title: Quantitative bioassays for measuring biologically functional gonadotropins based on eel gonadotropic receptors.
abstract: Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture.
authors.affiliation: Institute of Biology Leiden, Sylvius Laboratory, Leiden University, Leiden, The Netherlands. y.minegishi@biology.leidenuniv.nl
authors.forename: Y
authors.initials: Y
authors.lastname: Minegishi
authors.forename: R P
authors.initials: RP
authors.lastname: Dirks
authors.forename: D L
authors.initials: DL
authors.lastname: de Wijze
authors.forename: S A
authors.initials: SA
authors.lastname: Brittijn
authors.forename: E
authors.initials: E
authors.lastname: Burgerhout
authors.forename: H P
authors.initials: HP
authors.lastname: Spaink
authors.forename: G E E J M
authors.initials: GE
authors.lastname: van den Thillart
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000378:metabolism; D004524:Eels / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006062:Gonadotropins / Q000378:metabolism*; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism
chemical_list: D006063:Chorionic Gonadotropin; D006062:Gonadotropins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone