The fate of radiolabeled steroids in ovaries and eggs of the tobacco hornworm,Manduca sexta

Abstract

To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco hornworm,Manduca sexta. Following injection of [(14)C]cholesterol, 22,25[(14)C]dideoxyecdysome or [(3)H]ecdysone into female pupae (day 16), only [(14)C]cholesterol was incorporated and metabolized. It was converted to labeled nonecdysteroid and ecdysteroid conjugates, of which the latter in ovaries and 48- to 64-hr-old eggs is mainly 26-hydroxyecdysone 26-phosphate (>85% in ovaries). Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography (RP-HPLC) showed that the levels of 26-hydroxyecdysone 26-phosphate were 31 μg/g of ovaries from 4-day-old females and 25 μg/g and 17 μg/g of 48- to 64-hr-old and 72- to 88-hr-old eggs, respectively. The RP-HPLC of the conjugate fraction of 48- to 64-hr-old eggs showed an additional peak of radioactive material eluting about three min before the 26-hydroxyecdysone 26-phosphate. The quantity of this material increased in the 72-to 88-hr-old eggs, though it was not detected in the analyses of the conjugate fraction from ovaries. Additional peaks of radioactive material eluting before the 26-hydroxyecdysone 26-phosphate peak were observed in the chromatogram of the conjugates of 72- to 88-hr-old eggs. These radioactive materials need to be identified to determine the ultimate fate of ecdysteroids in the developing embryos of the tobacco hornworm. No radioactive free ecdysteroids were detected in either egg age group.

Isolation and identification of 26‐hydroxyecdysone 2‐phosphate: An ecdysteroid conjugate of eggs and ovaries of the tobacco hornworm, Manduca sexta

“…During embryogenesis, the level of radiolabeled 26-hydroxyecdysone 26-phosphate decreases, and there is a concurrent increase in the amount of a new labeled ecdysteroid conjugate [9]. The level, however, of this minor conjugate remains rather constant throughout development to 72-88-h-old eggs [9]. In this paper we report the isolation and unequivocal identification of this minor ecdysteroid conjugate from 48-64-h-old M .…”

“…By injection of [14C]cholesterol into M. sexta female pupae prior to synthesis of ovarian ecdysteroids, it was possible to obtain labeled ovarian and egg ecdysteroids [9]. The RP-HPLC and radioassay analyses of the ecdysteroid conjugates of ovaries showed a radioactive peak preceding the peak of 26-hydroxyecdysone 26-phosphate (Fig.…”

“…sexta pupae (day 16), [14C]cholesterol is largely converted to a radiolabeled nonecdysteroid glucoside conjugate [8] as well as ecdysteroid conjugates. RP-HPLC analyses indicate that the conjugates of ovaries of the 4-day-old adult females consist mainly of labeled 26-hydroxyecdysone 26-phosphate [9] and a less abundant ecdysteroid conjugate which is here identified as 26-hydroxyecdysone 2-phosphate (Fig. 1A).…”

“…Tobacco hornworm eggs were extracted and processed as described pre- or on an IBM Cg column (4.6 mm x 15 cm; Danbury, CT) by isocratic elution with 26% methanol in 0.03 M aqueous NaHzP04 solution [9]. The use of the NaH2P04 solution suppresses the ionization of the ecdysteroid conjugates so that they are in the form of the nonionic species.…”

“…As the moths emerged the females were kept in plastic crispers and allowed to develop until ovaries were excised, stored in methanol at -2O"C, and processed as previously described [9].…”