Genetic variability of foetal bovine myoblasts in primary culture

Selected strains of adult bovines and those which either have high muscle growth capacity or are double-muscled present particular characteristics of muscle fibres and collagen at slaughter that favour meat tenderness. For double-muscled bovines, it has been shown that these characteristics originated during foetal life. However, no studies have been done to determine the origin of muscle growth superiority in bovine with high muscle growth capacity compared to those with a low muscle growth capacity. Therefore, the objective of this study was to examine the proliferation and differentiation phases of myoblasts in primary culture taken from high and low muscle growth capacity foetuses at 110 days post-conception. These cultures were analysed on 1, 2, 3, 4, 6, 8, 10 days of culture. The proliferation phase was monitored by appropriate marker antibodies. The differentiation was studied by immunocytochemistry with specific antibodies for foetal, I, II (IIa and IIb), I and IIb, I and IIa myosin heavy chains (MHCs) and connectin respectively, and by immunoblotting with desmin antibody. A higher proliferation, a lower fusion and a delayed differentiation of the late markers namely MHCs fast (IIa and IIb) and connectin were shown in high muscle growth capacity foetuses compared to low capacity ones. The results indicate that the muscle growth superiority of high muscle growth capacity bovines seems, therefore, to have a similar foetal origin to that of double-muscled ones.     

Contractile differentiation of foetal cattle muscles: intermuscular variability

off actile differentiation was studied in six foetal muscles exhibiting different contractile characteristics in adult cattle: the Masseter, Diaphragma, Biceps femoris, Longissimus thoracis, Semitendinosus and Cutaneus trunci. These muscles were excised from foetuses aged 60-260 days. Fibre types were identified by immunohistochemistry using three monoclonal antibodies raised against types 1, 2a, 2b (or 2x) and foetal myosin heavy chains. The different myosin isoforms were also separated by electrophoresis, identified by immunoblotting and quantified by ELISA. At least two generations of cells were observed in all the muscles studied. The primary, early differentiated one, gave rise to type II fibres in Cutaneus trunci and type I fibres in all remaining muscles. The secondary generation of cells differentiated later than the first generation of cells. Its pattern of differentiation was more complex in particular from 150 to 210 days. It formed slow fibres in slow adult muscles, fast fibres in fast adult muscles and both types in mixed muscles. Precocity of differentiation was muscle-type dependent and related to muscle function at birth.     

Growth hormone receptor gene expression in the skeletal muscle of normal and double-muscled bovines during foetal development

The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a beta-actin cDNA probe, revealed lower beta-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.     

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.     

Immunocytochemical characterisation of two generations of fibers during the development of the human quadriceps muscle

We have carried out a comprehensive study of the formation of muscle fibers in the human quadriceps in a large series of well dated human foetuses and children. Our results demonstrate that a first generation of muscle fibers forms between 8-10 weeks. These fibers all express slow twitch myosin heavy chain (MHC) in addition to embryonic and foetal MHCs, vimentin and desmin. Between 10-11 weeks, a subpopulation of these fibers express slow tonic MHC, being the first primordia of muscle spindles. Extrafusal fibers of a second generation form progressively and asynchronously around the primary fibers between 10-18 weeks, giving the muscle a very heterogeneous aspect due to different degrees of organization of their proteins. By 20 weeks, these second generation fibers become homogeneous and thereafter undergo a process of maturation and differentiation when they eliminate vimentin, embryonic and foetal MHCs to express either slow twitch or fast MHC. The differentiation of these second generation fibers into slow and fast depends upon different factors, such as motor innervation or level of thyroid hormone. Around the intrafusal first generation fibers, additional subsequent generations of fibers are also progressively formed. Some differ from the extrafusal second generation fibers by expressing slow tonic MHC, others by continuous expression of foetal MHC. The differentiation of intrafusal fibers is probably under the influence of both sensory and motor innervation.     

Changes in the Metabolic and Contractile Characteristics of Muscle in Male Cattle Between 10 and 16 Months of Age

Samples of semitendinosus muscle from 28 male cattle (18 Salers and 10 Limousins) were taken at 10 months (biopsy) and at 16 months of age (at slaughter). The animals had received the same diet and were slaughtered after the same duration of fattening. The activities of isocitrate dehydrogenase and lactate dehydrogenase were measured in the muscle samples. The five lactate dehydrogenase isoenzymes were separated by electrophoresis under non-denaturing conditions and assayed by densitometry. Fibres were identified by histochemistry by myofibrillar ATPase and succinate dehydrogenase activities as SO (slow oxidative), FOG (fast oxidative glycolytic) or FG (fast glycolytic), and by immunohistochemistry by their reaction to monoclonal antibodies specific to slow and fast myosin heavy chain reactions in I, IIC, IIA, IIAB and IIB type fibres. The isocitrate dehydrogenase activity was not modified between 10 and 16 months of age; the lactate dehydrogenase activity decreased and was correlated with an increase in the proportion of the H isozyme to the detriment of the proportion of the M form. This period was characterized by an increase in fibre size, increased expression of MHC IIa, resulting in more IIA fibres, less IIB fibres, and an increase in the percentage of type IIAB fibres, however the proportions of SO, FOG and FG, when analysed statistically, were not modified between 10 and 16 months of age.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag and bag fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of "specialization" in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation. The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls. We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag and bag fibres.     

Comparison of foetal metabolic differentiation in three cattle muscles

Metabolic differentiation of Semitendinosus (ST), Cutaneus trunci (CT) and Masseter (MA) in cattle foetuses aged from 110 to 260 days was studied by measuring isocitrate dehydrogenase (ICDH, oxidative) and lactate dehydrogenase (LDH, glycolytic) activities. The five LDH isoenzymes were separated by electrophoresis and assayed by densitometry. ICDH activity increased from 210 days onwards in the three muscles but more intensively in MA (oxidative). LDH activity increased from 170 days onwards in ST, 180 days onwards in CT and only from 210 days onwards in MA and was higher in the glycolytic muscles (ST and CT). The proportion of the LDH-M subunit increased during foetal life in glycolytic muscles. At 110 days, it was higher in CT, intermediate in ST and lower in MA. These results show that 1) metabolic differentiation of bovine muscle begins during the last third of foetal life and 2) the proportion of the LDH-M subunit seems to be related to the contractile type of adult muscle from the first stages of foetal life.     

Endocrine and metabolic regulation of muscle growth and body composition in cattle

Muscle metabolism (in interaction with other organs and tissues, including adipose tissue) plays an important role in the control of growth and body composition. Muscle ontogenesis has been described in different genotypes of cattle for myofibres, connective tissue and intramuscular depots. The ontogenesis or the action of putatively important factors controlling muscle development (IGF-II expression, IGF receptors, growth hormone (GH) receptor, myostatin, basic fibroblast growth factor, transforming growth factor-β1, insulin and thyroid hormones) has also been studied on bovine foetal muscle samples and satellite cells. The glucose/insulin axis has been specifically studied in both the bovine adipose tissue and heart. Clearly, cattle, like sheep, are mature species at birth based on their muscle characteristics compared to other mammalian or farm animal species. The different myoblast generations have been well characterised in cattle, including the second generation which is liable to be affected by foetal undernutrition at least in sheep. Interesting genotypes, for example, double-muscled genotype, have been characterised by an altered metabolic and endocrine status associated with a reduced fat mass, specific muscle traits and different foetal characteristics. Finally, the recent development of genomics in cattle has allowed the identification of novel genes controlling muscle development during foetal and postnatal life. Generally, a high muscle growth potential is associated with a reduced fat mass and a switch of muscle fibres towards the glycolytic type. The possibility and the practical consequences of manipulating muscle growth and, hence, body composition by nutritional and hormonal factors are discussed for bovines based on our current biological knowledge.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

Summary In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag₁ and bag₂ fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of specialization in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation.The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls.We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag₁ and bag₂ fibres.     

Histological characteristics of longissimus dorsi muscle and their correlation with restriction fragment polymorphisms of calpastatin gene in F2 Jinghua × Piétrain crossbred pigs

In order to evaluate the genotype of the calpastatin (CAST) gene and its relationship to muscle histology and other post mortem traits in the Jinhua × Piétrain F2 pig family, 158 barrows and gilts were electrically stunned and exsanguinated. Both blood and muscle samples were collected, and both post mortem traits and meat qualities were recorded. Restriction fragment length polymorphism (RFLP) analysis, the periodic acid Schiff reaction (PAS) and myosin heavy-chain immunohistochemistry were employed to explore the relationship between genotype and muscle histology. Based on PAS reactivity, muscle fibres can be classified into three types: PAS (-), PAS (+) and PAS (++). Myosin heavy-chain immunohistochemistry can differentiate muscle fibres into either slow or fast fibres; the proportion of slow and fast fibres were 6% and 94%, respectively. When the amplification products of the CAST gene were digested with MspI, HinfI and RsaI, two different cleavage patterns could be discriminated from the endonuclease map detected using each enzyme. The results showed that the polymorphisms detected using these three endonucleases are identical. Only three genotypes (AA/CC/EE, AB/CD/EF and BB/DD/FF) were distinguished. Their frequencies were 0.1835, 0.5823 and 0.2342, respectively. Different genotypes had significant association with area and pH45m value of loin muscle, while showing no significant association with the water-holding capacity and conductivity of loin muscle. The results also revealed that the genotypes had a significant correlation with diameter, area, circularity and the aspect ratio of muscle fibres. It was also presented that the genotypes significantly correlated with the percentage of intramuscular connective tissue.     

Age-related changes and location of types I, III, XII and XIV collagen during development of skeletal muscles from genetically different animals

The ontogenesis of total collagen and of different collagen types was studied in four muscle types from genetically different cattle. Hydroxyproline content was 1.2-fold higher in muscles from cross-bred foetuses with normal muscle growth compared to those of the other genetic types (pure bred with different growth rates, double-muscled breed). A similar tendency was observed for type III collagen content. In all muscles of each animal studied, type XII and XIV collagens were colocated in perimysium. Immunolabelling obtained for type XII collagen was higher during foetal life than after birth, while for type XIV collagen, the opposite result was obtained. Whatever the muscle studied, but especially in semitendinosus muscle, during the foetal and the post-natal period until 15 months of age, immunolabelling with antibody anti-type XIV collagen tended to be more intense in muscles of animals from fathers selected for a low muscle growth capacity compared to those from fathers selected for a high muscle growth capacity. In conclusion, this study shows, that during foetal life, selection according to muscle growth capacity has no significant effect on the contents of total hydroxyproline or type III collagen, but minor effects on collagen localization.     

Rat embryonic myoblasts are restricted to forming primary fibres while later myogenic populations are pluripotent.

Three populations of myoblasts, embryonic, foetal and adult, appear sequentially during myogenesis. The present study uses retroviruses to mark myoblasts clones in vivo from these populations. Myoblasts labelled at E15 (embryonic) contributed to primary fibres only. The majority of marked primary fibres were slow but a small number of clones contained marked primaries which were no longer slow at E19. Myoblasts labelled at E17 (foetal) fused with both primary and secondary fibres and most clones contained both fast and slow fibres. Similarly, adult myoblasts marked at P0 fused with all fibre types. These results indicate that embryonic myoblasts are restricted to producing only primary fibres which are initially slow but which can convert to being fast. Clones of foetal and adult myoblasts fuse with both primary and secondary fibres which may be either fast or slow.     

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.     

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts.

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0-0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Abstract. Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

Regenerated rat fast muscle transplanted to the slow muscle bed and innervated by the slow nerve,exhibits an identical myosin heavy chain repertoire to that of the slow muscle

 The hypothesis that the limited adaptive range observed in fast rat muscles in regard to expression of the slow myosin is due to intrinsic properties of their myogenic stem cells was tested by examining myosin heavy chain (MHC) expression in regenerated rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The muscles were injured by bupivacaine, transplanted to the SOL muscle bed and innervated by the SOL nerve. Three months later, muscle fibre types were determined. MHC expression in muscle fibres was demonstrated immunohistochemically and analysed by SDS-glycerol gel electrophoresis. Regenerated EDL transplants became very similar to the control SOL muscles and indistinguishable from the SOL transplants. Slow type 1 fibres predominated and the slow MHC-1 isoform was present in more than 90% of all muscle fibres. It contributed more than 80% of total MHC content in the EDL transplants. About 7% of fibres exhibited MHC-2a and about 7% of fibres coexpressed MHC-1 and MHC-2a. MHC-2x/d contributed about 5–10% of the whole MHCs in regenerated EDL and SOL transplants. The restricted adaptive range of adult rat EDL muscle in regard to the synthesis of MHC-1 is not rooted in muscle progenitor cells; it is probably due to an irreversible maturation-related change switching off the gene for the slow MHC isoform. Accepted: 11 June 1996