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 micrograms/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 micrograms/microliter. 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