Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol

Abstract

AbstractThe levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1‐h old contain about 2.5 and 35 μ/g of the 2‐ and 26‐phosphates of 26‐hydroxyecdysone, respectively, and 1 μg/g of 26‐hydroxyecdysone. During embryogenesis of 26‐hydroxyedcdysone 26‐phosphate is hydrolyzed to 26‐hydroxyecdysone, which reaches a peak titer in 1–18‐h‐old eggs; the level of 26‐hydroxyecdysone 2‐phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26‐hydroxyecdysone 26‐phosphate decreases there is a progression of other ecdysteroids. C‐20 hydroxylation first appears in 24–40‐h‐old eggs and reaches peak activity in 48–64‐h‐old eggs, where 20‐hydroxyecdysone and 20, 26‐dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h‐old. C‐3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3‐epi‐26‐hydroxyecdysone and 3‐epi‐20, 26‐dihydroxyecdysone are the major free ecdysteroids in 96‐h‐old eggs. A new ecdysteroid conjugate, 26‐hydroxyecdysone 22‐glucoside, first appears at 24–40h and becomes the major conjugate in 72–80‐h‐old eggs; it represents an apparent end‐product as its peak titer is reached and maintained throughout the final embryonic stages. 20‐Hydroxyecdysonoic acid occurs in 48–64‐h‐old eggs, and along with 3‐epi‐20‐hydroxyecdysonoic and ecdysonoic acids in 72–88‐h‐old eggs. 20‐Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3‐epi‐20‐hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1‐h‐old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26‐hydroxyecdysone 26‐phosphate serves as source for 26‐hydroxyecdysone and the numerous metabolites; that 26‐hydroxyecdysone and 20,26‐dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26‐phosphate in the developing eggs of m. sexta is presented.