Study of the Chronology of Expression of Ten Extracellular Matrix Molecules during the Myogenesis in Cattle to Better Understand Sensory Properties of Meat

Abstract

The sensory properties of beef are known to depend on muscle fiber and intramuscular connective tissue composition (IMCT). IMCT is composed of collagens, proteoglycans and glycoproteins. The differentiation of muscle fibers has been extensively studied but there is scarcity in the data concerning IMCT differentiation. In order to be able to control muscle differentiation to improve beef quality, it is essential to understand the ontogenesis of IMCT molecules. Therefore, in this study, we investigated the chronology of appearance of 10 IMCT molecules in bovine Semitendinosus muscle using immunohistology technique at five key stages of myogenesis. Since 60 days post-conception (dpc), the whole molecules were present, but did not have their final location. It seems that they reach it at around 210 dpc. Then, the findings emphasized that since 210 dpc, the stage at which the differentiation of muscle fibers is almost complete, the differentiation of IMCT is almost completed. These data suggested that for the best controlling of the muscular differentiation to improve beef sensory quality, it would be necessary to intervene very early (before the IMCT constituents have acquired their definitive localization and the muscle fibers have finished differentiating), i.e., at the beginning of the first third of gestation.

Keywords: bovine; extracellular matrix; fetus; immunohistology; skeletal muscle.

Figure 1

Azorubine staining of transverse sections of foetal Semitendinosus muscle at 110 (A), 180 (B), 210 (C) and 260 (D) days post-conception (dpc). (ECM: ExtraCellular Matrix; MaN: perimysium major network, MiN: perimysium minor netork, Mt1: primary myotubes, Mt2: secondary myotube, MFiBu: Muscle Fiber Bundle).

Figure 2

Immunohistochemical labelling with antibodies against type I (A, B, C, D, E), and type VI (F, G, H, I, J) collagens of transverse sections of foetal Semitendinosus muscle at 60, 110, 180, 210 and 260 days post-conception (dpc) (Major (MaN) and minor (MiN) networks of perimysium; E: endomysium, Mt1: primary myotubes, Mt2: secondary myotubes).

Figure 3

Immunohistochemical labelling with antibodies against versican (A, B, C, D, E), decorin (F, G, H, I, J), chondroitin-4-sulfate (K, L, M, N, O), chondroitin-6-sulfate (P, Q, R, S, T) of transverse sections of foetal Semitendinosus muscle at 60, 110, 180, 210 and 260 days post-conception (dpc). (Major (MaN) and minor (MiN) networks of perimysium (P), E: endomysium, Mt1: primary myotubes, Mt2: secondary myotubes, F: muscle fiber).

Figure 4

Mmunohistochemical labelling with antibodies against tenascin-X (A, B, C, D, E), collagen XII (F, G, H, I, J), collagen XIV (K, L, M, N, O) of transverse sections of foetal Semitendinosus muscle at 60, 110, 180, 210 and 260 days post-conception (dpc). (Major (MaN) and minor (MiN) networks of perimysium).

Figure 5

(a) Collagen XII and (b) Collagen XIV Western blot analysis of foetal Semitendinosus muscle at 110, 180, 210 and 260 days post-conception (dpc). The monoclonal antibodies against collagen XII and XIV, two disulphide bonded polypeptides, recognized three bands on Western blots of bovine muscle extracts, one band at 220 kDa and two others at about 290 kDa. The presence of these three bands was due to the fact that migration conditions were reducing. The relative amounts of collagen XII and XIV (least square means ± standard error of the mean) were expressed in arbitrary units. Different letters on the same graph indicated that relative amounts of collagen XII and XIV differed significantly between foetal stages (p < 0.05).

Figure 6

(a) The immunohistochemical labeling with antibodies directed against collagen IV (A, B, C, D, E) of transverse sections of fetal Semitendinosus muscle at 60, 110, 180, 210 and 260 days post-conception (dpc). (P: perimysium, E: endomysium, Mt1: primary myotubes, Mt2: secondary myotubes, F: muscle fiber). (b) Schematic representation of differentiation of muscle fibers at 60, 110, 180, 210 and 260 days post-conception (dpc). At 60 and 110 dpc, muscle fibers are present as primary (cells schematically represented by an oval grey form with a central lumen surrounded by a dark grey line representing endomysium) myotubes (Mt1) and secondary myotubes (Mt2) (cells schematically represented by a brown form without central lumen). At 60 dpc, Mt2 are not individualized; they are wrapped in the same endomysium than Mt1. At 110 dpc, almost all Mt2 are individualized and they have their own endomysium (dark grey line). At 180 and 210 dpc, some Mt2, individualized or not, are still present, but the majority of muscle cells are fibers (schematically represented by a yellow form surrounded by a dark grey line representing endomysium). At 260 dpc, all myotubes are muscle fibers. (c) Schematic description of localization of different molecules of ExtraCellular matrix. At each stage, the localization of ECM molecules (Col I, IV, VI, XII and XIV: collagen of type I, IV, VI, XII and XIV; DCN: decorin, TN-X: tenascin-X, C4 and 6S: chondroitin 4 and 6 sulfate; VCN: versican) is indicated in the major and minor networks of perimysium and in the endomysium. From 110 dpc, only the modifications of localization of ECM molecules are indicated.