Ecdysteroids in Eggs of Periplaneta Americana: Rapid Analysis Using 3µ C18 Columns and the Binding of Ecdysteroids to Vitellin

Abstract

The titres of free, polar and apolar conjugates of ecdysteroids during embryonic development of the cockroach, Periplaneta americana, were determined by radioimmunoassay (RIA). The different classes of ecdysteroid were resolved by both open column chromatography and reversed-phase high performance liquid chromatography (HPLC). HPLC using short 3µ C18 column and a gradient of acetonitrile in water permitted the rapid analysis of crude methanolic extracts without the need for prior clean-up procedures. Apolar ecdysteroid esters present in newly-laid oothecae were metabolized during early embryogenesis to release free ecdysteroids and to generate polar conjugates. Titres of free and polar compounds declined at around day 20 with the polar conjugates remaining at a low level for the remainder of embryogenesis. A large peak of free ecdysteroid (mainly 20-hydroxyecdysone) appeared at day 33 which, being 8 days before hatching, is likely to be involved in the formation of the first larval cuticle. At this late stage of development a new group of apolar ecdysteroids, probably inactivation metabolites, appeared and increased in importance as the free hormone titre declined. The maternal apolar esters present at day 0 were bound to vitellin which was degraded to low molecular weight polypeptides by day 20. This is coincident with the metabolism of the maternal esters to release free hormone.