Muscle differentiation in blackspot seabream (Pagellus bogaraveo, Brunnich): histochemical and immunohistochemical study of the fibre types.

The aim of this work was to gain insights into the mechanism of muscle differentiation and growth in Pagellus bogaraveo, by studying muscle fibre phenotypes identified by immunohistochemistry. At hatching, several layers of deep fast-white fibres were covered by a superficial fibre monolayer. At 5 days, slow-red fibres appeared near the lateral line nerve. At 40 days, the intermediate-pink muscle became visible, and in the slow-red and fast-white muscle layers transitions from larval myosin isoforms to the isoforms typical of adult muscle occurred. Between 70 and 100 days, small fibres with a distinct ATPase profile appeared throughout the fast-white muscle, marking the onset of "mosaic" hyperplasia. The myosin of the original superficial monolayer fibres underwent two myosin transformations, before being slowly replaced by an adult slow-red isoform. In juveniles and adults, the slow-red muscle layer could be resolved into two distinct types. The analysis of fibre phenotypes indicated that post-larval muscle growth occurred by two distinct stages of hyperplasia. This study offers a basis for further comparative and experimental studies with this economically relevant species, namely for identifying factors influencing its muscle growth dynamics and disclosing underlying mechanisms.

Long-term culture of muscle explants from Sparus aurata.

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.

Organization of motor units in the axolotl: a continuously growing animal.

The characteristics of motor units in the iliotibialis posterior muscle of the axolotl hindlimb are described. Tension recording and intracellular electrophysiological methods demonstrate that the physiological properties of the population of motor units are continuously distributed rather than grouped into a series of discrete types. Overlap between motor units occurs and this is positively correlated with motor unit size but negatively correlated with differences in time to peak tension. Immunocytochemical staining with antimyosin antibodies combined with histochemical demonstration of actomyosin ATPase activity revealed at least four types of muscle fibre which were distributed asymmetrically within iliotibialis posterior. The results are discussed in terms of the continuous growth of the muscle and the interactions between muscle and nerve in the formation of the axolotl motor system.

Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle.

Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.

Fibre type classification and myosin isoforms in the human masseter muscle.

Human masseter muscle is highly unusual since it contains relatively large numbers of fibres with variable myofibrillar ATPase staining as well as fibres that express neonatal and alpha-cardiac myosin heavy chain (MHC). These findings however, have not been organised together into a fibre type classification scheme. Biopsies from the anterior superficial area of masseter were collected from a large sample of healthy young adults. Biopsies were sectioned and stained for myofibrillar ATPase reactivity and the presence of MHC isoforms as detected by a series of antibodies. The MHC composition of the same biopsies was also analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). A series of rectus abdominis muscle biopsies were analysed similarly to serve as a control for type I, IIA and IIB fibres and isoforms. From the histochemical, immunohistochemical and biochemical experiments we found the masseter to contain type I, IM, IIC, IIA and IIB fibres as previously classified, but in addition there were type neonatal, alpha-cardiac, and 'other' (three or more myosins including neonatal and alpha-cardiac). The percentage of each fibre type was highly variable in masseter biopsies, but generally type I fibres were most common, and the proportion of IIB, neonatal, alpha-cardiac and 'other' fibres was low. Even in biopsies that contained relatively large amounts of these last three fibre types, the amount of neonatal and/or alpha-cardiac MHC detected on SDS-PAGE was limited, suggesting that these MHCs are a minor component in the fibres in which they are expressed.

Myosin isoform transitions during development of extra-ocular and masticatory muscles in the fetal rat.

The late fetal development of rat extra-ocular and masticatory muscles was examined by myosin immunohistochemistry. The pattern of slow and neonatal myosin isoform expression in primary and secondary myotubes in these muscles was generally similar to that seen by others in limb muscles. We observed a consistent difference between the Sprague-Dawley and Wistar rats in the degree of maturity reached by all muscles studied at a particular age. In both strains, extra-ocular muscles were also about one day in advance of the masticatory muscles. Thus, secondary myotubes were first seen at E17 in Wistar extraocular muscles, at E18 in Sprague-Dawley extra-ocular muscles and Wistar masticatory muscles, and at E19 in Sprague-Dawley masticatory muscles. There was a strikingly early and complete type differentiation of primary myotubes in extraocular muscles, and tonic myosin first appeared before birth in presumptive extrafusal tonic fibres in the orbital layer of the oculorotatory muscles. Throughout the late fetal period, retractor bulbi was composed of fast myotubes only, but these myotubes were not arranged in classical clusters. In the masticatory muscles at E17/E18 some slow primary myotubes started to express tonic myosin, and these presumptive spindle bag2 fibres were located only in regions of the muscles known to contain spindles in the adult. Presumptive bag1 fibres appeared about a day later (initially without tonic myosin), and in the region of the spindle cluster in anterior deep masseter extrafusal secondary myotube production appeared to be suppressed.

Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study.

The myosin composition of lateral muscle in Poecilia reticulata from birth to adult was studied by ATPase histochemistry and immunostaining with myosin isoform-specific antibodies. At birth the muscle consists of two layers containing developmental isoforms of myosin. In deep layer fibres the developmental myosin is replaced by the adult fast-white isoform soon after birth. In the epaxial and hypaxial monolayer fibres the myosin composition present at birth (J1) is replaced within 3 days by another (J2). In some fibres, this J2 composition is retained in the adult, but in others it is slowly replaced by the adult slow-red muscle isoform. Close to the lateral line, all monolayer fibres are already in transition between the J2 myosin and the adult slow-red form at birth, and rapidly complete the transition to slow-red form. These fibres, together with others generated de novo in an underlying hyperplastic zone, form the red muscle layer of the adult. The pink muscle develops during the first month after birth, and by 31 days it consists of an outer, middle and inner layer. A few middle layer fibres are already present at birth, while the outer layer fibres first appear 3 days after birth. The thin inner layer is probably a transitional form between the middle pink and adult white types, and appears at about 31 days. A morphometric analysis showed that growth of the white muscle occurs principally by hypertrophy. Even at the magnification level of the electron microscope, no satellite cells or myoblasts which could give rise to new fibres were found in the white muscle, except in the far epaxial and hypaxial regions and only in the first 10 days. A zone of hyperplastic growth was also found lying just under the superficial monolayer close to the lateral line, and this presumably contributes fibres to the red and pink muscle layers.

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.). An ultrastructural and morphometric study.

In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers. In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance. Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life. The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a "flattened" profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.

Myosin expression in the jaw-closing muscles of the domestic cat and American opossum.

Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), glycerol SDS-PAGE, two-dimensional electrophoresis, and protein immunoblotting techniques were used to identify myosin heavy chain (MHC) and light chain (MLC) isoforms in limb and masticatory muscles of the cat and American opossum. The fibre types in which these isoforms are expressed were identified by histochemistry and immunohistochemistry. Antibodies specific for the type IIM MHC isoform characteristic of cat jaw-closing muscles and the type I MHC isoform were produced and characterized. The IIM antibody stained the majority of fibres found in the jaw-closing muscles of both species. These IIM-containing fibres characteristically had a histochemical ATPase that remained active after both acid and alkali pre-incubations. A minority of type I fibres was also present in cat jaw-closing muscles, and these reacted positively with antibody specific for type I MHC. It was confirmed that the vast majority of fibres in the cat jaw-closing muscles contained only the characteristic masticatory MHC (IIM) and masticatory MLCs (LC1m and LC2m). These muscles did not contain either the type II fibre isoforms of limb muscles or the atrial cardiac (alpha-cardiac) MHC. The type IIM MHC could also be identified in jaw-closing muscles of the opossum. Two-dimensional gel electrophoresis was used to identify the MLC composition of single, histochemically defined, type I fibres in the cat soleus and deep masseter. The type I fibres of limb muscle contained the usual slow MLCs, but type I fibres from the jaw-closing muscles contained only the masticatory light chains.

Preparation of type-specific antimyosin antibodies and determination of their specificity by biochemical and immunohistochemical methods.

This paper reports the preparation of specific anti-slow myosin antibodies (anti-I) and anti-fast myosin antibodies (anti-IIA) raised against myosins from sheep and guinea pig masseter muscles. The specificity of the antibodies has been studied by immunodiffusion in agar and by the GEDELISA test using slow-twitch (type I), fast-twitch red (type IIA) and fast-twitch white (type IIB) myofibrils isolated from guinea pig muscles. The principal specificity of the anti-I and anti-IIA antibodies was for the heavy chains of type I and IIA myosins, respectively. A smaller reaction with the corresponding light chains was also detected. Immunohistochemical staining of muscle sections using these antibodies confirmed their fibre type specificity.

Tertiary myotubes in postnatal growing pig muscle detected by their myosin isoform composition.

The postnatal development of skeletal muscles was studied in growing pigs from 8 to 210 d of age. Indirect immunoperoxidase staining of frozen sections of porcine semimembranosus muscle and longissimus muscle revealed a distinct population of small fibers (tertiary myotubes) that were stained specifically by an antibody (anti-NE) selective for the developmental (embryonic and neonatal) isoforms of muscle myosin. At 8 d of age the other larger fibers were already anti-NE negative and differentiated into Types I and II. A gradual decrease in the number of anti-NE positive fibers together with a gradual increase in area of the remaining positive fibers was observed throughout the pigs' growth. These results may indicate that hyperplastic growth does not cease at birth. Possible mechanisms to explain the origin of these tertiary myotubes containing developmental isoforms of myosin are suggested.

Masseter function and skeletal malocclusion.

The aim of this work is to review the relationship between the function of the masseter muscle and the occurrence of malocclusions. An analysis was made of the masseter muscle samples from subjects who underwent mandibular osteotomies. The size and proportion of type-II fibers (fast) decreases as facial height increases. Patients with mandibular asymmetry have more type-II fibers on the side of their deviation. The insulin-like growth factor and myostatin are expressed differently depending on the sex and fiber diameter. These differences in the distribution of fiber types and gene expression of this growth factor may be involved in long-term postoperative stability and require additional investigations. Muscle strength and bone length are two genetically determined factors in facial growth. Myosin 1H (MYOH1) is associated with prognathia in Caucasians. As future objectives, we propose to characterize genetic variations using "Genome Wide Association Studies" data and their relationships with malocclusions.

Developmental programming of skeletal muscle phenotype/metabolism.

Skeletal muscle is a highly dynamic and malleable tissue that is able to adapt to different stimuli placed upon it, both during gestation and after birth, ultimately resulting in anatomical changes to muscle fibre composition. Variation in nutrient supply throughout gestation is common, whether in livestock or in the human. The specific effects of maternal nutrition on foetal development are at the forefront of scientific research. However, results describing how different maternal feeding strategies affect skeletal muscle fibre development in the offspring are not fully consistent, even where the same time windows during gestation have been examined. The aim of this study is to determine the effects of increased maternal nutrition (above the recommended levels) on the Musculus semitendinosus phenotype of progeny. In all, 24 pregnant sows were assigned to one of four feeding regimes during gestation; T1 (control group): 30 MJ digestible energy per day (MJ DE/day) throughout gestation, T2: same as that for T1 but increased to 60 MJ DE/day from 25 to 50 days of gestation (dg), T3: same as that for T1 but increased to 60 MJ DE/day from 50 to 80 dg, T4: same as that for T1 but increased nutrition to 60 MJ DE/day from 25 to 80 dg. Light- and heavy-weight littermate pairs of the same sex were selected at birth and individually fed to slaughter (c. 158 days). Histochemical and immunohistochemical staining were used to identify the predominantly oxidative (deep) and less oxidative (superficial) regions of the M. semitendinosus, and to determine total fibre number and proportions of fibre types. The results demonstrate that increased maternal nutrition alters skeletal muscle phenotype in the offspring by changing fibre-type proportions, leading to an increased oxidative capacity due to an increase in Type IIA fibres. No change in total muscle area, total muscle fibre number, or fibre cross-sectional area is observed. The precise molecular mechanism(s) by which these findings occur is being investigated.

Natural involution of muscle in the proximal sesamoidean ligament in sheep.

In sheep, the muscle component of the proximal sesamoidean ligament, which is well developed at birth, undergoes a progressive involution postnatally. The development of muscle fibres in the proximal sesamoidean ligament was compared with masseter and semimembranosus muscles from before birth into adult life, using histochemical, immunohistochemical and biochemical methods. Neonatal myosin (a marker for developmental immaturity) disappeared earlier, and the adult pattern of myosin expression and fibre type composition was reached earlier in the proximal sesamoid ligament than masseter and semimembranosus. Proximal sesamoid ligament muscle fibres therefore complete normal development, but with a faster time course than the other muscles. Invasion of fibrous connective tissue between muscle fibres of the proximal sesamoidean ligament adjoining the tendinous component (one feature of the involution) was found to begin perinatally, eventually resulting in a marked fibrosis and atrophy of peripheral fibres. Regeneration of muscle fibres was absent or abortive, even near areas of fibre necrosis.

Motor units of juvenile rat lumbrical muscles and fibre type compositions of the glycogen-depleted component.

1. Fourth deep lumbrical muscles were dissected out, with their nerve supply, from juvenile rats aged 8-15 days (a period corresponding to maximal rate of decline of polyneuronal innervation), and aged 28, 29 and 30 days (when developmental synapse elimination is complete). Preparations were superfused with rat Ringer solution at 25 degrees C. 2. Isometric twitches and tetani were recorded from the whole muscles and from a single motor unit in the muscle. Unit isolation was by partial section of the sural or lateral plantar nerves. The axon of a single unit occurred naturally in the sural nerve in some cases. 3. Fibres in single units were depleted of glycogen by repetitive stimulation, and studied histologically in frozen midbelly sections of the muscle, stained for glycogen with periodic acid-Schiff's reagent (PAS). Most fibre counts were based on transmittance measurements made with an image analysis system. Contralateral muscles were unstimulated and acted as controls. 4. Motor unit sizes were estimated from tetanic tensions and from muscle fibre cross-sectional area measurements. Comparison of the two methods indicated that in most units glycogen depletion was not complete. This effect was maximal at 8 and 10 days postnatally. It is suggested that this is due to weak neuromuscular transmission at synapses in the process of natural elimination during development. 5. Other sections (serial and semi-serial) were immunostained with a polyclonal antibody raised against slow myosin. Fibres staining for the antibody (slow; S-fibres) contribute about 12-9% of muscle fibres depending on age. In some muscles, fibre types were determined by myosin ATPase staining following alkali pre-incubation. Fast fibres (F-fibres) contained no slow myosin. 6. Some units had no S-fibres (i.e. they were homogeneous), and many units had a small proportion of S-fibres, though less than in the whole muscle (i.e. they were heterogeneous but composition was biased in favour of F-fibres). 7. One unit from a 10-day-old rat contained more S- than F-fibres. Many of the F-fibres were small. It is proposed that this was a developing IIC/IIA unit, a type known to occur in adults. 8. It is concluded that mismatched connections in developing motor units possibly become weak early (by 8 days postnatally) in the process of synapse elimination (which is complete by 20 days postnatally), but that the time course of actual withdrawal cannot be followed by the technique of glycogen depletion.

Muscle spindles in the jaw-closer muscles of the domestic cat.

The objectives of this study were to identify the exact location of spindles in jaw-closer muscles of the cat, to count the total number of spindles and to compare their distribution with the distribution of slow extrafusal fibres. The jaw-closer muscle group with all the skeletal attachments intact was fixed in a modified Carnoy solution, decalcified and processed through to wax. Complete series of sections were cut transverse, sagittal and perpendicular to the anterior temporalis muscle. At regular intervals, serial sections were stained by the Weigert-van Gieson method or immunostained for myosin isoforms. Spindle counts were made only from muscle areas where fibres were cut in transverse section, and the spindles were followed individually. The extrafusal fibres were identified by indirect immunoperoxidase staining with antibodies specific for the slow (type I) and fast (type IIM) isoforms of myosin found in jaw-closer muscles. The mean numbers and locations of spindles found were 13 in medial pterygoid (close to inferior border), 123 (one count only) in the deep anterior portion of the temporalis muscle (between the coronoid process of the mandible and the cranium), 50.5 in a small deep zone of superficial masseter anterior to the temporomandibular joint, and 50 in zygomaticomandibularis (the deepest portion of masseter). Most of the spindles were simple spindles. Spindle complexes (4 or more spindle units fused in parallel) were rare and were found only in zygomaticomandibularis and in masseter. Most parts of the jaw-closer muscles had no spindles and contained only fast fibres. Cosegregation of muscle spindles with slow fibres was found in most parts of this muscle group, but the distribution of spindles was more restricted than that of slow fibres.

Pink lateral muscle in the carp (Cyprinus carpio L.): histochemical properties and myosin composition.

The pink muscle of the carp differs from the white (and red) muscle not only histochemically but also in its myosin isoform, as shown by peptide maps of the myosin heavy chains. Results of an electrophoretic analysis of myosins are discussed in the light of their immunohistochemical properties and histochemical ATPase activity.

Muscle fibre types in the external eye muscles of the pigeon, Columba livia.

Fibre typing with antisera raised against specific myosin types from muscles of known physiological properties were used to characterise the fibre types within the oculorotatory muscles of pigeons. Fibres reacting strongly to antiserum anti-ALD (specific for tonic fibre myosin) were found lying along the global margin of the muscle and also in a layer lying immediately beneath a discrete band of fibres running along the orbital margin. These fibres resembled those of the skeletal muscle ALD in their type properties. Using another antiserum, anti-I, specific for slow twitch and to a lesser extent, slow tonic myosins, it was possible to identify another slow fibre type which formed the orbital layer and also lay scattered randomly through the body of the muscle. No equivalent to this type was found in the skeletal muscles ALD or iliofibularis. The remaining fibres which did not react with either anti-ALD or anti-I formed 58% of the fibre population and reacted with an antiserum specific for fast myosin. However, their response to alkali preincubation suggests that the fast fibres of eye muscles also contain a myosin which is different from those in skeletal muscle.

Skeletal fiber types and spindle distribution in limb and jaw muscles of the adult and neonatal opossum, Monodelphis domestica.

The South American opossum, Monodelphis domestica, is very immature at birth, and we wished to assess its potential for studies of jaw muscle development. Given the lack of prior information about any Monodelphis fiber types or spindles, our study aimed to identify for the first time fiber types in both adult and neonatal muscles and the location of spindles in the jaw muscles. Fiber types were identified in frozen sections of adult and 6-day-old jaw and limb muscles by using myosin ATPase and metabolic enzyme histochemistry and by immunostaining for myosin isoforms. The distribution of fiber types and muscle spindles throughout the jaw-closer muscles was identified by immunostaining of sections of methacarnoy-fixed, wax-embedded heads. Most muscles contained one slow (type I) and two fast fiber types (equivalent to types IIA and IIX), which were similar to those in eutherian muscle, and an additional (non-IIB) fast type. In jaw-closer muscles, the main extrafusal fiber type was IIM (characteristic of these muscles in some eutherians), and almost all spindles were concentrated in four restricted areas: one in masseter and three in temporalis. Six-day neonatal muscles were very immature, but future spindle-rich areas were revealed by immunostaining and corresponded in position to the adult areas. Extrafusal and spindle fiber types in Monodelphis share many similarities with eutherian mammalian muscle. This finding, along with the immaturity of myosin isoform expression observed 6 days postnatally, indicates that Monodelphis could provide a valuable model for studying early developmental events in the jaw-closer muscles and their spindles.