Evidence for the involvement of 3-oxo-Delta(4) intermediates in ecdysteroid biosynthesis

Although the involvement of 3-oxo-Delta(4) compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-Delta(4)-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-Delta(4) compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-Delta(4)-steroid 4,7-cholstadien-14 alpha-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-Delta(4) substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-Delta(4)-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.