Skip to main content

Advertisement

SpringerLink
Log in

Primary culture of medaka (Oryzias latipes) testis: a test system for the analysis of cell proliferation and differentiation

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

An in vitro test system using primary testis cells of the medaka (Oryzias latipes) was established that provides quantitative data on cell proliferation and spermatocyte differentiation. The primary cultures were characterised over a period of 2 days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained at a dynamic equilibrium in vitro for several days. The proliferating cells were predominantly present amongst the clusters of suspended cells as determined by BrdU labelling (cytological identification and quantification by ELISA). Based on cytological criteria the proliferating cells were mostly spermatogonia and preleptotene spermatocytes. Differentiation of spermatocytes to spermatids or spermatozoa was also observed mainly amongst suspended cells. Quantification of cell proliferation and cell differentiation by flow cytometry was achieved by labelling the primary cells with carboxyfluorescein diacetate N-succinimidyl ester, which allowed the identification and quantification of meiotically or mitotically dividing primary cells. Addition of the flavonoid genistein (10 µg/ml) to the primary cultures inhibited both cell proliferation and cell differentiation significantly. The test system is suitable for the study of the effect of substances which interfere with spermatogenesis in the vertebrate medaka model.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3a, b.
Fig. 4.
Fig. 5.
Fig. 6.

Similar content being viewed by others

References

  • Adams NR (1998) Natural and anthropogenic environmental oestrogens: the scientific basis for risk assessment, clover phyto-oestrogens in sheep in western Australia. Pure Appl Chem 70:1855–1862

    CAS  Google Scholar 

  • Adlercreutz H, Mazur W (1997) Phytooestrogens and Western diseases, the Finnish Medical Society DUODECIM. Ann Med 29:95–120

    CAS  PubMed  Google Scholar 

  • Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific kinases. J Biol Chem 262:5592–5595

    CAS  PubMed  Google Scholar 

  • Arai N, Ström A, Rafter JJ, Gustafsson JA (2000) Estrogen receptor β mRNA in colon cancer cells: growth effects of estrogen and genistein. Biochem Biophys Res Commun 270:425–431

    Article  CAS  PubMed  Google Scholar 

  • Barrett J (1996) Phytoestrogens. Friends or foes ? Environ Health Perspect 104:478–482

    Google Scholar 

  • Blottner S, Roelants H (1998) Quantification of somatic and spermatogenic cell proliferation in the testes of ruminants, using proliferation marker and flow cytometry analysis. Theriogenology 49:1275–1287

    Article  CAS  PubMed  Google Scholar 

  • Cappelletti V, Fioravanti L, Miodini P, Fronzo GD (2000) Genistein blocks breast cancer cells in the G2M phase of the cell cycle. J Cell Biochem 79:594–600

    Article  CAS  PubMed  Google Scholar 

  • Casanova M, You L, Gaido KW, Archibeque-Engle S, Janszen DB, Heck Hd'A (1999) Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors α and β in vitro. Toxicol Sci 51:236–244

    Google Scholar 

  • Chang CJ, Geahlen RL (1992) Protein-tyrosine kinase inhibition: mechanism-based discovery of antitumor agents. J Natl Prod 55:1529–1560

    CAS  Google Scholar 

  • Dey P, Mondal AK, Singh SK, Vohra H (2000) Quantification of spermatogenesis by DNA flow cytometry from fine-needle aspiration cytology material. Diagn Cytopathol 23:386–387

    Article  CAS  PubMed  Google Scholar 

  • Flouriot G, Vaillant C, Salbert G, Pelissero C, Guiraud JM, Valotaire Y (1993) Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates. J Cell Sci 105:407–416

    CAS  PubMed  Google Scholar 

  • Foran CM, Peterson BN, Benson WH (2002) Transgenerational and developmental exposure of Japanese medaka (Oryzias latipes) to ethinylestradiol results in endocrine and reproductive differences in the response to ethinylestradiol as adults. Toxicol Sci 68:389–402

    Article  CAS  PubMed  Google Scholar 

  • Han DH, Denison MS, Tachibana H, Yamada K (2002) Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogen and suppression by flavonoids. Biosci Biotechnol Biochem 66:1479–1487

    Article  CAS  PubMed  Google Scholar 

  • Kang IJ, Yokota H, Oshima Y, Tsuruda Y, Yamaguchi T, Maeda M, Imada N, Tadokoro H, Honjo T (2002) Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes). Chemosphere 47:71–80

    Article  CAS  PubMed  Google Scholar 

  • Kime DE (1998) Endocrine disruption in fish. Kluwer Academic, Boston

  • Kordes C, Rieber EP, Gutzeit HO (2002) An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes). Aquat Toxicol 58:151–164

    Article  CAS  PubMed  Google Scholar 

  • Kumi-Diaka J, Rodriguez R, Goudaze G (1998) Influence of genistein (4',5,7-trihydroxyisoflavone) on the growth and proliferation of testicular cell lines. Biol Cell 90:349–354

    Article  CAS  PubMed  Google Scholar 

  • Loir M, Sourdaine P (1994) Testes cells: isolation and culture. In: Hochachka PW, Mommsen TP (eds) Biochemistry and molecular biology of fishes, vol 3. Analytical techniques. Elsevier, Amsterdam, pp 249–272

  • Malkov M, Fisher Y, Don J (1998) Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol Reprod 59:84–92

    CAS  PubMed  Google Scholar 

  • Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M (2002) DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559–563

    Article  CAS  PubMed  Google Scholar 

  • Matsukawa Y, Marui N, Sakai T, Satomi Y, Yoshida M, Matsumoto K (1993) Genistein arrests cell cycle progression at G2-M. Cancer Res 53:1328–1331

    CAS  PubMed  Google Scholar 

  • Milligan SR, Kalita JC, Pocock V, Van de Kauter V, Stevens JF, Deinzer ML, Rong H, De Keukeleire D (2000) The endocrine activities of 8-prenylnarigenin and related hop (Humulua lupulus L.) flavonoids. J Clin Endocrinol Metab 85:4912–4915

    CAS  PubMed  Google Scholar 

  • Miura T, Miura C, Ohta T, Nader MR, Todo T, Yamauchi K (1999) Estradiol-17beta stimulates the renewal of spermatogonial stem cells in males. Biochem Biophys Res Commun 264:230–234

    Article  CAS  PubMed  Google Scholar 

  • Molteni A, Brizio-Molteni L, Persky V (1995) In vitro hormonal effects of soybean isoflavones. J Nutr Mar 125:751–756

    Google Scholar 

  • Parish CR (1999) Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 77:499–508

    Article  CAS  PubMed  Google Scholar 

  • Price KR, Fenwick GR (1985) Naturally occurring oestrogens in foods—a review. Food Addit Contam 2:73–106

    CAS  PubMed  Google Scholar 

  • Rickard SE, Thompson LU (1995) Phytoestrogens and lignans: effects on reproduction and chronic disease. In: Shahidi F (ed) Antinutrients and phytochemicals in food, chap 16. ACS Symposium Series 662. American Chemical Society, Washington, DC

  • Saiki A, Tamura M, Matsumoto M, Katowgi J, Watanabe A, Onitake K (1997) Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes. Dev Growth Differ 39:337–344

    Google Scholar 

  • Sakai N (2002) Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture. Development 129:3359–3365

    CAS  PubMed  Google Scholar 

  • Scholz S, Gutzeit HO (2000) 17-α-ethynylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes). Aquat Toxicol 50:363–373

    Article  CAS  PubMed  Google Scholar 

  • Scholz S, Gutzeit HO (2001) Lasting effects of xeno- and phytoestrogens on sex differentiation and reproduction of fish. Environ Sci 8:57–73

    CAS  Google Scholar 

  • Seki M, Yokota H, Matsubara H, Tsuruda Y, Meada M, Tadokoro H, Kobayashi K (2002) Effect of ethinylestradiol on the reproduction and induction vitellogenin and testis-ova in medaka (Oryzias latipes). Environ Toxicol Chem 21:1692–1698

    CAS  PubMed  Google Scholar 

  • Setchell KDR, Zimmer-Nechemias L, Cai J, Heubl JE (1997) Exposure of infants to phyto-oestrogens from soy-based infant formular. Lancet 350:23–27

    PubMed  Google Scholar 

  • Shinomiya A, Hamaguchi S, Shibata N (2001) Sexual differentiation of germ cell deficient gonads in the medaka, Oryzias latipes. J Exp Zool 290:402–410

    Article  CAS  PubMed  Google Scholar 

  • Shioda T, Wakabayashi M (2000) Effect of certain chemicals on the reproduction of medaka (Oryzias latipes). Chemosphere 40:239–243

    Article  CAS  PubMed  Google Scholar 

  • Song M, Gutzeit HO (2003) Effect of 17-α-ethynyloestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis. Dev Growth Differ (in press)

  • Spinozzi F, Cristina M, Migliorati G, Moraca R, Grignani F, Riccardi C, Nicoletti I (1994) The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in jurkat T-leukemia cells. Leukemia Res 18:431–439

    CAS  Google Scholar 

  • Tres LL, Kierszenbaum AL (1983) Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Dev Biol 80:3377–3381

    CAS  Google Scholar 

  • Wang C, Kurzer MS (1997) Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells. Nutr Cancer 28:236–247

    CAS  PubMed  Google Scholar 

  • Weinbauer GF, Schubert J, Yeung C-H, Rosiepen G, Nieschlag E (1998) Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry. J Endocrinol 156:23–34

    CAS  PubMed  Google Scholar 

  • Weston SA, Parish CR (1990) New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J Immunol Methods 133:87–97

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto TO (1975) Medaka (Killifish): biology and strains. Kaigaku Publishing Co., Tokyo

Download references

Acknowledgements

We wish to thank D. Zschernig, Y. Henker, and N. Bachmann for their technical assistance. The help of Dr. S.V. Tokalov with the flow cytometry analysis is acknowledged. The flow cytometer was provided with a grant from the State of Saxony.

Author information

Authors and Affiliations

  1. Institute of Zoology, TU Dresden, Mommsenstrasse 13, 01062, Dresden, Germany

    Miyeoun Song & Herwig O. Gutzeit

Authors
  1. Miyeoun Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Herwig O. Gutzeit
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Herwig O. Gutzeit.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Song, M., Gutzeit, H.O. Primary culture of medaka (Oryzias latipes) testis: a test system for the analysis of cell proliferation and differentiation. Cell Tissue Res 313, 107–115 (2003). https://doi.org/10.1007/s00441-003-0729-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-003-0729-x

Keywords

Search

Navigation