Impaired ovarian ecdysone synthesis of <i>Drosophila melanogaster</i> insulin receptor mutants

Deficient juvenile hormone synthesis is thought to extend adult longevity in insects, including Drosophila melanogaster, the Monarch butterfly and several species of Mediterranean grasshopper (Pener, 1972; Herman & Tatar, 2001; Tatar & Yin, 2001). In the monarch and the grasshopper, surgical removal of the adult JH synthetic tissue (corpora allata) mimics adult diapause while it eliminates reproduction and increases longevity. In D. melanogaster, adult diapause also is favoured by reduced JH and is associated with suppressed reproduction and slow aging (Tatar et al., 2001a). Aspects of this diapause syndrome are recapitulated in D. melanogaster mutant for the insulin-like receptor (InR), which are conditionally non-reproductive, long-lived and deficient in JH (Tatar et al., 2001b). In each system sketched above, JH, a mere sesquiterpenoid, was demonstrated to have a direct effect upon aging because treatment with the hormone restored reproductive activity and normal longevity. Juvenile hormone is but one of the major endocrine factors found in adult insects. Other endocrines include the steroids α-ecdysone and its active form 20-hydroxyecdysone (20-HE). Both JH and 20-HE are best understood as regulators of development, but in the adult they have been studied primarily as they affect oogenesis and vitellogenesis (recent reviews in Kozlova & Thummel, 2000; Truman & Riddiford, 2002). These detailed studies show that JH and ecdysone work in tandem. Indeed, the nuclear hormone receptor for ecdysone, EcR, dimerizes with the nuclear receptor ultraspirical (USP) which has JH as its most likely ligand (Jones & Sharp, 1997; Jones et al., 2002). Although the potential impact of ecdysone upon aging has yet to be reported, from basic studies of insulin function we predict that ovaries of long-lived D. melanogaster mutant for InR will produce little ecdysone. In D. melanogaster, insulin signal was found to mediate ovarian stem cell proliferation in response to diet (Drummond-Barbosa & Spradling, 2001), and with fewer eggs, total ecdysone production may be reduced. In another dipteran, the mosquito, ovaries produce ecdysone following a blood meal and this synthesis is insulin dependent; ovaries of sugar-fed females produce ecdysone when stimulated by insulin, and they do so in a PI3-kinase, PKB/AKT, PIP3-dependent manner (Riehle & Brown, 1999). Through either avenue, hypomorphism of InR should affect titres of adult ecdysone. In the initial course of characterizing the longevity-extending genotypes of InR (Tatar et al., 2001b), we investigated the ecdysone production of ovaries from females possessing InR+, InRE19 and InRp5545 alleles. Here we report our preliminary findings. Although limited in scope, the data clearly show that reduced ecdysone synthesis is a phenotype of InR mutant alleles. From these simple data we hope to stimulate, perhaps in others, an effort toward detailed investigation of ecdysone action upon aging. In adult females the active form of ecdysone, 20-HE, is produced by follicle cells of the egg, and to a lesser extent by other tissues (Delbecque et al., 1990). We assessed ovarian synthesis of total ecdysteroids (α-ecdysone and 20-HE) as a function of InR genotype: InR+/+, InR+/p5545, InR+/E19 which have normal growth and longevity, InRE19/E19 which has reduced growth but normal life span, and InRE19/p5545 which is dwarf and long-lived. At the time of these measures, contemporaneous with the work reported in Tatar et al. (2001b), the alleles had been back-crossed 10 generations to the wild-type isogenic control (ri, red, e, InR+). Ovaries in sets of 20 were dissected from virgin females at 6-h intervals within the first 72 h post-eclosion and incubated in buffered Ephrussi–Beadle Ringers media for 5 h (Warren & Mahowald, 1979). In cases where low ecdysteroids were synthesized, 40–80 pairs of ovaries were pooled together for measurement. In general, three to six replicate sets were assessed for each time point and genotype. Edysteroids were measured by RIA following Bollenbacher et al. (1975); anti-ecdysone antiserum was kindly provided by Gilbert and Bollenbacher (University of North Carolina). The assay was linear in the range of 10–1000 pg using α-ecdysone (Sigma) as standard. Ecdysteroid synthesis (Fig. 1) was minimal in the ovaries of both dwarf genotypes and could be detected only after 48 h post-eclosion (timing evaluated with InRp5545/E19). Ovaries of wild-type and heterozygotes, in contrast, produced up to 90 pg per incubation, and with strong detection as early as 18 h post-eclosion. The age-dependent kinetics of ecdysone synthesis from ovaries of heterozygotes was significantly delayed relative to the wild-type females, despite the homogeneity of body size and developmental rate among these genotypes (Tatar et al., 2001b). Ovarian production of ecdysteroids from virgin Drosophila melanogaster females. At each datum, ecdysteroid synthesis was determined relative to α-ecdysone standard (Sigma) after 5 h incubation of at least 20 ovaries. Replicate means with one standard deviation are plotted. Factorial anova was used to assess differences in the cumulative and the time distribution of ecdysteroid produced by InR+/+ relative to InR−/+ (InRE19 and InRp5545 data combined). The genotypes differed only by 6.84 pg in overall ecdysteroid synthesis (P = 0.054); genotype-by-hour interaction was large and significant (F = 105.0, P < 0.0001). Ecdysteroid synthesis is reduced in ovaries of InR mutant females. Egg maturation is retarded or almost absent in the dwarf genotypes (Tatar et al., 2001b), perhaps as a direct result of hypomorphic insulin signal within the ovary (Drummond-Barbosa & Spradling, 2001). Few ecdysone-capable follicles are produced. InR hypomorphism may also directly impede activation of follicle hormone synthesis (Riehle & Brown, 1999). It would be useful at this stage to challenge the ovaries of dwarf genotypes with insulin in an in vitro assay, as was done for