Characterisation and location of insulin-like-growth factor (IGF) receptors in the foetal bovine Semitendinosus muscle.

We characterised IGFI and IGFII receptors and located them in bovine muscle during foetal growth. Semitendinosus muscle samples were taken from foetuses ranging from 80 to 270 days post-conception. The relative affinities of 125I-IGFII and 125I-IGFI mark the presence of typical type II and type I receptors in foetal muscle membranes. IGFII-specific binding is consistently five times greater than that of IGFI. The patterns of 125I-IGFII- and 125I-IGFI-specific binding are similar. They increase up to 110 and 170 days post-conception, respectively (P < 0.05); thereafter, they decrease (P < 0.05). This decrease was due to a fall in the number of receptors without any change in affinity. At the adult stage the specific binding of both the 125I-IGF is very low. In foetal muscle, type II receptors are located both in the muscle bundles and in the connective tissue while type I receptors are only located in the muscle bundles.

Developmental control of cathepsin B expression in bovine fetal muscles.

Expression of lysosomal cysteine proteinases was studied during fetal calf muscle development. The peptide cleaving activities of cathepsins B and L decreased strongly from 80 to 250 days of fetal age. This decrease in cathepsin activities occurred similarly in three muscles exhibiting different metabolic and contractile properties in adult animals. Cathepsin B from adult or fetal muscle revealed similar enzymatic properties, but presented a five- to sixfold lower concentration in adult muscle as indicated by active-site titration with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guani din o)butane. During fetal growth, decreases in muscle cathepsin B specific activity and active enzyme concentration were associated with a parallel drop of cathepsin B mRNA levels. Bovine cathepsin B is encoded by two different transcripts resulting from alternative polyadenylation [Mordier, S. B., Béchet, D. M., Roux, M. P., Obled, A., and Ferrara, M. (1995) Eur. J. Biochem. 229, 35-44]. As revealed by ribonuclease protection assays, the two mRNAs encoding cathepsin B declined similarly during fetal muscle growth. This study indicates that lysosomal proteinases in skeletal muscle are under developmental control. The decrease of muscle cathepsins during fetal development appears sufficient to account for the low levels of these enzymes in adult muscles. In fetuses, high activities of lysosomal cysteine proteinases might be important for remodeling muscles during early development.

Comparison of the foetal development of muscle in normal and double-muscled cattle.

Muscle differentiation was studied in foetal Semitendinosus muscle from normal cattle and those with the 'culard' gene of muscular hypertrophy sampled at 90, 130, 170 and 210 days of foetal life. The different fibre types were characterized by immunohistochemistry with antibodies specific to different isoforms of myosin heavy chains. The isoforms were separated by electrophoresis, identified by immunoblotting and quantified by ELISA. In double-muscled animals, there was a slower rate of differentiation in the first generation of cells, most markedly apparent at 90 days. At 130 days, differentiation was retarded mainly in the second generation, while at 170 days there was no longer any difference between the two animal types in the differentiation of first generation cells, which were totally slow in both. At the same stage however, type IIC fibres in double-muscled animals were much slower in appearing and continued to be so at 210 days, albeit to a lesser extent. These findings show that differentiation of the muscle fibres occurs at a slower rate in double-muscled foetuses particularly during the first two-thirds of foetal life.

Study of the influence of age and weaning on the contractile and metabolic characteristics of bovine muscle.

Weaning is an interesting period for the study of the nutritional regulation of muscle energy metabolism, since during this stage the nature of the substrates supplied to the muscle and their energy balance are profoundly changed. The aim of this study was to determine the effect of these modifications on the contractile and metabolic characteristics of bovine muscle. Two similar groups of 7 male Montbéliard calves were used with the same age and weight, and with the same energy intake. One group consisted of milk-fed calves, the other of weaned animals. The latter were progressively weaned over a period between 107 and 128 d. The average age at slaughter in the 2 groups was 170 d. Biopsy specimens of semitendinosus (ST) muscle were taken at the ages of 66 d, 94 d (before the beginning of weaning) and 136 d (at the end of weaning) to follow the evolution of muscle characteristics. Samples of longissimus thoracis (LT) muscle were taken 24 h after slaughter and used to study the changes in protein and DNA content. The proportion and area of the different types of fiber, I (slow, oxidative), IIA (fast, oxido-glycolytic), IIB (fast, glycolytic) and IIC (fast/slow, oxidoglycolytic) were measured by immunohistochemistry and image analysis. The metabolism of the muscles was determined by studying isocitrate dehydrogenase (ICDH, oxidative) and lactate dehydrogenase (LDH, glycolytic) activity. The results obtained between 2 and 6 months of life showed an overall increase in the area of the fibers (I, IIA, IIB and IIC) and a conversion of type IIA fibers into type IIB accompanied by a shift in the energy metabolism towards a glycolytic type. Weaning caused temporary stress, whose main consequences were to decrease overall muscle fiber area and the percentage of type IIB fibers, and increase the proportion of type IIC fibers in weaned animals. These effects may have been due to the nutritional and behavioral disturbances that accompany weaning, because 42 d after the end of weaning there was no difference in the size of ST and LT fibers between the 2 groups whereas the proportion of type IIA fibers was still higher in weaned animals.

Enzyme-linked immunosorbent assay for myosin heavy chains in the horse.

The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a human atrium specimen that reacted specifically against MHC 1. Mab 2 was prepared from myosin of rabbit psoas muscle and reacted against MHC 2. The masseter muscle contained solely MHC 1 (100%) and this was confirmed by electrophoresis and immunohistochemistry. By contrast, the cutaneus trunci was very poor in MHC 1 (1.3%) and was entirely composed of MHC 2 (98.7%) which was confirmed by the other techniques. The diaphragm, tensor fasciae latae and semitendinosus contained 89, 40 and 2% of MHC 1, respectively. It was concluded that this ELISA method made it possible to measure a wide range of MHC contents in equine muscles with a good reproducibility. The results were consistent with those of the other fibre typing techniques. Moreover, this immunoenzymatic method is less time consuming than histological techniques and therefore offers new perspectives for muscle fibre typing in the horse.

Inter-animal variation in the biological characteristics of muscle tissue in male limousin cattle.

The biochemical, metabolic and contractile characteristics of semitendinosus (ST) and longissimus thoracicus (LT) muscles were studied in 147 male Limousin cattle. The animals, which were slaughtered at 16 months, were the offspring of 15 different sires. Weight gain and carcass tissue composition (proportion of muscle and adipose tissue) were also measured. The biochemical characteristics of the two muscles studied had coefficients of variation between 13 and 30% markedly higher than for proximate characteristics of the carcass, in which coefficients were all lower than 18%. There were significant differences between the two muscles: ST had a higher protein/DNA ratio and greater lactate dehydrogenase (LDH) activity than LT, also a lower isocitrate dehydrogenase (ICDH) activity and lower slow myosin heavy chain (MHCI) content. The percentage of MHC 1 was positively correlated with ICDH activity and negatively with LDH activity and protein/DNA ratio. These muscle characteristics were also correlated with weight gain: animals with greater weight gain had a higher protein/DNA ratio and lower oxidative activity, with differences in the level of significance of the correlations according to muscles. Selection of animals on the basis of muscle growth may therefore be a means of improving the quality of meat.

Post-natal changes in the biological characteristics of Semitendinosus muscle in male Limousin cattle.

Samples of semitendinosus muscle from 45 male Limousin cattle reared under similar conditions were taken at 1, 6 and 12 months of age (biopsy) and at 16 months (at slaughter). The development of biochemical (DNA, protein/DNA), enzymic (lactic dehydrogenase, isocitric dehydrogenase), and histochemical (ATPase, succunic dehydrogenase) characteristics was studied. Muscle fibres were classified according to their contractile and metabolic properties (SO = slow oxidative; FOG = fast oxidative glycolytic; FG = fast glycolytic). DNA accumulation or hyperplasia was observed up to the age of 16 months. The period of growth and development, between 1 and 12 months of age, was characterized by increased glycolytic metabolism, as evidenced by the conversion of FOG fibres into FG fibres, a decrease in ICDH activity and a rise in LDH activity. The period between 12 and 16 months, was characterized by a slowing down of the conversion of FOG fibres into FG fibres and an increase in ICDH activity. The presence of type IIC fibres at the four ages studied is discussed. Of the muscle characteristics studied, LDH activity seemed to be a discriminating factor between animals from an early age. It could thus be used as to predict animal muscle characteristics at birth.

Presence of an unidentified myosin isoform in certain bovine foetal muscles.

Genetic variation in the establishment of bovine muscle fibre types was studied by comparing muscle differentiation at 210 days of foetal life in normal cattle and in 'culard' animals, which have muscular hypertrophy. The different fibre types were determined by histochemical and immunohistochemical analyses with monoclonal antibodies specific to different myosin heavy chain isoforms. The isoforms were separated by electrophoresis and quantified by the ELISA method. Four muscles with different contractile and metabolic characteristics were studied: Semitendinosus, Longissimus thoracis, Masseter (slow) and Cutaneus trunci (fast). Muscle fibres recognized by none of the antibodies used were observed in 'culard' foetuses in all the muscles studied and also in the Cutaneus trunci of normal animals. Electrophoretic analysis showed no particular myosin isoform in these muscles. It is possible therefore that the fibres contained a mysosin isoform until now unidentified in cattle, with a molecular weight the same as that of known isoforms. This newly observed isoform seems to be specific to muscles rich in IIB fibres such as Cutaneus trunci and to the muscles of adult 'culard' cattle.

Comparison of the foetal development of fibre types in four bovine muscles.

The pattern of expression of different types of myosin heavy chains and the development of different generations of muscle cells during foetal life were studied in four bovine muscles with widely varying characteristics, the Masseter, Longissimus thoracis, Cutaneus trunci and Diaphragma. Different complementary techniques were performed: immunocytochemistry, electrophoresis, immunoblotting and ELISA. Monoclonal antibodies against different myosin heavy chain isoforms were used. The results confirmed the existence of at least two generations of cells during foetal development in cattle. A first generation, which appeared at a very early stage, gave rise to adult type I fibres. A second generation, made up of different cell populations, gave rise to adult fast type IIA and IIB fibres, and to type IIC. In the slow muscles, it also seemed to give rise to type I fibres. The beginning of myogenesis was characterized in the different cell generations by the expression of transitory myosin forms that are not found in the adult. Type 1 myosin heavy chain was observed from 90 days whereas the fast types, 2a and 2b, were present from 210 to 230 days, at which stage the foetal form disappeared. Muscles that have greatly different contractile characteristics in the adult exhibit also different profiles of differentiation: the Diaphragma was the first to develop, followed by Cutaneus trunci, Longissimus thoracis and Masseter.

In situ localization of muscle insulin-like growth factor-II mRNA in developing bovine fetuses.

Insulin-like growth factor-II (IGF-II) modulates myogenesis in muscle cell cultures, in utero. IGF-II gene expression is developmentally regulated in several tissues including muscle. Determining whether IGF-II is expressed by developing muscle cells or by neighbouring cells in developing muscle tissue is crucial for determining whether IGF-II exerts a paracrine or an autocrine affect on myogenesis. Semitendinosus muscle samples from 12 bovine fetuses ranging from 60 to 274 days post conception (pc) were analysed for the amount and localization of muscle IGF-II mRNA using Northern, dot blot and in situ hybridization analyses. Northern blot analysis revealed multiple IGF-II transcripts of 5.1, 3.7, 2.6, 2.0, 1.7 and 1.1 kb in developing bovine muscle tissue. The relative amount of muscle IGF-II mRNA increased (P < 0.05) until 162 days pc, then decreased (P < 0.01) to near undetectable levels by the end of gestation (approximately 284 days pc). Between 60 and 162 days pc, in situ hybridization revealed that the majority of the IGF-II transcripts were localized to developing muscle cells rather than connective tissue. After 162 days pc the IGF-II hybridization signal shifted away from muscle cells and greater accumulation was observed in the connective tissue at 274 days pc. These data confirm that the expression of IGF-II in developing bovine muscle tissue is primarily localized in muscle cells and support the claim that IGF-II acts as an autocrine-acting growth factor during myogenesis in vivo.

Quantitative determination of type I myosin heavy chain in bovine muscle with anti myosin monoclonal antibodies.

Bovine type I muscle fibers were characterized by enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for slow myosin heavy chains (MHC 1). Two bovine muscles, the Masseter and Cutaneus trunci, were analyzed by different complementary techniques: electrophoresis, immunoblotting and immunohistiology. The results showed that the two muscles have extreme characteristics. The Masseter contains only slow MHC and the Cutaneus trunci is composed solely of rapid MHC (MHC 2a and 2b). A standard for this ELISA was obtained by mixing the two muscles and was used as a reference in the determination of the percentage of MHC 1 in a given muscle. In this study, the Longissimus thoracis of 27 Charolais cattle were examined. The different conditions under which assays were carried out were described and the accuracy of the measurement was calculated. In view of the results, ELISA was chosen for the analysis of muscle fiber types in large numbers of animal specimens. This technique could be used in several research projects to study the muscle characteristics that determine beef quality.

Myosin expression in semitendinosus muscle during fetal development of cattle: immunocytochemical and electrophoretic analyses.

The pattern of expression of different types of myosin and the development of different muscle cell populations were studied in the semitendinosus muscle of cattle from 39 d of gestation to 30 d of post-natal life. Monoclonal antibodies specific to different myosin heavy chains were used. Two cell generations were identified during myogenesis. They appeared successively and were characterized by different patterns of expression of myosins. The first population, which was present from the first stage studied (39 d of gestation), gave rise to type I fibers, which, in the mature animal, express only slow myosin. A second generation became differentiated at about 120 d of fetal life and then developed into type II fibers (IIa, IIb or IIc). The beginning of differentiation was characterized in all the cell populations by the expression of specific types of embryonic or fetal myosins. A comparison of these results with findings from previous works shows a marked similarity between species in the pattern of myogenesis but great differences in the length of the different stages of development. In this respect, myogenesis in cattle closely resembles that in man.

Muscle differentiation in the bovine fetus: a histological and histochemical approach.

The chronology of muscle fiber differentiation was analysed in 37 fetal calves of 69 to 266 days of age. Semitendinosus muscle weight was measured throughout the experimental period and biochemical, histological and histochemical investigations were made to determine respectively the protein and DNA content of the muscle, the size and the number of the fibers and their ATPase and SDH activity. The relative growth of all the quantitative characteristics (muscle weight, protein and DNA content) was much greater in the early stages of gestation than in the new-born animal. In the younger fetuses DNA relative growth was faster than protein relative growth, whereas at the end of gestation the reverse progression was observed. Before 90 days, the muscle tissue was composed of myotube-like cells without any clear organization. The organization of muscle tissue into clear bundles occurred around 120 days of age, and about 30 days later the large myotubes transformed into myofibers. The myotubes reacted positively for acid-ATPase activity, whereas the large population of smaller cells which developed in parallel did not. The number of muscle cells increased up to 240 days of age, as did the percentage of fibers positive for acid-ATPase activity. Finally, oxidative differentiation occurred around 260 days of age, with the appearance of a population of cells characterized by increased SDH activity. A comparison of these results with previous findings suggests that the muscular tissue differentiates through similar stages in various species, but over different lengths of time. The percentage of mature weight might provide a better inter-species time scale than chronological age.

Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells.

We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly; these mRNAs disappeared in cells differentiated into myotubes. 10(-7)M 12-O-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process.

[Mathematical model of daily increase of lipids and proteins in bovines]. / Modèle de calcul du croît journalier de lipides et de protéines chez les bovins.

A mathematical model for predicting body gain composition of cattle is presented. Daily lipid, protein or tissue gains are predicted according to body weight and body weight gain, for male or female cattle of various breeds. The aim and the biological basis of this model are described.

Iodothyronine 5'-deiodinase activity as an early event of prenatal brown-fat differentiation in bovine development.

Iodothyronine 5'-deiodinase activity appears to be a type I enzyme in bovine brown adipose tissue, on the basis of its high Km for 3,3',5'-tri-iodothyronine ('reverse T3') (in the micromolar range) and sensitivity to propylthiouracil inhibition. This enzyme activity is already detectable in perirenal adipose tissue of bovine fetuses in the second month of gestation, reaches peak values around the seventh month of fetal life, declines before birth, becomes lower after parturition and finally undetectable in the adult cow. Iodothyronine 5'-deiodinase activity is present in the pericardic, peritoneal and intermuscular adipose depots of the neonatal calf, but it is always undetectable in the subcutaneous adipose tissue. It is concluded that iodothyronine 5'-deiodinase is a specific feature of brown fat in the bovine species that is not shared by white adipose tissue. white adipose tissue. Peak values of 5'-deiodinating activity appear as an early event in the prenatal differentiation programme of bovine brown-fat cells as they occur when uncoupling-protein-gene expression first starts.

Short-term and long-term effects of early nutritional deprivation on adipose tissue growth and metabolism in calves.

Two groups of 10 Friesian calves received, respectively, 819 and 1380 g of milk replacer daily between birth and 95 d of age. After weaning, both groups were pair-fed until slaughter at 533 d of age. Body composition, cellularity, and lipogenic activity of kidney and omental fat were determined at 95 and 533 d of age. Milk intake restriction produced a 40% reduction of growth rate and a 68% decrease in lipid deposition between birth and 95 d of age. This was accompanied by a reduced adipose cell hypertrophy without any effect on adipose cell number. Acetate incorporation in isolated cells, glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase, and lipoprotein lipase activities were lower in restricted animals. Glucose incorporation in isolated fat cells was very slight in both groups. Acetate incorporation and lipogenic enzyme activities were more than 10 times higher in 533-d-old animals that have larger adipocytes than in younger calves. Moreover, kidney fat presented a higher rate of de novo fatty acid synthesis than omental fat. The reverse order was observed for lipoprotein lipase activity. Early postnatal nutrition had no significant effect on lipid deposition between 95 and 533 d of age. There were no significant differences in body composition, adipose tissue cellularity, or metabolism at slaughter.

Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth.

Samples of adipose tissue were obtained from different sites in bovine and ovine foetuses and newborns. RNA was isolated and analysed using bovine cDNA and ovine genomic probe for uncoupling protein (UCP), cDNA for subunits III and IV of cytochrome c oxidase and cDNA for ADP/ATP carrier. UCP mRNA was characterized for the first time in foetal bovine and ovine adipose tissue. It appeared later than mRNA of cytochrome c oxidase subunit III, and increased dramatically at birth (10-fold). ADP/ATP carrier mRNA was expressed at a lower level but also increased 10-fold at birth. It was demonstrated that UCP mRNA reached its highest level at birth in all bovine adipose tissues studied, except subcutaneous tissue. It disappeared quickly afterwards, being no longer detectable two days after birth. Similar variations were observed in newborn lambs. ADP/ATP carrier mRNA showed the same pattern of expression as UCP mRNA; although it was still lightly expressed two days after birth, it disappeared soon afterwards. Only mRNAs for cytochrome c oxidase subunits III and IV remained at the same level during the first postnatal week. On the basis of these data and of observations reported in the literature a sequence of events for the development of brown adipose cells in vivo is proposed. Soon after birth the perirenal adipose tissue of ruminants, which still contains mitochondria of typical brown adipose tissue morphology and high levels of cytochrome c oxidase mRNA, lacks UCP mRNA. Can it still be considered as brown fat? Ruminant species appear to be attractive models to study both the differentiation of brown adipose tissue and its possible conversion to white fat in large animals.

[Utilization of urea as a marker for body water in the cow, in comparison with heavy water]. / Utilisation de l'urée comme marqueur de l'eau corporelle chez la vache, en comparaison avec l'eau lourde.

Urea dilution space in 5 cows was compared to deuterium oxide dilution space and to direct determination of body water after slaughter. Urea and deuterium oxide dilution spaces were, respectively, close to empty body water and total body water weights.

Regulation of growth hormone release in fetal calves.

Plasma GH and IGF1 concentrations were measured during the last 2 months of gestation in 9 chronically catheterized fetal calves under basal conditions or following growth-hormone-releasing factor (GRF), thyrotropin-releasing hormone (TRH) or SRIF intravenous cotyledonnary injections. Plasma GH concentrations were higher in fetuses (1.40 +/- 0.10 nmol/l) than in dams (0.14 +/- 0.01 nmol/l). Plasma GH secretory profile was pulsatile. The number of secretory pulses, as well as their magnitude and mean baseline values decreased from 220 to 270 days of gestation. Synthetic 1-29 GRF or TRH increased fetal plasma GH concentration at 250 and 270 days of gestation but was devoid of any significant effect at 220 days. SRIF injection decreased plasma GH concentration in 270-day-old fetuses. Plasma IGF1 concentrations were lower in fetuses than in dams. No treatment had a significant effect on fetal and maternal IGF1 levels.