Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, Clarias batrachus (L.)
Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E2)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml−1 for Vg1 and 40 ng ml−1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E2 induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.
KeywordsVitellogenin ELISA Catfish Clarias batrachus
This research was supported in part by grants from ICAR (F. No. 4(20)2002-ASR-I) and DST (F. No. SR/SO/AS-31/2003), New Delhi sanctioned to Dr. P. Nath and by the UGC sponsored CAS program to Department of Zoology, Visva-Bharati University, Santiniketan.
Compliance with ethical standards
The experiments conducted on the live fish were performed following all the rules and regulations of the Institutional Animal Ethics Committee of Visva-Bharati University.
- Debnath S (2011) Clarias batrachus, the medicinal fish: an excellent candidate for aquaculture & employment generation. International Conference on Asia Agriculture and Animal, IPCBEE, IACSIT Press, Singapoore vol. 13Google Scholar
- Ding JL, Hee PL, Lam TJ (1989) Two forms of vitellogenin in the plasma and gonads of male Oreochromis aureus. Comp Biochem Physiol 93B:363–370Google Scholar
- Hiramatsu N, Cheek AO, Sullivan CV, Matsubara T, Hara A (2005) Vitellogenesis and endocrine disruption. In: Mommsen TP, Moon T (eds) Biochemistry and molecular biology of fishes. Elsevier Science Press, Amsterdam, pp. 431–471Google Scholar
- LaFleur GJ Jr, Byrne BM, Haux C, Greenburg RM, Wallace RA (1995a) Liver-derived cDNAs: vitellogenins and vitelline envelope protein precursors (choriogenins). In: Goetz WF, Thomas P (eds) Reproductive physiology of fish. The University of Texas at Austin, Texas, pp. 505–521Google Scholar
- Matsubara T, Ohkubo N, Andoh T, Sullivan CV, Hara A (1999) Two form of vitellogenin yielding two distinct lipovitellins, play vital roles during oocyte maturation early development of barfin flounder, Verasper moseri, a marine teleost that spawns pelagic eggs. Dev Biol 213:18–32CrossRefPubMedGoogle Scholar
- Mommsen TP, Walsh PJ (1988) Vitellogenesis and oocyte assembly. In: Hoar WS, Randall DJ (eds) Fish physiology. Academic, New York, pp. 347–406Google Scholar
- Nath P (1999) Some aspects of teleost vitellogenesis. In: Saksena DN (ed) Ichthyology: recent advances. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi, pp. 249–259Google Scholar
- Ndiaye P, Forgue J, Lamothe V, Cauty C, Tacon P, Lafon P, Davail B, Fostier A, Le Menn F, Nunez J (2006) Tilapia (Oreochromis niloticus) vitellogenins: development of homologous and heterologous ELISAs and analysis of vitellogenin pathway through the ovarian follicle. J Exp Zool 305A:576–593CrossRefGoogle Scholar
- Sawaguchi S, Kagawa H, Ohkubo N, Hiramatsu N, Sullivan CV, Matsubara T (2006) Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs. Mol Reprod Dev 73:719–736CrossRefPubMedGoogle Scholar
- Sawaguchi S, Koya Y, Yoshizaki N, Ohkubo N, Andoh T, Hiramatsu N, Sullivan CV, Hara A, Matsubara T (2005) Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional vg genes and their circulating and yolk protein products. Biol Reprod 72:1045–1060CrossRefPubMedGoogle Scholar
- Sawaguchi S, Ohkubo N, Amano H, Hiramatsu N, Hara A, Sullivan CV, Matsubara T (2008) Controlled accumulation of multiple vitellogenins into oocytes during vitellogenesis in the barfin flounder, Verasper moseri. Cybium: Int J Ichthyol 32(suppl 2):262Google Scholar
- Selman K, Wallace RA (1989) Cellular aspects of oocyte growth in teleosts. Zool Sci Tokyo 6:211–231Google Scholar
- Snedecor W, Cochran G (1957) Statistical methods. Iowa University Press, Ames, IA, pp. 481–488Google Scholar
- Specker JL, Sullivan CV (1994) Vitellogenesis in fish: status and perspectives. In: Davey KG, Peter RE, Tobe SS (eds) Perspectives in comparative endocrinology. National Research Council of Canada, Ottawa, pp. 304–315Google Scholar
- Umbreit WN, Burris RH, Stauffer JF (1958) Manometric techniques, 3rd edn. Burgess Publishing Company, Minneapolis, pp. 272–279Google Scholar