Grahame E. Pratt

Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro.

Abstract When corpora allata from adult female Schistocerca gregaria are incubated in vitro with either 3H- trans , trans farnesenic acid or 3H- trans , trans , cis bishomo-farnesenic acid and [ methyl -14C] methionine, they fabricate large quantities of the corresponding double labelled methyl 10, 11-epoxy esters. Radio GLC of these products indicates retention of geometric configuration at the C-2 and C-6 double bonds. Separate analyses of the contents of the glands and medium after incubation show that the epoxy esters are rapidly released from the glands into the medium and that only the glands contain the corresponding unepoxidized esters. We suggest that unepoxidized esters are the intracellular intermediates in the formation of juvenile hormones from the unsaturated acids. Gel filtration shows that the epoxy esters are not released as stable protein complexes but as simple solutes into the medium. Using this method of promoting the synthesis of juvenile-hormone-active compounds, rates of biosynthesis of epoxy esters of up to 33 ng. per pair of glands per hour have been achieved.

Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaneta americana

Abstract Isolated pairs of corpora allata (CA) from adult females of both Schistocerca gregaria and Periplaneta americana incorporate radioactivity from [methyl- 14 C] methionine into C 16 juvenile hormone (JH) when incubated for up to several hours, such that the rate of JH synthesis can be accurately determined. Analysis of the medium shows that CA from P. americana continue to release newly synthesised JH at constant rates for at least 5 hr, whereas those from S. gregaria may show a marked decrease in rate of JH release after 3 hr, particularly in the case of glands having high initial rates of synthesis and release. In both cases the rates of release of JH are strictly proportional to the rates of JH synthesis when measured over a period of 3 hr incubation, independent of the rate of synthesis. It is concluded that the spontaneous level of endocrine activity in glands from both these species can be faithfully quantified by precise radiochemical methods using the short-term incubation procedures described.

The effect of enforced virginity and subsequent mating on the activity of the corpus allatum of Periplaneta americana measured in vitro, as related to changes in the rate of ovarian maturation

ABSTRACT. Female P. americana, reared with males from the time of adult emergence, mated on the 4th–5th day after metamorphosis, produced the first ootheca on the 8th or 9th day, and then produced successive oothecae at intervals of 3.0 days, whereas, only 50% of virgin females had produced their first ootheca by the 28th day after adult emergence. Examination of the ovaries indicated that oocyte development is normal in virgins until shortly after the time when they first become receptive to males. When mating was not allowed there was a dramatic reduction in the rate of vitellogenic growth of the terminal batch of oocytes which persisted until mating was allowed, and was often accompanied by resorption of a percentage of the oocytes.

Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30-40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 microM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other development stages.

Effects of starvation and feeding upon corpus allatum activity and oöcyte growth in adult female Periplaneta americana

Abstract Direct radiochemical determinations of juvenile hormone (JH) biosynthesis by corpora allata (CA) isolated from starved and re-fed Periplanteta americana have been employed to elucidate the humoral mechanisms involved in the modulation of reproductive activity in response to food availability. When starvation was initiated in mature adult females at the time of formation of an ootheca the next ootheca was normally deposited 5 to 6 days later, a delay of 2–3 days, and a third ootheca was formed by only 50% of starved females. The terminal oocytes in the remaining females were either resorbed or maintained in an arrested state. Ovarian development had effectively ceased after 2 weeks of starvation but recommenced within 3 days of re-feeding. The CA of most starved females exhibited 2 activity cycles following food withdrawal. The first peak occurred on day 1 of the starvation period and was coincident with the timing for fed controls. The second peak was delayed by about 2 days and the activity of the CA then declined to the extent that glands from animals starved for more than 11 days were completely inactive. Feeding, after 10 or 16 days starvation, resulted in a resumption of CA activity which was detectable in some animals within 24 hr, and very high rates of JH biosynthesis were found 4 or 5 days later. The results suggest that P. americana can readily and efficiently modulate egg production in response to food supply, and that control is effected through alterations in JH production by the CA. The use of farnesenic acid as a biochemical probe indicates that CA inactivity after long periods of starvation does not arise because malnutrition has caused complete metabolic shut-down in the glands, and that JH biosynthesis is basically modulated at a control point prior to the last two enzymic stages in the pathway.

The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocerca gregaria

Abstract We have compared, on an individual basis, the volume of the corpora allata with their ability to synthesize and release juvenile hormone (JH) using glands taken at daily intervals throughout the period of sexual maturation and the first two ovarian cycles in Schistocerca gregaria . A standard in vitro radiochemical assay was used to measure the rates of both spontaneous JH biosynthesis from [methyl-14C]-methionine, and of JH biosynthesis stimulated by optimal concentrations of [C-2 3H]-farnesenic acid. Computation of results showed that there are, during this period, changes of up to 250-fold in the rate of spontaneous JH biosynthesis per unit volume corpora allata. It is concluded that the volume of the corpora allata is of no value as an indicator of the spontaneous synthetic activity of the glands in this species, and that the overall rate of JH synthesis is regulated by mechanisms that do not involve large changes in the volume of the gland cells. However, in the presence of farnesenic acid, there is a corelation between stimulated JH synthesis and glandular volume, suggesting that the volume of the gland reflects the maximum activity of the final two stages in JH biosynthesis.

Use of automated capillary column radio gas chromatography in the identification of insect juvenile hormones

Automated capillary column radio gas chromatography used in conjunction with high-resolution liquid chromatography has been used for the identification of biosynthesised insect hormones and their immediate precursors. The method affords high resolution plus high sensitivity and discrimination through quantitation by liquid scintillation counting. Picogram quantities of the hormones are produced by corpora allata cultured in vitro with medium containing different 3H- and 14C-labelled precursors.

Precocene sensitivity of corpora allata in adult female Locusta migratoria after electrocoagulation of the pars intercerebralis neurosecretory cells

Destruction, by electrocoagulation, of the median neurosecretory cells of the pars intercerebralis of 2-day old adult female Locusta migratoria completely suppressed normal juvenile hormone-biosynthetic activity of the corpora allata in most animals. For example, 6 days after electrocoagulation the rates of spontaneous juvenile hormone biosynthesis, measured by radiochemical assay of freshly isolated glands, showed a median value of less than 1100 that of the corresponding sham operated controls, which were then in mid-vitellogenesis. Injection of graded doses (200–1600 μg) of precocene I at this time, followed by assays five days later, resulted in a similar decline of both corpus allatum volume and precocene epoxidase activity (measured by radiochemical assays of precocene I dihydrodiol formation in vitro) in pars intercerebralis-coagulated and sham-operated animals, when expressed as a percentage of their own zero precocene controls. Electrocoagulation of the pars intercerebralis largely prevented the normal increase in both corpus allatum volume and its epoxidase activity, so that by age 13 days these parameters were about 2.5-fold lower in coagulated vs control (sham operated) animals. In fact, electrocoagulation had no marked effect on the value of epoxidase activity per unit corpus allatum volume. It is concluded that the corpora allata from this species and stage are sensitive to precocene irrespective of whether they are active in biosynthesis of juvenile hormone.

Inhibition of juvenile hormone carboxyesterase of locust haemolymph by organophosphates in vitro

Abstract A rapid electrophoretic method is described for estimating the rate of hydrolysis of [C-23H]C16 juvenile hormone by haemolymph carboxyesterases from 5th instar Schistocerca gregaria. Maximum reaction rate is observed at a total substrate concentration of 40 μm, which is only slightly higher than that of a saturated aqueous solution of hormone. The following concentrations of organophosphates yielded 50% inhibition when assayed at substrate saturation: Paraoxon 1.7 × 10 −7 m ; Dichlorvos, 6 × 10 −6 m ; bis-4- nitrophenyl phosphate, 4 × 10 −4 m ; tris- butyl phosphorotrithiolate, 5 × 10 −3 m . Enzyme activity could be quantitatively recovered after micro-preparative thin layer gel filtration. The enzyme-inhibitor complexes formed from both Paraoxon and bis-4- nitrophenyl phosphate were separated from free inhibitor by gel filtration and found to be stable for at least 22 hr at 30°C.

The absolute configuration of precocene I dihydrodiols produced by metabolism of precocene I by corpora allata of locusta migratoria, in vitro

The corpora allata from adult female Locusta migratoria metabolize precocene I (7-methoxy-2,2-dimethyl-2H-benzo [b]pyran to cis- & trans-precocene I dihydrodiols (3,4-dihydro-7-methoxy-2,2-dimethyl-2H-benzo [b]pyran-3,4-diol). Derivatization of the dihydrodiols with (-)menthoxy acetyl chloride allowed complete resolution of all four optical isomers. When [4-3H]-precocene I was incubated in vitro with Locusta migratoria corpora allata, it was metabolized stereospecifically to (-)trans-(3R,4S) and (+)cis-(3R,4R) dihydrodiols. Approximately half the precosyl residues bound to cellular macromolecules were discharged by heating to 95 degrees C at neutral pH, as dihydrodiols of the same stereochemistry.