Development and validation of enzyme-linked immunosorbent assays specific for follicle-stimulating hormone and luteinizing hormone in Japanese eel
Two types of gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), are primary regulators of reproduction in vertebrates, including teleost fish. In this study, recombinant Japanese eel Gths (reGth; reFsh and reLh), and recombinant chimeric Gths (rcGth; rcFsh and rcLh), consisting of an eel β-subunit with rabbit glycoprotein hormone α-subunit, were produced as single-chain proteins, using a mammalian expression system and subsequently highly purified. Antibodies raised against rcGths were affinity purified with the corresponding reGth, and specific enzyme-linked immunosorbent assays (ELISAs) for Japanese eel Fsh and Lh were developed using these antibodies for the first time. The ELISAs were sensitive and parallelism was seen between the standard curve and serial dilutions of Japanese eel serum and pituitary extract (PE). Contents of Fsh and Lh protein in the pituitaries of the female Japanese eel at the pre-vitellogenic stage were comparable and the Lh contents at the migratory nucleus stage were highly induced during artificial induction of maturation by injections of salmon PE. Furthermore, the accumulated Lh was released at a high level by administration of a Lh-releasing hormone analogue and pimozide. Recombinant Gths and homologous ELISAs established for Japanese eel may be useful tools for studying reproductive eel biology.
KeywordsGonadotropin Reproductive regulation Anguilla japonica Maturation
We would like to express our appreciation to the staff of the National Research Institute of Aquaculture for the maintenance of the fish. This research was supported by the Ministry of Science and Technology (grant no. 22580219).
Funding was received from the Ministry of Science and Technology (grant no. 22580219).
- Dufour S, Lopez E, Le Menn F, Le Belle N, Baloche S, Fontaine YA (1988) Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol. Gen Comp Endocrinol 70:20–30CrossRefGoogle Scholar
- Kazeto Y, Kohara M, Miura T, Miura C, Yamaguchi S, Trant JM, Adachi S, Yamauchi K (2008) Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology. Biol Reprod 79:938–946CrossRefGoogle Scholar
- Kim D, Suzuki Y, Aida K (2011) The control mechanism of gonadotropin-releasing hormone and dopamine on gonadotropin release from cultured pituitary cells of rainbow trout Oncorhynchus mykiss at different reproductive stages. Fish Aquat Sci 14:379–388Google Scholar
- Matsuyama M, Kagawa H, Takeuchi H, Kashiwagi M, Iwai T, Hirose K (1992) Effect of LHRH-a cholesterol pellet on gonadal development, maturation and spawning in Pagrus major during winter season. Aquacult Sci 40:159–165Google Scholar
- Montero M, Le Belle N, Vidal B, Dufour S (1996) Primary cultures of dispersed pituitary cells from estradiol-pretreated female silver eels (Anguilla anguilla L.): immunocytochemical characterization of gonadotropic cells and stimulation of gonadotropin release. Gen Comp Endocrinol 104:103–115CrossRefGoogle Scholar
- Nagae M, Todo T, Gen K, Kato Y, Young G, Adachi S, Yamauchi K (1996) Molecular cloning of the cDNAs encoding pituitary glycoprotein hormone alpha- and gonadotropin II beta-subunits of the Japanese eel, Anguilla japonica, and increase in their mRNAs during ovarian development induced by injection of chum salmon pituitary homogenate. J Mol Endocrinol 16:171–181CrossRefGoogle Scholar
- Saito K, Lokman PM, Young G, Ozaki Y, Matsubara H, Okumura H, Kazeto Y, Yoshiura Y, Aida K, Adachi S, Yamauchi K (2003) Follicle-stimulating hormone beta, luteinizing hormone beta and glycoprotein hormone alpha subunit mRNA levels in artificially maturing Japanese eel Anguilla japonica and naturally maturing New Zealand longfinned eel Anguilla dieffenbachii. Fish Sci 69:146–153CrossRefGoogle Scholar
- Tachiki H, Nakgawa T, Tamura K, Hirose K (1997) Effects of oral Administration of estradiol-17β to young on gonadal sex and growth of Japanese eel (Anguilla japonica). Suisanzoshoku 45:61–66 (In Japanese with English abstract) Google Scholar
- Yamauchi K, Yamamoto K (1982) Experiments on artificial maturation and fertilization of the Japanese eel (Anguilla japonica). In: Richer CJ, Goos GJTh (eds) Reproductive physiology of fish. Pudoc Press, Wargeningen, pp 185–189Google Scholar