Radioimmunoassay: Ecdysteroids

Warren, James T.; Gilbert, Lawrence I.

doi:10.1007/978-1-4612-3798-3_6

Cited by 7 publications

(7 citation statements)

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“…Methods for directly measuring the juvenile hormone (JH) titer, JH titer regulators, or the ecdysteroid (ECD) titer in endocrine studies of evolution and development (see text for additional information and discussion) JH TITER; RADIOIMMUNOASSAY (RIA) 1 Measurement: 10-100s ml hemolymph (H) required from adults (A) or juveniles (J); titers often can be measured in single individuals (I), especially A Advantage over mass spectrometry/gas chromatography (GC-MS): rapid, esp. if sample purification not required; very useful in populational studies; expensive equipment not required Disadvantage: individual JHs typically cannot be distinguished by the antibody; not recommended for whole-body (WB) titers; no antibody to main JHs of some groups 2 Caveats: potential artifacts due to lack of specificity of antibody or impurities (especially lipids) in sample (background studies essential) Comments: JH RIA technically more difficult to perform than ECD RIA References: Goodman et al (1993), Trumbo et al (1995), Cisper et al (2000), Zhao and Zera (2004a), Goodman and Granger (2005) JH TITER: GC-MS 3 Measurement: typically 100s ml H, or grams WB required; titers typically measured on H pooled from several individuals or WB samples (however see Westerlund and Hoffmann 2004 for assay requiring smaller amount of biological material) Advantage over RIA: most reliable method to measure JH titer; versatileFindividual JHs and JH metabolites can be measured in H or WB Disadvantage: expensive equipment required (mass spectrometer); more labor intensive than RIA; requires prior sample purification Comments: less useful than RIA when many samples must be measured (e.g., population studies) References: Baker (1990), Botens et al (1997), Tawfik et al (2000), Westerlund and Hoffmann (2004), Goodman and Granger (2005) ECDYSTEROID (ECD) TITER RIA 4 Measurement: H (few microliters or less); WB or individual tissues (mg); A, J; I titers often possible Comments: widely used; very rapid, technically simple; less potential for experimental artifacts than JH RIA; References: Warren and Gilbert (1988), Porcheron et al (1989), , Rachinsky and Hartfelder (1990), Koch et al (1996)…”

Section: Jh Titermentioning

confidence: 99%

Endocrine analysis in evolutionary‐developmental studies of insect polymorphism: hormone manipulation versus direct measurement of hormonal regulators

Zera

2007

Evolution and Development

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"Hormone manipulation" is being used increasingly in evo-devo studies as the sole or primary technique to investigate the regulation of insect polymorphism by hormones, most notably juvenile hormone (JH). This manuscript critically evaluates the limitations and strengths of this indirect method for inferring aspects of endocrine regulation, and conclusions derived from recent endocrine studies of evolution and development in which data have been obtained primarily or exclusively by this method. The main conclusions of this critique are as follows: first, when used alone, or as the primary empirical technique, hormone manipulation is a superficial method that is fraught with problems with respect to identifying a hormone that regulates developmental-morphological variation, let alone identifying its mode of action. Second, conclusions reported in studies using this technique as the exclusive, or nearly exclusive experimental approach, most notably recent studies of JH regulation of horn polymorphism in dung beetles, and some studies of wing polymorphism should be considered, at best, weakly supported until substantiated by well-validated, direct methods. Finally, there are many reliable and well-validated techniques that can be used to directly and accurately quantify JH levels, and activities of JH regulators, in many insects, even in small, nonmodel species. Some of the most important of these assays will be briefly described and their strengths and weaknesses will be discussed.

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Section: Jh Titermentioning

confidence: 99%

Endocrine analysis in evolutionary‐developmental studies of insect polymorphism: hormone manipulation versus direct measurement of hormonal regulators

Zera

2007

Evolution and Development

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“…RIAs were performed according to Warren & Gilbert (1988) with minor modifications (Sakurai et al 1998, Takaki & Sakurai 2003 (approximately 10 000 c.p.m. in 100 µl borate buffer) and the 6000-fold-diluted 0-6 anti-ecdysone antiserum (Yokoyama et al 1996) in 100 mM borate buffer (100 mM boric acid, 50 mM borax and 60 mM NaCl, pH 8·4) containing 0·02% sodium azide, 0·05% rabbit IgG (Miles, Kankakee, IL, USA) and 0·2% BSA (fraction V; Sigma) were added to aliquots of the extracts.…”

Section: E Titrementioning

confidence: 99%

Nutritional status affects 20-hydroxyecdysone concentration and progression of oogenesis in Drosophila melanogaster

Terashima¹,

Takaki²,

Sakurai³

et al. 2005

Journal of Endocrinology

149

117

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Drosophila egg production depends upon the nutritional available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. We have shown that expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone induces and suppresses the apoptosis, respectively. In this study, we investigated which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. We measured ecdysteroid levels in the whole body, ovaries and haemolymph samples by RIA and found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. We suggest there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake.

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“…The pooled solvents were evaporated, and the residues, along with the selected residues of samples after their RP-HPLC or TLC purification, were subjected to RIA. The H22 antibody was used for embryo titers (13), and the SHO3 antibody for chromatography residues (14). Results are expressed in ecdysone equivalents.…”

Section: Riamentioning

confidence: 99%

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster

Warren

Petryk

Marqués

et al. 2002

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

Self Cite

303

243

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Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C 22-and C 2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

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Radioimmunoassay: Ecdysteroids

Cited by 7 publications

References 59 publications

Endocrine analysis in evolutionary‐developmental studies of insect polymorphism: hormone manipulation versus direct measurement of hormonal regulators

Endocrine analysis in evolutionary‐developmental studies of insect polymorphism: hormone manipulation versus direct measurement of hormonal regulators

Nutritional status affects 20-hydroxyecdysone concentration and progression of oogenesis in Drosophila melanogaster

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster

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