Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
권호 표지
DOI QR코드

DOI QR Code

Validation of Methods for Isolation and Culture of Alpaca Melanocytes: A Novel Tool for In vitro Studies of Mechanisms Controlling Coat Color

  • Bai, Rui (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Sen, Aritro (Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University) ;
  • Yu, Zhihui (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Yang, Gang (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Wang, Haidong (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Fan, Ruiwen (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Lv, Lihua (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Lee, Kyung-Bon (Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University) ;
  • Smith, George W (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Dong, Changsheng (College of Animal Science and Technology, Shanxi Agricultural University)
  • Received : 2009.09.01
  • Accepted : 2009.10.30
  • Published : 2010.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

Keywords

References

  1. Aberdam, E., C. Bertolotto, E. V. Sviderskaya, V. Thillot, T. J. Hemesath, D. E. Fisher, D. C. Bennett, J. P. Ortonne and R. Ballotti. 1998. Involvement of microphthalmia in the inhibition of melanocyte lineage differentiation and of melanogenesis by agouti signal protein. J. Biol. Chem. 273: 19560-19565 https://doi.org/10.1074/jbc.273.31.19560
  2. Andersson, L. 2003. Melanocortin receptor variants with phenotypic effects in horse, pig, and chicken. Ann. NY Acad. Sci. 994:313-318 https://doi.org/10.1111/j.1749-6632.2003.tb03195.x
  3. Bennett, D. C., P. J. Cooper, T. J. Dexter, L. M. Devlin, J. Heasman and B. Nester. 1989. Cloned mouse melanocyte lines carrying the germline mutations albino and brown: Complementation in culture. Development 105:379-385
  4. Clarkson, K. S., I. C. Sturdgess and A. J. Molyneux. 2001. The usefulness of tyrosinase in the immunohistochemical assessment of melanocytic lesions: A comparison of the novel t311 antibody (anti-tyrosinase) with s-100, hmb45, and a103 (anti-melan-a). J. Clin. Pathol. 54:196-200 https://doi.org/10.1136/jcp.54.3.196
  5. Eisinger, M and O. Marko. 1982. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc. Natl. Acad. Sci. USA. 79:2018-2022 https://doi.org/10.1073/pnas.79.6.2018
  6. Gregor, M. B. 2006. Production, attributes and relative value of alpaca fleeces in southern australia and implications for industry development. Small Rumin. Res. 61:93-111 https://doi.org/10.1016/j.smallrumres.2005.07.001
  7. Hemesath, T. J., E. R. Price, C. Takemoto, T. Badalian and D. E. Fisher. 1998. Map kinase links the transcription factor microphthalmia to c-kit signalling in melanocytes. Nature 391:298-301 https://doi.org/10.1038/34681
  8. Iijima, S. and K. Watanabe. 1956. Studies on dopa reaction. I. A simple technique for dopa reaction. J. Invest. Dermatol. 26:235
  9. Jule, S., P. Bosse, G. Egidy and J. J. Panthier. 2003. Establishment and characterization of a normal melanocyte cell line derived from pig skin. Pigment Cell Res. 16:407-410 https://doi.org/10.1034/j.1600-0749.2003.00071.x
  10. Kadekaro, A. L., H. Kanto, R. Kavanagh and Z. A. Abdel-Malek. 2003. Significance of the melanocortin 1 receptor in regulating human melanocyte pigmentation, proliferation, and survival. Ann. NY Acad. Sci. 994:359-365 https://doi.org/10.1111/j.1749-6632.2003.tb03200.x
  11. King, R., K. N. Weilbaecher, G. McGill, E. Cooley, M. Mihm and D. E. Fisher. 1999. Microphthalmia transcription factor. A sensitive and specific melanocyte marker for melanomadiagnosis. Am. J. Pathol. 155:731-738 https://doi.org/10.1016/S0002-9440(10)65172-3
  12. Levy, C., M. Khaled and D. E. Fisher. 2006. Mitf: Master regulator of melanocyte development and melanoma oncogene. Trends Mol. Med. 12:406-414 https://doi.org/10.1016/j.molmed.2006.07.008
  13. Li, Q., L. J. Bakke, J. R. Pursley and G. W. Smith. 2004. Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles. Reproduction 128:555-564 https://doi.org/10.1530/rep.1.00282
  14. Lin, Y. P., F. L. Hsu, C. S. Chen, J. W. Chern and M. H. Lee. 2007. Constituents from the formosan apple reduce tyrosinase activity in human epidermal melanocytes. Phytochemistry 68: 1189-1199 https://doi.org/10.1016/j.phytochem.2007.02.001
  15. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. Methods 25:402-408 https://doi.org/10.1006/meth.2001.1262
  16. Lupton, C. J., A. McColl and R. H. Stobart. 2006. Fiber characteristics of the huacaya alpaca. Small Rumin. Res. 64: 211-224 https://doi.org/10.1016/j.smallrumres.2005.04.023
  17. Rees, J. L. 2000. The melanocortin 1 receptor (mc1r): More than just red hair. Pigment Cell Res. 13:135-140
  18. Renieri, C. 1995. Pigmentation in domestic mammals, with reference to fine fiber producing species In: Genetic improvement of fine fiber producing animals No. 1 (Ed. J. P. Laker and S. C. Bishop). p 113-136. EFFN, Peebles, UK
  19. Rouzaud, F., J. P. Annereau, J. C. Valencia, G. E. Costin and V. J. Hearing. 2003. Regulation of melanocortin 1 receptor expression at the mrna and protein levels by its natural agonist and antagonist. FASEB. J. 17:2154-2156
  20. Shibahara, S., K. Yasumoto, S. Amae, T. Udono, K. Watanabe, H. Saito and K. Takeda. 2000. Regulation of pigment cell-specific gene expression by mitf. Pigment Cell Res. 13 (Suppl 8):98-102 https://doi.org/10.1034/j.1600-0749.13.s8.18.x
  21. Sponenberg, D. P., S. Ito, K. Wakamatsu and L. A. Eng. 1988. Pigment types in sheep, goats, and llamas. Pigment Cell Res. 1:414-418 https://doi.org/10.1111/j.1600-0749.1988.tb00145.x
  22. Steingrimsson, E., K. J. Moore, M. L. Lamoreux, A. R. Ferré-D'Amare, S. K. Burley, D. C. Zimring, L. C. Skow, C. A. Hodgkinson, H. Arnheiter and N. G. Copeland. 1994. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat. Genet. 8:256-263 https://doi.org/10.1038/ng1194-256
  23. Suzuki, I., A. Tada, M. M. Ollmann, G. S. Barsh, S. Im, M. L. Lamoreux, V. J. Hearing, J. J. Nordlund and Z. A. Abdel-Malek. 1997. Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin. J. Invest. Dermatol. 108:838-842 https://doi.org/10.1111/1523-1747.ep12292572
  24. Tsatmali, M., J. Ancans and A. J. Thody. 2002. Melanocyte function and its control by melanocortin peptides. J. Histochem. Cytochem. 50:125-133 https://doi.org/10.1177/002215540205000201
  25. Vancoillie, G., J. Lambert and J. M. Nayaert. 1999. Melanocyte biology and its implications for the clinician. Eur. J. Dermatol. 9:241-251
  26. Wakamatsu, K., R. Kavanagh, A. L. Kadekaro, S. Terzieva, R. A. Sturm, S. Leachman, Z. Abdel-Malek and S. Ito. 2006. Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin. Pigment Cell Res. 19:154-162 https://doi.org/10.1111/j.1600-0749.2006.00293.x
  27. Yamaguchi, Y. and V. J. Hearing. 2009. Physiological factors that regulate skin pigmentation. Biofactors 35:193-199 https://doi.org/10.1002/biof.29

Cited by

  1. Nitric oxide enhances melanogenesis of alpaca skin melanocytes in vitro by activating the MITF phosphorylation vol.352, pp.1-2, 2011, https://doi.org/10.1007/s11010-011-0761-1
  2. Expression and Distribution of the Guanine Nucleotide-binding Protein Subunit Alpha-s in Mice Skin Tissues and Its Association with White and Black Coat Colors vol.29, pp.10, 2016, https://doi.org/10.5713/ajas.15.0711
  3. The effects of IGF1 on the melanogenesis in alpaca melanocytes in vitro vol.52, pp.8, 2016, https://doi.org/10.1007/s11626-016-0052-y
  4. Role of microRNA508-3p in melanogenesis by targeting microphthalmia transcription factor in melanocytes of alpaca vol.11, pp.02, 2017, https://doi.org/10.1017/S1751731116001294
  5. Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos)1 vol.93, pp.4, 2015, https://doi.org/10.2527/jas.2014-8404
  6. MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation and melanogenesis vol.27, pp.2, 2018, https://doi.org/10.1111/exd.13480
  7. MicroRNA-5110 regulates pigmentation by cotargeting melanophilin and WNT family member 1 vol.32, pp.10, 2018, https://doi.org/10.1096/fj.201800040R
  8. Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to α-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor vol.396, pp.4, 2010, https://doi.org/10.1016/j.bbrc.2010.05.001
  9. miR-148a-3p inhibits alpaca melanocyte pigmentation by targeting MITF vol.177, pp.None, 2010, https://doi.org/10.1016/j.smallrumres.2019.06.004
  10. miR-380-3p regulates melanogenesis by targeting SOX6 in melanocytes from alpacas (Vicugna pacos) vol.20, pp.1, 2010, https://doi.org/10.1186/s12864-019-6343-4
  11. Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation vol.20, pp.1, 2019, https://doi.org/10.1186/s12867-019-0137-8
  12. MicroRNA‐379 mediates pigmentation, migration and proliferation of melanocytes by targeting the insulin‐like growth factor 1 receptor vol.29, pp.5, 2010, https://doi.org/10.1111/exd.14095