Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts Md. Abul Hashem a , Dilip P. Bhandari a , Sung Keun Kang a,b , Byeong Chun Lee a,b, * , Hwang Woo Suk a,b,c a Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea b The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, South Korea c School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea Received 5 December 2005; revised 25 February 2006; accepted 26 April 2006 Abstract The present study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. Seven experiments were performed, each with a one-way completely randomized design involving three replicates. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved, and fully confluent stages of Goral cells. In Experiments II and III, the effects of different antioxidants like b-mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 24 h and 4 h, respectively. In Ex- periments IVand V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ml), were used as in Experiment II. In Experiments VI and VII, the effect of different levels of dimethylsulphoxide (DMSO) at 0%, 0.5%, 1.0% and 2.5% were tested by flow cytometry (FACS). In Experiment I, 68.7% of Goral skin fibroblasts reached the G 0 /G 1 stage (2C DNA content) when subjected to the serum-starved medium, which was more than the cycling (64.9%) and fully confluent groups (61.0%) (P > 0.05). Among the chemically treated group, b-ME, cysteine and DMSO showed better results for synchronization of G 0 þ G 1 phases than cycling, serum-starved and fully confluent groups. It can thus be concluded that b-ME, cysteine and DMSO at certain concentrations can synchronize the cell cycle effectively, which could have a positive impact on somatic cell nuclear transfer in the goral. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Goral; Cell cycle; Synchronization 1. Introduction Gorals are listed as endangered animals in CITES Appendix I. The grey goral is classified as being at low risk, or nearly threatened, by the 1996 IUCN report (Shackleton, 1997). Go- rals derived from the subfamily caprinae have been classified into three tribes, Rupicaprini, Ovibovini and Caprini (Geist, 1987; Gentry, 1992). Within the tribe Rupicaprini, serows and gorals have been assigned to two separate genera (Mead, 1989; Nowak, 1999), Capricornis and Nemorhaedus, respec- tively, despite being closely related. The genus Nemorhaedus of Rupicaprini includes three species, N. caudatus (long-tailed goral or Chinese goral), N. goral (Himalayan goral) and N. bai- leyi (red goral). Nemorhaedus species’ range from northern Pakistan to Northeastern Asia including Korea (Mead, 1989). The long- tailed goral (or Chinese goral), N. caudatus, is one of the most endangered mammalian species in Korea (Ministry of Environment of Korea, 2000) and has been designated as a Natural Monument species by the Cultural Properties Ad- ministration of the Korean Government. The goral population in South Korea is assumed to be less than 250 (Cultural Prop- erties Administration of Korea, 1999), and their habitats are * Corresponding author. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea. Tel.: þ82 2 880 1269; fax: þ82 2 884 1902. E-mail address: bclee@snu.ac.kr (B.C. Lee). 1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.04.008 Cell Biology International 30 (2006) 698e703 www.elsevier.com/locate/cellbi