Biosynthesis of α-Ecdysone by Prothoracic Glands in vitro

Chino, Haruo; Sakurai, Shigeki; Ohtaki, Tetsuya; Ikekawa, Nobuo; Miyazaki, Hiroshi; Ishibashi, Masataka; Abuki, Hideo

doi:10.1126/science.183.4124.529

Cited by 162 publications

(19 citation statements)

References 16 publications

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“…The medium was then analyzed for ma- ¶ Under assay conditions, synthesis is maximal [12][13][14][15][16][17][18] hr after hormone stimulation (Bohm, unpublished observation). [3H]Phenylalanine was then added for a further 3 hr and synthesized vitellogenin was assayed as described in Fig.…”

Section: Resultsmentioning

confidence: 99%

The ovary as a source of alpha-ecdysone in an adult mosquito.

Hagedorn¹,

O’Connor²,

Fuchs³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

317

154

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The ovaries of the mosquito Aedes aegypti cultured in vitro secrete material that behaves like ecdysone in a radioimmunoassay. The material was identified as aecdysone by high-resolution liquid and gas-liquid chromatography. Secretion reached a maximum 16 hr after a blood meal as shown by bioassay and direct determination. Ovariectomy reduced the concentration of ecdysone in the adult after a blood meal. Qualitative analysis of whole-body extracts indicated fi-ecdysone to be the principal species present. Thus the ovaries appear to secrete a prohormone, a-ecdysone, which is converted to,-ecdysone. ,-ecdysone plays a significant role in stimulating egg development in the adult mosquito and may have reproductive roles in other insects.In mosquitoes the blood meal triggers egg development. This process, which has been found to be quite complex (1), involves the elaboration of yolk for later use by the developing embryo. The major proteins which become yolk are synthesized by the fat body, secreted into the hemolymph, and selectively taken up by the developing oocyte (2, 3). These proteins have been termed vitellogenins (4). Recent investigations into the control of this process showed that, in contrast with most insects, the mosquito ovary was the source of a hormonal factor which activated and maintained vitellogenin synthesis by the fat body (5). Thus synthesis by the fat body was abolished by ovariectomy and restored by reimplantation. Further, when ovaries from blood-fed females were incubated in vitro with the inactive fat body from unfed females, synthesis of vitellogenin was activated. This ovarian hormone was designated the vitellogenin stimulating hormone, VSH (1).The discovery that injected f3-ecdysone stimulates egg development (6) and dopa decarboxylase activity (7) in the absence of a blood meal led us to investigate the possibility that VSH might be f3-ecdysone. Our results indicated that f3-ecdysone mimicked VSH in vivo and in vitro (8) and that material with ecdysone-like activity could be detected in mosquitoes after a blood meal (9). These lines of evidence strongly suggested that VSH was fl-ecdysone or a closely related steroid. We here describe experiments designed to identify the secretory product of the ovary. METHODSMosquitoes (Aedes aegypti) were reared at 27 + 0.50 using standard techniques (8, 9). The assay for vitellogenin synthesis has been described in detail (1, 8). The radioimmunoassay (RIA) was performed as previously described (10, 11).Ovaries were removed from females 15 hr after a rabbit blood meal and incubated at 250 with gentle shaking in groups of 100 in 100,ul of a defined medium (8). After 24 hr the medium was removed and the ovaries were rinsed with saline. An equal volume of methanol was added to the combined medium and rinse. Particulates were removed by filtration. The extract was evaporated to dryness under reduced pressure, redissolved in chloroform:methanol (2:1) and partitioned against water according to Folch et al. (12). The epiphase was dried under vacuum ...

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Section: Resultsmentioning

confidence: 99%

The ovary as a source of alpha-ecdysone in an adult mosquito.

Hagedorn¹,

O’Connor²,

Fuchs³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

317

154

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“…The binding curve for solutions of crystalline a-ecdysone (Simes, Milano) was log-linear between 25 and 500 pg of ecdysone (r = 0.999). Although the product of PG is a-ecdysone (21,22), the antibody was crossreactive with other ecdysteroids. Consequently, all data are expressed as a-ecdysone equivalents.…”

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confidence: 99%

Spontaneous and evoked release of prothoracicotropin from multiple neurohemal organs of the tobacco hornworm

Carrow

Calabrese

Williams

1981

Proc. Natl. Acad. Sci. U.S.A.

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Release ofneurohormone from putative cephalic neurohemal organs was directly demonstrated in an insect. The prothoracicotropic hormone (PTTH) of the tobacco hornworm, Manduca sexta, was measured indirectly by its ability to stimulate the secretion of a-ecdysone by inactive prothoracic glands; the ecdysone was measured by radioimmunoassay. The PTTH released spontaneously from intact brain-retrocerebral complexes was localized to the retrocerebral complex by placing a waxy barrier across the nerves connecting the corpora cardiaca to the brain. Isolated corpora allata spontaneously released much more PTTH than did either isolated corpora cardiaca or isolated brains. Media containing 100 mM potassium stimulated PTTH release from both isolated corpora allata and isolated corpora cardiaca. In calcium-free media, spontaneous PTTH release was diminished and release could not be stimulated by high potassium. These results indicate that depolarization ofthe neurosecretory cells is correlated with calcium-dependent neurohormone release and that there are multiple neurohemal organs for PITH. The biological activities of stored and circulating PTTH are compared.

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“…Recent studies have established the role of the prothoracic glands in the secretion of ecdysone during larval development of insects [1,2]. This steroid is however not the major moulting hormone of insect larvae.…”

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confidence: 99%

Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

Durst

1978

Eur J Biochem

128

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Ecdysone 20-monooxygenase, an enzyme which convem ecdysone to ecdysterone (the major moulting hormone of insects) has been characterized in cell-free preparations of tissues from African migratory locust. The product of the reaction has been identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods.Ecdysone 20-monooxygenase activity is located primarily in the microsomal fraction which also carries NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation.Optimal conditions for the ecdysone 20-monooxygenase assay have been determined. The enzyme has a K,,, for ecdysone of 2.7 x lo-' M and is competitively inhibited by ecdysterone Ecdysone 20-monooxygenase is a typical cytochrome P-450 linked monooxygenase : the reaction requires O2 and is inhibited by CO, an effect partially reversed by white light. The enzyme is effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicits a type I difference spectrum when added to oxidized microsomes.NADPH acts as preferential electron donor. The transfer of reducing equivalents proceeds through NADPH cytochrome c (P-450) reductase : ecdysone 20-monooxygenase is inhibited by cytochrome c. Both NADPH cytochrome c reductase and' ecdysone 20-monooxygenase are inhibited by NADP' and show a similar K, for NADRH.The Malpighian tubules have the highest specific activity of ecdysone 20-monooxygenase, while fat body contain most of the cytochrome P-450 and NADPH cytochrome c reductase.( K~ = 7.5 x 10-7 M).Recent studies have established the role of the prothoracic glands in the secretion of ecdysone during larval development of insects [1,2]. This steroid is however not the major moulting hormone of insect larvae. Indeed, ecdysone serves in peripheral tissues as a precursor to ecdysterone (20-hydroxyecdysone) [3] which is also biologically more potent than ecdysone in vitro in several tests of moulting hormone activity [4].Enzymes. Ecdysone 20-monooxygenase, or ecdysone, reduced flavoprotein : oxygen oxidoreductase (20-hydroxylating) (EC 1.14.14.-); NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4).Trivial Names. Ecdysone, 2fi,3fl,14~~,22R,25-pentahydroxy-5p-cholest-7-en-6-one; ecdysterone or 20-hydroxy-ecdysone, 2P,3/j14a,20R,22R,25-hexahydroxy-S~-cholest-7-en-6-one. (The use of 'a-ecdysone' for ecdysone and '8-ecdysone' for ecdysterone is an abuse and should be avoided.)While it appears that the secretion of ecdysone by the prothoracic glands and the inactivation of ecdysteroids vary during larval development [5,6], it has also become clear that the relative amounts of ecdysone and ecdysterone (and other ecdysteroids?) are fluctuating [7,8]. The enzyme responsible for the conversion of ecdysone to ecdysterone, ecdysone 20-monooxygenase, thus plays a central role in the delicate hormonal balance which controls the moulting process. It was felt that a precise characterization of this enzyme was a prerequisite to studies...

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Biosynthesis of α-Ecdysone by Prothoracic Glands in vitro

Cited by 162 publications

References 16 publications

The ovary as a source of alpha-ecdysone in an adult mosquito.

The ovary as a source of alpha-ecdysone in an adult mosquito.

Spontaneous and evoked release of prothoracicotropin from multiple neurohemal organs of the tobacco hornworm

Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

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