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Endothelin-1 increases melanin synthesis in an established sheep skin melanocyte culture

  • Yamiao Pang
  • Jianjun GengEmail author
  • Yilong Qin
  • Haidong Wang
  • Ruiwen Fan
  • Ying Zhang
  • Hongquan Li
  • Shan Jiang
  • Changsheng Dong
Article

Abstract

The aims of the study were to establish a culture system for sheep skin melanocytes and uncover the effects of endothelin-1 on melanin synthesis in cultured melanocytes in order to provide an optimal cell system and a theoretical basis for studying the regulatory mechanism of coat color in sheep. In this study, skin punch biopsies were harvested from the dorsal region of 1–3-yr-old sheep, and skin melanocytes were then obtained by the two-step digestion using dispase II and trypsin/ethylene diamine tetraacetic acid (EDTA). The primary cultures of the melanocytes were established and characterized by dopa-staining, immunocytochemical localization of melanocyte markers, and RT-polymerase chain reaction (PCR) analysis of coat color genes. To determine the effect of endothelin-1 on proliferation and melanin synthesis of melanocytes, the cultured cells were treated with different doses of endothelin-1 (10−7, 10−8, 10−9, 10−10, and 0 mol/L), and the growth rate of melanocytes, production of melanin, expression of related genes, and location of related protein in cultured cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ultraviolet spectrophotometry, qRT-PCR, and immunocytochemical localization, respectively. The results showed that the established melanocyte culture functions properly. Endothelin-1 treatment increased markedly the number of melanocytes and melanin content. In responding to this treatment, expressions of microphthalmia-associated transcription factor (MITF), melanocortin 1 receptor (MC1R), tyrosinase (TYR), and endothelin receptor B (EDNRB) in the melanocytes were significantly up regulated (P < 0.05). Immunocytochemical localization revealed that TYR was mainly localized in the cytoplasm. Positive staining of TYR in the melanocytes was significant. The findings demonstrated that the culture system of sheep skin melanocytes was established successfully in vitro, and endothelin-1 promotes the melanogenesis in sheep skin melanocytes.

Keywords

Melanocyte Endothelin-1 Melanin Skin color Sheep 

Notes

Acknowledgments

This work was supported by the Shanxi Natural and Science Foundation (grant number 2012011046-6) and Special Fund for Agro-scientific Research in the Public Interest (grant number 201303119). Shan Jiang and Yilong Qin carried out the cell dispersion and culture. Haidong Wang participated in the determination of melanocyte growth rate. Hongquan Li and Changsheng Dong participated in the DOPA reaction and immunocytochemical localization. Ruiwen Fan and Ying Zhang participated in the determination of melanocyte proliferation and detection of melanin content. Jianjun Geng conceived the study and performed the statistical analysis. Yamiao Pang participated in RT-PCR and qRT-PCR and drafted the manuscript. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests. We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

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Copyright information

© The Society for In Vitro Biology 2016

Authors and Affiliations

  • Yamiao Pang
    • 1
  • Jianjun Geng
    • 1
    Email author
  • Yilong Qin
    • 1
  • Haidong Wang
    • 1
  • Ruiwen Fan
    • 1
  • Ying Zhang
    • 1
  • Hongquan Li
    • 1
  • Shan Jiang
    • 1
  • Changsheng Dong
    • 1
  1. 1.College of Animal Science and TechnologyShanxi Agricultural UniversityTaiguChina

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