Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB ₃ juvenile hormone III bisepoxide     

Some old concepts and new findings on hormonal control of insect morphogenesis

By means of the artificially induced heterochronic developmental deviations represented by local prothetelies and metathetelies it has been possible to investigate the individual developmental fates of ontogenetically different tissues, such as larval, pupal, and adult epidermal cells, in one and the same body and under the identical concentration of juvenile hormone (JH) in the haemolymph.In contrast to the widely accepted hormonal theories which claim that the kind of morphogenesis is determined by large, intermediate, and low titres of JH, the heterochronic character of the tissues never developed into a uniform population of homomorphic epidermal cells. Instead, in the presence of effective amounts of JH the heterochronic pattern has been fully preserved and carried on into the next developmental instar. Moreover, in the absence of the effective JH amounts the ontogenetically different tissues, such as larval and pupal epidermal cells, simultaneously undergo their respective morphogenetic developments, i.e. larval-pupal and pupal-adult morphogenesis in the same hormonal milieu. It is concluded that the selective factor in determination of the kind of morphogenetical changes is not an altered JH titre but the extant, previously attained degree of ontogenetic structural differentiation. It has been demonstrated that JH can temporarily and reversibly inhibit the morphogenetic progress at quite different ontogenetic levels but it cannot cause a ‘reversal of metamorphosis’ at any of these levels.Under specific experimental conditions the larval epidermal cells can undergo pupal and adult morphogenesis without secreting the pupal cuticle. However, the pupal morphogenetic interstage, whether with the cuticle or without the pupal cuticle, constitutes an obligatory developmental step. Further, it appears that an absence of JH may represent an important condition but not a real cause of insect metamorphosis, as presumed in some other hormonal concepts. Thus, chromosomal duplications or cellular divisions in the absence of JH have not committed the cells to morphogenesis unless provided by an additional stimulus of endogenous prothoracic gland hormone or exogenous ecdysterone. An important factor in understanding the hormonal control of insect morphogenesis is the critical timing of the respective morphogenetic steps. This corresponds closely with the duration of the pharate phases in insect development. Possible hormonal mechanisms concerned in the regulation of morphogenesis in endopterygote insects have been outlined.     

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.     

Identification and characterization of a juvenile hormone (JH) response region in the JH esterase gene from the spruce budworm, Choristoneura fumiferana

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.