375 Animal Science Journal (2002) 73, 375–381 ORIGINAL ARTICLE Sequential expression of myogenic regulatory factors in bovine skeletal muscle and the satellite cell culture Susumu MUROYA, Ikuyo NAKAJIMA and Koichi CHIKUNI National Institute of Livestock and Grassland Science, Tsukuba Norin Kenkyu Danchi, Ibaraki-ken, Japan ABSTRACT Myogenic regulatory factors (MRFs) are important in the control of skeletal muscle development. To understand myogenic regulation by MRFs in bovine adult muscle cells, their expressions, namely that of Myf5, MyoD, myogenin, and MRF4 in the biceps femoris muscle (BF) and in the satellite cell culture, were analyzed by RT-PCR. In the BF, all four MRFs were expressed and in particular, myogenin and MRF4 were strongly expressed, whereas Myf5 was faintly expressed. The satellite cells pre- pared from the BF expressed Myf5, but only a trace of MyoD, at day 9 of culture. During the growth of the cells to day 14, the MyoD and myogenin expressions gradually increased, and that of MyoD expression reached its maximum at the con- fluence of the culture. After induction of myogenic differentiation by a serum-free medium at day 14, Myf5 expression gradually decreased, and the up-regulated expression of MyoD was suppressed, whereas myogenin expression continued to increase sharply. Following the myogenin expression, MRF4 also drastically increased toward the myotube formation of the cells. When huge myotubes were formed at day 18, Myf5 was expressed at a low level, whereas the MyoD expression remained at a moderate level. KEYWORDS: cattle, myogenic regulatory factors, RT-PCR, satellite cell, skeletal muscle. and/or Myf5 genes revealed that they are responsible for myogenic commitment, but can compensate for the lack of each other (Braun et al. 1992; Rudnicki et al. 1992, 1993). Myogenin gene disruption provided the evidence for its essential role in myotube formation (Hasty et al. 1993; Nabeshima et al. 1993), whereas dis- ruption of the MRF4 gene did not show a decisive role of MRF4 in myotube maturation (Braun & Arnold 1995; Patapoutian et al. 1995). On the other hand, the forced expression of any of the four proteins in non- muscle cell lines results in a myogenic differentiation that is essentially indistinguishable among the factors introduced (Davis et al. 1987; Edmondson & Olson 1989; Rhodes & Konieczny 1989; Wright et al. 1989). INTRODUCTION The myogenic regulatory factors (MRFs), MyoD, Myf5, myogenin and MRF4 are skeletal muscle- specific transcription factors that bind to the regula- tory regions of muscle-specific genes via their basic helix-loop–helix domain forming a heterodimer with other transcription factors such as E proteins (Arnold & Braun 2000). In situ hybridization and RT-PCR studies have demonstrated that the expression pattern of each MRF is restricted spatio-temporally. In the mouse embryo, myoblasts first express Myf5 (Ott et al. 1991), then MyoD and myogenin in the somites (Sassoon et al. 1989), and terminally differentiated myotubes express MRF4 in the later stage, despite subtle differences in the order between somites and limb buds (Bober et al. 1991; Hinterberger et al. 1991). Gene knockout studies have demonstrated the stage-specific roles of MRFs in vivo predicted from their expression patterns. The disruption of the MyoD Correspondence: Susumu Muroya, National Institute of Livestock and Grassland Science, Tsukuba Norin Kenkyu Danchi, Ibaraki-ken 305–0901, Japan. (Email: muros@affrc. go.jp) Received 25 March 2002; accepted for publication 8 May 2002.