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Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

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An Erratum to this article was published on 08 January 2013

Abstract

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.

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Acknowledgments

The authors thank Dr. Koji Ninomiya and Munezi Uziie, Shiga Prefectural Fisheries Experimental Station, for providing Honmoroko (Gnathopogon caerulescens). This work was funded in part by a Grant-in-Aid for challenging Exploratory Research (23651248 to T.T.), Scientific Research on Priority Area (21028021 to T.T.), and Young Scientists (B) (23700511 to S.H.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. This study was also supported by NIG Collaborative Research Program (2010-A41, 2011-A36, 2012-A28 to T.T.) and Ritsumeikan Global Innovation Research Organization (R-GIRO).

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Authors and Affiliations

  1. Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, Shiga, 525-8577, Japan

    Shogo Higaki, Yoshie Koyama & Tatsuyuki Takada

  2. Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, Shiga, 525-8577, Japan

    Emi Shirai, Tomoya Yokota & Tatsuyuki Takada

  3. Lake Biwa Museum, Oroshimo 1091, Kusatsu, Shiga, 525-0001, Japan

    Yasuhiro Fujioka

  4. Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan

    Noriyoshi Sakai

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  1. Shogo Higaki
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  2. Yoshie Koyama
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Correspondence to Tatsuyuki Takada.

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Higaki, S., Koyama, Y., Shirai, E. et al. Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens). Fish Physiol Biochem 39, 701–711 (2013). https://doi.org/10.1007/s10695-012-9733-y

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