In vitro induction of vitellogenin by estradiol 17 β in isolated hepatocytes of catfish, Clarias gariepinus

  • Rajendra Phartyal
  • L. Bibekananda Singh
  • S. V. Goswami
  • Neeta Sehgal
Research Article

Abstract

Vitellogenin is a female-specific calcium-binding glycolipophosphoprotein synthesized in the hepatocytes of fishes. Its synthesis can be induced in fishes of either sex by estradiol or by xenoestrogens. To study the in vitro synthesis of vitellogenin, different culture conditions were set up using the hepatocytes of Clarias gariepinus. The present study reports on a non-enzymatic procedure for isolation and culture of hepatocytes from the liver of the catfish Clarias gariepinus, in order to study the effects of estradiol on vitellogenin synthesis in vitro. The procedure employs chelating properties of ethylenediamine tetracetic acid to achieve cell viability in excess of 95%. Equal numbers of isolated cells were incubated in different culture media viz. RPMI F1640, Medium-199, and Williams’ Medium E. At 36 h, cell attachment and monolayer formation is faster in M-199 and Williams’ Medium E than in RPMI. In order to study the effects of estradiol on vitellogenin synthesis, the isolated hepatocytes were seeded in Williams’ Medium E in 24-well cell culture plates. 17 β-estradiol (E2) was introduced in the culture plates at different concentrations and for different time periods. The media were assayed for vitellogenin using competitive ELISA. Vitellogenin appeared in the medium after 48 h of incubation with 10−5 M estradiol whereas after 72 h of incubation 5×10−7 M E2 could elicit the synthesis.

Keywords

Hepatocytes Non-enzymatic isolation Primary culture Vitellogenin Clarias gariepinus 

References

  1. 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 105:407–416PubMedGoogle Scholar
  2. Flouriot G, Pakdel F, Valotaire Y (1996) Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression. Mol Cell Endocrinol 124:173–183CrossRefPubMedGoogle Scholar
  3. Flouriot G, Pakdel F, Ducouret B, Ledrean Y, Valotaire Y (1997) Differential regulation of two genes implicated in fish reproduction: vitellogenin and estrogen receptor genes. Mol Reprod Dev 48:317–323CrossRefPubMedGoogle Scholar
  4. Hayashi S, Ooshiro Z (1985) Primary culture of the freshly isolated liver cells of eel. Bull Jpn Soc Sci Fish 51:765–771Google Scholar
  5. Islinger M, Pawlowski S, Hollert H, Völkl A, Braunbeck T (1999) Measurement of vitellogenin-mRNA expression in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blot/RNAse protection-assay. Sci Total Environ 233:109–122CrossRefPubMedGoogle Scholar
  6. Kim BH, Takemura A (2002) In vitro vitellogenin synthesis in primary cultures of tilapia hepatocytes. Fish Sci 68:123–131CrossRefGoogle Scholar
  7. Kim BH, Takemura A (2003) Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes. Comp Biochem Physiol 135B:231–239CrossRefGoogle Scholar
  8. Komatsu M, Hayashi S (1998) Optimal conditions for estradiol-17β induced vitellogenin synthesis by cultured hepatocyte of eel Anguilla japonica. Fish Sci 64:658–659Google Scholar
  9. Kwon HC, Mugiya Y (1994) Involvement of growth hormone and prolactin in the induction of vitellogenin synthesis in primary hepatocyte culture in the eel, Anguilla japonica. Gen Comp Endocrinol 93:51–60CrossRefPubMedGoogle Scholar
  10. Kwon HC, Hayashi S, Mugiya Y (1993) Vitellogenin induction by estradiol-17β in primary hepatocyte culture in the rainbow trout, Oncorhynchus mykiss. Comp Biochem Physiol 104B:381–386Google Scholar
  11. Lethimonier C, Flouriot G, Valotaire Y, Kah O, Ducouret B (2000) Transcriptional interference between glucocorticoid receptor and estradiol receptor mediates the inhibitory effect of cortisol on fish vitellogenesis. Biol Reprod 62:1763–1771CrossRefPubMedGoogle Scholar
  12. Maitre J, Valotaire Y, Guguen-Guillouzo C (1986) Estradiol 17-β stimulation of vitellogenin synthesis in primary culture of male rainbow trout hepatocytes. In Vitro Cell Dev Biol 22:337–343CrossRefGoogle Scholar
  13. Mommsen TP, Moon TW, Walsh PJ (1994) Hepatocytes: isolation, maintenance and utilization. In: Hochachka PW, Mommsen TP (eds) Biochemistry and Molecular Biology of Fishes, vol 3. Elsevier Science, Amsterdam, pp 354–373Google Scholar
  14. Mori T, Matsumoto H, Yokota H (1998) Androgen-induced vitellogenin gene expression in primary cultures of rainbow trout hepatocytes. J Steroid Biochem Mol Biol 67:133–141CrossRefPubMedGoogle Scholar
  15. Peyon P, Baloche S, Burzawa-Gérard E (1997) Investigation into the possible role of androgens the induction of hepatic vitellogenesis in the European eel; in vivo and in vitro studies. Fish Physiol Biochem 16:107–118CrossRefGoogle Scholar
  16. Peyon P, Calvayrac R, Baloche S, Burzawa-Gérard E (1998) Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17β-estradiol and growth hormone. Comp Biochem Physiol 121A:35–44Google Scholar
  17. Seddon WL, Prosser CL (1999) Non-enzymatic isolation and culture of channel catfish hepatocytes. Comp Biocehm Physiol 123A:9–15CrossRefGoogle Scholar
  18. Segner H (1998) Isolation and primary culture of teleost hepatocytes. Comp Biochem Physiol 120A:71–81CrossRefGoogle Scholar
  19. Sehgal N, Goswami SV (1994) Steroidal effects on plasma vitellogenin levels in intact and hypophysectomized Indian freshwater murrel, Channa punctatus (Bloch). Ind J Exp Biol 32:387–392Google Scholar
  20. Smeets JMW, Rankouhi TR, Nichols KM (1999) In vitro vitellogenin production by carp (Cyprinus carpio) hepatocytes as a screening method for determining (anti)estrogenic activity of xenobiotics. Toxicol Appl Phamacol 157:68–76CrossRefGoogle Scholar
  21. Sumpter JP, Jobling S (1995) Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspect 103:173–178CrossRefPubMedGoogle Scholar
  22. Takemura A, Kim BH (2001) Effects of estradiol-17beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia. Comp Biochem Physiol 129A:641–651CrossRefGoogle Scholar
  23. Tyler CR, Sumpter JP, Bromage NR (1988) In vivo ovarian uptake and processing of vitellogenin in the rainbow trout, Salmo gairdneri. J Exp Zool 246:171–179CrossRefGoogle Scholar
  24. Vaillant C, Le Guellec C, Pakdel F, Valotaire Y (1988) Vitellogenin gene expression in primary culture of male rainbow trout hepatocytes. Gen Comp Endocrinol 70:284–290CrossRefPubMedGoogle Scholar
  25. Wallace RA, Selman K (1981) Cellular and dynamic aspects of oocyte growth in teleosts. Am Zool 21:325–343Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Rajendra Phartyal
    • 1
  • L. Bibekananda Singh
    • 1
  • S. V. Goswami
    • 1
  • Neeta Sehgal
    • 1
  1. 1.Department of ZoologyUniversity of DelhiDelhiIndia

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