Eugene Spaziani

5-HYDROXYTRYPTAMINE MEDIATES RELEASE OF MOLTINHIBITING HORMONE ACTIVITY FROM ISOLATED CRAB EYESTALK GANGLIA

Neurosecretory cells in crustacean eyestalk ganglia produce a putative molt-inhibiting hormone (MIH) which directly suppresses production of the steroid molting hormone, ecdysone, by the peripheral Y-organs. Neurotransmitter mediation of MIH release from isolated eyestalk ganglia of the crab, Cancer antennarius, was explored using an MIH bioassay based upon in vitro inhibition of Y-organ ecdysteroid production by eyestalk ganglion-conditioned saline. The conditioned saline (0.01-1.0 eyestalk equivalent) inhibited Y -organ ecdysteroid production dose-dependently and reversibly, and the effect of the saline was specific as to conditioning tissue. Isolated ganglia released a significant portion of their MIH activity in 2-h incubations, but also retained a significant portion. 5-hydroxytryptamine (5-HT) enhanced MIH release at concentrations of 10-10 M to 10-6 M. Acetylcholine, dopamine, octopamine, norepinephrine, or gamma-aminobutyric acid (10-7 M-10-6 M) did not alter basal MIH release. The 5-HT precursor,...

Biosynthesis of ecdysteroids from cholesterol by crab Y-organs, and eyestalk suppression of cholesterol uptake and secretory activity, in vitro

Precursor incorporation studies were conducted in vitro with activated Y-organs from 48-hr de-eyestalked Cancer antennarius donors. When the glands were prelabeled in vivo by systemic injection of [3H]cholesterol 12 hr prior to removal, and subsequently incubated 24 hr in label-free medium, the glands secreted 3H-labeled ecdysone. The glands also secreted an unidentified ecdysteroid with comigrating 3H-label with characteristic retention time on normal-phase HPLC of 4 min (4-min unknown). The compound is less polar, and is secreted in a quantity and apparent specific activity approximately fivefold greater, than ecdysone. Compared with chromatographic retention times and competitive binding curves of authentic standards, the 4-min unknown was determined not to be ponasterone A, inokosterone, makisterone, or several other possible products or intermediates. In contrast with Y-organs from intact donors, those from de-eyestalked crabs exhibit greatly increased cholesterol uptake and secretion of both ecdysone and 4-min unknown in vitro. All three responses were suppressed in dose-dependent manner by eyestalk extract in the dose range, 1-4 eyestalk equivalents/Y-organ. Secretion of 4-min unknown was the response most sensitive to eyestalk extract (requiring the least dose for 50% inhibition).

Evidence that Y-organs of the crab Cancer antennarius secrete 3-dehydroecdysone

Y-organs are paired glands in crustaceans that secrete a class of steroid hormones (ecdysteroids) that regulate growth, molting and development. The glandular secretion has been assumed to be solely the ecdysteroid, ecdysone, a polyhydroxylated derivative of cholesterol. We previously reported that Y-organs of a crab (Cancer antennarius) additionally secreted an ecdysteroid that is less polar than ecdysone. Evidence is presented here that the other secretion product is 3-dehydroecdysone (3-dhE). The compound co-chromatographed with authentic 3-dhE in both normal-phase, and reversed-phase, high-performance liquid chromatography. Mass spectrometry of the ecdysteroid gave results consistent with its identity as 3-dhE. The putative 3-dhE was radiolabeled by injecting crabs with [3H]cholesterol and then incubating the Y-organs. The putative [3H]3-dhE secretion was then subjected to chemical reduction. The reaction yielded labeled products that co-chromatographed with authentic ecdysone and 3-epiecdysone. Results of other experiments gave the following results: (1) Putative 3-dhE was not altered (chromatographic criteria) by incubations with snail hydrolases. (2) Putative [3H]3-dhE, added to incubations of Y-organ halves or homogenates, was not significantly converted to ecdysone; also, no conversion was evident after incubation in medium alone in which the hemolymph serum supplement was raised to 50% of the volume. (3) [3H]Ecdysone was not converted to putative 3-dhE in vitro by Y-organ halves or homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and turnover of cholesterol-14C in Y-organs of the crab Hemigrapsus as a function of the molt cycle ☆

Abstract The uptake of cholesterol- 14 C and incorporation into cholesterol pools and derivatives in vivo were studied in Y-organs, hemolymph, and whole bodies of male crabs. Tissues and serum, removed 24 hr after injection of 1–4 μCi per crab, were extracted for total lipid and analyzed by chromatography (thin-layer, gas-liquid), autoradiography, and scintillation counting. Y-organs of late intermolt crabs took up 650 times more labeled cholesterol per unit weight of tissue than the general body mass of the same stage, and the order of 100 times more than Y-organs of premolt or postmolt animals. Uptake of label occurred against a 70:1 Y-organ:serum concentration gradient of total cholesterol. Since Y-organ total cholesterol did not change markedly with stage, the increased uptake at intermolt reflected rapid turnover of cholesterol pools. Labeled derivatives of cholesterol, cochromatographing through three solvent systems with α- and β-ecdysone standards, were found in all tissues and sera. Absolute amounts of the labeled products in Y-organs were too low for additional physicochemical analyses and their identity as ecdysones is provisional. Concentrations (cpm/unit weight tissue), however, were distinctly higher in Y-organs than in carcasses. Among Y-organs, the largest concentrations were found in premolt which contained 60 and 260 times more labeled “β-ecdysone” than intermolt or postmolt glands, respectively. The occurrence of accelerated cholesterol uptake and turnover in Y-organs of summer intermolt, followed by accumulation in premolt of newly synthesized ecdysone-like metabolites, supports physiological evidence that these organs are involved in the molting process. The data suggest that they function as sources of a form of sterol molting hormone.

Effects of eyestalk removal on cholesterol uptake and ecdysone secretion by crab (Cancer antennarius) Y-organs in vitro☆

Y-Organs and control tissues from intact (intermolt) and 48-hr de-eyestalked Cancer antennarius donors were cultured for 12 and 24 hr in crustacean saline supplemented 10% with crab serum and containing [14C]cholesterol. Under these conditions, Y-organs took up significantly more [14C]cholesterol than ovary or muscle, and Y-organs from 48-hr de-eyestalked crabs took up threefold more than Y-organs from intact crabs. The labeled cholesterol of the culture medium was observed to bind rapidly to the lipoproteins of the serum supplement; subcellular fractionation of the activated Y-organs after incubation showed 59% of the label localized in the cytosolic fraction. The increase in cholesterol uptake did not result from a change in extracellular volume, and was not accompanied by a change in Y-organ total cholesterol. It was, however, accompanied by a greater than threefold increase in ecdysone secretion.

Cyclic AMP mediates the negative regulation of Y-organ ecdysteroid production ☆

The putative neuropeptide, molt-inhibiting hormone (MIH), regulates crustacean growth by periodically suppressing the secretion of ecdysteroid molting hormone from peripheral glands (Y-organs). A mediating role for cyclic AMP (cAMP) in MIH action was evaluated with isolated Y-organs of the crab, Cancer antennarius. MIH activity in eyestalk extracts inhibited ecdysteroid secretion but increased cAMP levels dose-dependently in 24-h incubations. The cAMP rise preceded the onset of ecdysteroid suppression. Dibutyryl cAMP, activators of adenylate cyclase (forskolin, choleragen), and an inhibitor of phosphodiesterase (IBMX), but not AMP or cGMP, mimicked the inhibitory action of MIH.

Regulation of crab Y-organ steroidogenesis in vitro: evidence that ecdysteroid production increases through activation of cAMP-phosphodiesterase by calcium-calmodulin

In decapod crustaceans steroidogenic glands (Y-organs) produce the molting hormone, ecdysone. A putative neuropeptide, molt-inhibiting hormone (MIH), released from eyestalk neurosecretory cells, directly regulates Y-organs by suppressing steroidogenesis; the effect is mediated by an increase in cAMP. We explored calcium-cAMP interactions in the regulation of Y-organs in vitro of the crab, Cancer antennarius. Basal ecdysteroid production was enhanced by extracellular calcium (EC). MIH suppression did not require EC but its action was blocked by high EC. The inhibitors of Ca2+ flux, lanthanum and ruthenium red, mimicked and enhanced MIH action. Calcium ionophore A23187 raised basal steroidogenesis dose-dependently (10(-6) to 10(-4) M) and with time course (effect evident after 2 h) similar to that of suppression by MIH. Low EC enhanced the suppressive effects on steroidogenesis of forskolin and dibutyryl cyclic AMP ((Bu)2cAMP) but not of MIH, lysine vasopressin (LVP), or 3-isobutyl-1-methyl-xanthine (IBMX); basal Y-organ cAMP levels were elevated by low EC and reduced by A23187. A23187 reduced the steroidogenic-suppressive effects of MIH, LVP, forskolin and (Bu)2cAMP but not of IBMX; rises in cAMP induced by MIH, LVP, and forskolin but not by IBMX were blunted by A23187. These findings suggested a stimulatory action of calcium on phosphodiesterase (PDE). The calmodulin (CM) inhibitor trifluoperazine (TFP; 10(-5) to 10(-4) M) reduced basal and A23187-stimulated steroidogenesis, enhanced the inhibitory effects of MIH and (Bu)2cAMP on ecdysteroid production, enhanced the stimulatory effects of MIH and forskolin on cAMP, and blocked the inhibition of cAMP by A23187. Y-organ PDE activity was enhanced by increasing free Ca2+ (10(-7) to 10(-5) M) and inhibited by TFP (10(-5) to 10(-4) M). Adenylate cyclase activity of Y-organ cell particulate fraction was unaffected by Ca2+ or TFP. Calcium stimulates steroidogenesis, apparently by activating a calcium-CM-dependent cAMP-PDE: the action is counter to the cAMP-mediated MIH-inhibitory system. Ca2+ fluxes were measured with dispersed Y-organ cells, in the presence and absence of agents that alter cAMP levels. The ionophore A23187, but not MIH or forskolin, increased 45Ca2+ entry by 45% over untreated control cells. Efflux from 45Ca2+-preloaded cells was increased 30% by MIH and forskolin, but not A23187. These data, together with those further above, suggest that MIH suppresses steroidogenesis in part by fostering Ca2+ depletion, and that the effect is mediated by cAMP.

Ovarian and haemolymph titres of ecdysteroid during the gonadotrophic cycle in Diploptera punctata

Abstract The free (non-conjugated) ecdysteroid in the ovaries during the first gonadotrophic cycle of Diploptera punctata was identified as 20-hydroxyecdysone. The hormone, quantified by radioimmunoassay and by ultraviolet absorbance, was detectable in the ovary toward the end of vitellogenesis; the quantity increased rapidly during chorion formation. Ovaries with chorionated eggs contained 67 μg of 20-hydroxyecdysone per g fresh weight. The haemolymph free-ecdysteroid, not identified physicochemically, was quantified by radioimmunoassays. The highest concentration was observed at adult emergence; the titre declined between days 1–3 and then remained at a relatively constant level through oviposition (which occurs between day 7 and 8); titres in pregnant females were higher. Ovariectomized females exhibited the same pattern of ecdysteroid titres in the haemolymph as the sham operated controls throughout the period corresponding to the first gonadotrophic cycle. Thus the ovary may not be the only source of haemolymph ecdysteroid related to reproduction in adult females.

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.

Rapid Isolation of Ecdysteroids from Crustacean Tissues and Culture Media Using Sep-Pak C18 Cartridges

Abstract A liquid chromatographic method for isolating ecdy-steroids from crab tissues and crustacean tissue culture media is reported. The method employs commercially-available Sep-Pak C18 cartridges containing a reversed phase packing that retains ecdysteroids. The technique is simple and rapid; it typically yields recoveries in the range of 85–90% for extractions of tissues, and 90–95% for extractions of media.