In vitro induction of vitellogenin by estradiol
17 β in isolated hepatocytes of catfish, Clarias gariepinus |
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Authors: | Rajendra Phartyal L Bibekananda Singh S V Goswami Neeta Sehgal |
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Institution: | (1) Department of Zoology, University of Delhi, Delhi, 110 007, India |
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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. |
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Keywords: | Hepatocytes Non-enzymatic isolation Primary culture Vitellogenin Clarias gariepinus |
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