Purification of follicle-stimulating hormone from immature Japanese eel, Anguilla japonica, and its biochemical properties and steroidogenic activities

Abstract

Follicle-stimulating hormone (FSH) was purified, for the first time, from immature Japanese eel, Anguilla japonica, and its biochemical properties were investigated. FSH was extracted from immature eel pituitaries and purified by gel-filtration on Sephadex G-100, and two step anion-exchange chromatography: stepwise elution on DE-52, followed by gradient elution on TSK-gel Super-Q using HPLC. Purification was performed using its molecular mass and the positive reaction with anti-Japanese eel (je) FSHβ antiserum. Purified eel FSH was detected as a single band after separation by SDS–PAGE under a non-reducing condition, showing positive reaction with both anti-je glycoprotein (GP) α and anti-jeFSHβ antisera. The molecular mass of purified eel FSH was estimated to be approximately 33 kDa. After separation by SDS–PAGE under reducing condition, the intact molecule was detected as distinct proteins, whose N-terminus amino acid sequences coincided with those predicted from cDNA sequences for jeGPα and jeFSHβ mature peptides. Deglycosylation of these subunits led to a decrease in their molecular mass. These results suggest that eel FSH is a heterodimeric molecule which consists of distinct glycoprotein subunits, GPα and FSHβ. Cells reacting with anti-jeFSHβ antiserum were observed in the proximal pars distalis of an immature eel pituitary, while jeLHβ-immunoreactive cells were not detected. Gonadotropic activities of eel FSH were demonstrated in vitro by stimulating testosterone and 11-ketotestosterone secretions in immature eel testes. Purified eel FSH stimulated the secretion of both androgens from the immature eel testis in a dose-dependent manner, similar to immature eel pituitary homogenate and recombinant eel FSH produced by yeast. These results show that endogenous and recombinant FSH in this species possess similar activities, presumably stimulating the gametogenesis through the sex steroid secretion during the early stages of gonadal development.