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

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.