Steroid Effects on Cell Proliferation, Differentiation and Steroid Receptor Gene Expression in Adult Bovine Satellite Cells

  • Lee, Eun Ju ;
  • Choi, Jinho ;
  • Hyun, Jin Hee ;
  • Cho, Kyung-Hyun ;
  • Hwang, Inho ;
  • Lee, Hyun-Jeong ;
  • Chang, Jongsoo ;
  • Choi, Inho
  • Received : 2006.08.08
  • Accepted : 2006.10.16
  • Published : 2007.04.01


The present study was conducted to establish primary bovine muscle satellite cell (MSC) culture conditions and to investigate the effects of various steroid hormones on transcription of the genes involved in muscle cell proliferation and differentiation. Of three different types of proteases (type II collagenase, pronase and trypsin-EDTA) used to hydrolyze the myogenic satellite cells from muscle tissues, trypsin-EDTA treatment yielded the highest number of cells. The cells separated by hydrolysis with type II collagenase and incubated on gelatin-coated plates showed an enhanced cell attachment onto the culture plate and cell proliferation at an initial stage of cell growth. In this study, the bovine MSCs were maintained in vitro up to passage 16 without revealing any significant morphological change, and even to when the cells died at passage 21 with decreased or almost no cell growth or deformities. When the cells were incubated in a steroid-depleted environment (DMEM(-)/10% CDFBS (charcoal-dextran stripped FBS)), they grew slowly initially, and were widened and deformed. In addition, when the cells were transferred to an incubation medium containing steroid (DMEM(+)/10% FBS), the deformed cells resumed their growth and returned to a normal morphology, suggesting that steroid hormones are crucial in maintaining normal MSC morphology and growth. The results demonstrated that treatments with 19-nortestosterone and testosterone significantly increased AR gene expression (p<0.05), implying that both testosterone and 19-nortestosterone bind with AR and that the hormone bound-AR complex up-regulates the genes of its own receptor (AR) plus other genes involved in satellite cell growth and differentiation in bovine muscle.


Bovine Muscle Satellite Cell;Steroid;Primary Cell Culture


  1. Allen, R. E., R. A. Merkel and R. B. Young. 1979. Cellular aspects of muscle growth: Myogenic cell proliferation. J. Anim. Sci. 49:115-127.
  2. Campion, D. R. 1984. The muscle satellite cell: Int. Rev. Cytol. 87:225-251.
  3. Cornelison, D. D. and B. J. Wold. 1997. Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev. Biol. 191:270-283.
  4. Johnson, B. J., N. Halstead, M. E. White, M. R. Hathaway and W. R. Dayton. 1998. Activation state of muscle satellite cells isolated from steers implanted with a combined trenbolone acetate and estradiol implant. J. Anim. Sci. 76:2779-2786.
  5. Kahlert, S., C. Grohe, R. H. Karas, K. Lobbert, L. Neyses and H. Vetter. 1997. Effects of estrogen on skeletal myoblast growth. Biochem. Biophys. Res. Commun. 232:373-378.
  6. Lindquist, D. L. and P. A. de Alarcon. 1987. Charcoal-dextran treatment of fetal bovine serum removes an inhibitor of human CFU-megakaryocytes. Exp. Hematol. 15:234-238.
  7. Tricarico, C., P. Pinzani, S. Bianchi, M. Paglierani, V. Distante, M. Pazzagli, S. A. Bustin and C. Orlando. 2002. Quantitative realtime reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal. Biochem. 309:293-300.
  8. Chen, J. C. J. and D. J. Goldhamer. 2003. Skeletal muscle stem cells. Reproductive Biol. Endocrinol. 1:101-107.
  9. McFarland, D. C., M. E. Doumit and R. D. Minshell. 1988. The turkey myogenic satellite cell: Optimization of in vitro proliferation and differentiation. Tissue and Cell. 20:899-908.
  10. Greene, E. A. and R. H. Raub. 1992. Procedures for harvesting satellite cells from equine skeletal muscle. Equine. Nut. Phys. Soc. 12:33-35.
  11. Lay, S. L., Lefrere, C. Trautwein, I. Dugail and S. Krief. 2002. Insulin and sterol-regulatory element-binding protein-1c (SREBP-1C) regulation of gene expression in 3T3-L1 adipocytes. J. Biol. Chem. 277:35625-35634.
  12. Oh, Y. S., S. B. Cho, K. H. Beak and C. B. Choi. 2005. Effects of Testosterone, $17{\beta}$-estradiol, and Progesterone on the Differentiation of Bovine Intramuscular Adipocytes. Asian-Aust. J. Anim. Sci. 18:1589-1593.
  13. Dodson, M. V., E. L. Martin, M. A. Brannon, B. A. Mathison and D. C. McFarland. 1987. Optimization of bovine satellite cellderived myotube formation in vitro. Tissue and Cell. 19:159-166.
  14. Doumit, M. E. and R. A. Merkel. 1992. Conditions for the isolation and culture of porcine myogenic satellite cells. Tissue and Cell. 24:253-262.
  15. Thompson, S. H., L. K. Boxhorn, W. Y. Kong and R. E. Allen. 1989. Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I. Endocrinol. 124:2110-2117.
  16. Allen, R. E. and L. L. Rankin. 1990. Regulation of satellite cell during skeletal muscle growth and development. PSEMB. 194:81-86.
  17. Kamanga-Sollo, E., M. S. Pampusch, G. Xi, M. E. White, M. R. Hathaway and W. R. Dayton. 2004. IGF-I mRNA levels in bovine satellite cell cultures: effects of fusion and anabolic steroid treatment. J. Cell Physiol. 201:181-189.
  18. Katzenellenbogen, B. S., I. Choi, R. Delage-Mourroux, T. R. Ediger, P. G. Martini, M. Montano, J. Sun, K. Weis and J. A. Katzenellenbogen. 2000. Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology. J. Steroid Biochem. Mol. Biol. 74:279-285.
  19. Shen, H. C. and G. A. Coetzee. 2005. The androgen receptor: unlocking the secrets of its unique transactivation domain. Vitam Horm. 71:301-319.
  20. Dodson, M. V., B. A. Mathison and B. D. Mathison. 1990. Effects of medium and substratum on ovine satellite cell attachment, proliferation and differentiation in vitro. Cell. Diff. Dev. 29:59-66.
  21. Hunt, D. W., D. M. Henricks, G. C. Skelley and L. M. Grimes. 1991. Use of trenbolone acetate and estradiol in intact and castrate male cattle: Effects on growth, serum hormones, and carcass characteristics. J. Anim. Sci. 69:2452-2462.
  22. Johnson, B. J., P. T. Anderson, J. C. Meiske and W. R. Dayton. 1996. Effect of a combined trenbolone and estradiol implant on steroid hormone levels, feedlot performance, carcass characteristics and carcass composition of feedlot steers. J. Anim. Sci. 74:363-371.
  23. Seale, P., L. A. Sabourin, A. Girgis-Gabardo, P. Gruss and M. A. Rudnicki. 2000. Pax7 is required for the specification of myogenic satellite cells. Cell. 102:777-786.
  24. Duclos, M. J., B. Chevalier, C. Goddard and J. Simon. 1993. Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by IGF-I. J. Cell. Physiol. 157:650-657.
  25. Sinha-Hikim, I., W. E. Taylor, N. F. Gonzalez-Cadavid, W. Zheng and S. Bhasin. 2004. Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. J. Clin. Endocrinol. Metab. 89:5245-5255.
  26. Apple, J. K., M. E. Dikeman, D. D. Simms and G. Kuhl. 1991. Effects of synthetic hormone implants, singularly or in combinations, on performance, carcass traits, and longissimus muscle palatability of Holstein steers. J. Anim. Sci. 69:4437-4448.
  27. Dodson, M. V., D. C. McFarland, E. L. Martin and M. A. Brannon. 1986. Isolation of satellite cells from ovine skeletal muscle. J. Tiss. Cul. Meth. 10:233-237.
  28. Bonavaud, S., O. Agbulut, G. D'Honneur, R. Nizard, V. Mouly and G. Butler-Browne. 2002. Preparation of isolated human muscle fibers: a technical report. In vitro Cell. Dev. Biol. Anim. 38:66-72.<0066:POIHMF>2.0.CO;2
  29. Doumit, M. E., D. R. Cook and R. A. Merkel. 1996. Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinol. 137:1385-94.
  30. Choi, I., L. J. Gudas and B. S. Katzenellenbogen. 2000. Regulation of keratin 19 gene expression by estrogen in human breast cancer cells and identification of the estrogen responsive gene region. Mol. Cell Endocrinol. 164:225-237.
  31. Herschler, R. C., A. W. Olmsted, A. J. Edwards, R. L. Hale, T. Montgomery, R. L. Preston, S. J. Bartle and J. J. Sheldon. 1995. Production responses to various doses and ratios of estradiol benzoate and trenbolone acetate implants in steers and heifers. J. Anim. Sci. 73:2873-2881.
  32. Bischoff, R. 1974. Enzymatic liberation of myogenic cells from adult rat muscle. Anat. Rec. 180:645-662.
  33. Dodson, M. V., D. C. McFarland, A. L. Grant, M. E. Doumit and S. G. Velleman. 1996. Extrinsic regulation of domestic animalderived satellite cells. Dom. Anim. Endocrin. 13:107-126.

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