Regulation of Ovarian Ecdysteroid Production in the Housefly, Musca domestica

Highly purified Musca ecdysteroidogenin (ESG) was obtained by extracting housefly heads with an acid-ethanol-urea procedure followed by desalting on a Sephadex G25F column and fractionation on a Sephadex G50F column. Further fractionation was done on a semi-preparative C18 reverse-phase HPLC column with an acetonitrile gradient in 0.1% trifluoroacetic acid in water. Ecdysteroidogenic activity eluted from the column with 35% acetonitrile. I-125 size exclusion HPLC revealed activity in a fraction with a molecular mass of 8.1 kDa. ESG from the C18 column at a concentration of 1.1 μg/fly stimulated ovarian development to late vitellogenesis when injected into flies without the corpus allatum–cardiacum complex and stimulated maximal ovarian ecdysteroid production in vitro at a concentration of 40 μg/μl. Concentrations of ESG outside these ranges were less effective. Ovaries produced about 500 pg each of ecdysone, 20-hydroxyecdysone, and makisterone A when exposed to ESG in vitro. However, control ovaries produced about a tenth the amount of ecdysone and 20-hydroxyecdysone and half the amount of makisterone A. An exposure of 4 h or more to ESG was required by ovaries before they would make ecdysteroid, with the greatest rate of ecdysteroid production occurring in the 12–24 h interval of exposure. Ecdysteroid production ceased from 36–50 h after ESG was removed from the medium. A juvenile hormone analog (JHA) did not stimulate ovarian ecdysteroid production, but ovaries from flies without the corpus allatum–cardiacum complex required JHA to produce high levels of ecdysteroid during incubation with ESG. ESG activity was recovered from both male and female heads. Arch. Insect Biochem. Physiol. 35:135–148, 1997. © 1997 Wiley-Liss, Inc.