres, plus pink intermediate fibres) in a nerve-dependent process. During larval life the muscle growth takes place mainly due to hyperplastic growth at the level of specific proliferative zones of the myomeres, from which slow, pink and white muscle fibres are derived. In those species that reach a large adult size a new typical hyperplastic process disseminated throughout the fast white muscle layer takes place during post-larval life. In contrast, hypertrophic growth occurs in all stages, but is the dominant mechanism of muscle growth only in juvenile and adult. The suitable recruitment of the different fibre types enables the fish to optimize its performances according to specific functional and metabolic requirements related to the swimming behaviour and hydrodynamic regimes. The different mechanisms of growth are here analysed in their detailed structural and ultrastructural aspects in order to interpret their adaptive significance in the light of the fish life cycle, with particular reference to locomotion and feeding behaviour.