Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays
2004
Lin Yu-Wai Peter | Petrino Teresa | Wallace Robin
Abstract Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (M<sub>r </sub>~ 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E<sub>2</sub>)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E<sub>2 </sub>synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E<sub>2</sub>-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.
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