Y-organ Cells Research Articles

Stage-specific changes in calcium concentration in crustacean (Callinectes sapidus) Y-organs during a natural molting cycle, and their relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is suppressed by molt-inhibiting hormone (MIH). The suppressive effect of MIH on ecdysteroidogenesis is mediated by one or more cyclic nucleotide second messengers. In addition, existing data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular Ca++. Despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca++ in Y-organ cells has not been previously measured during a natural molting cycle for any crustacean species. In studies reported here, a fluorescent calcium indicator (Fluo-4) was used to measure Ca++ levels in Y-organs during a molting cycle of the blue crab, Callinectes sapidus. Mean calcium fluorescence increased 5.8-fold between intermolt (C4) and stage D3 of premolt, and then dropped abruptly, reaching a level in postmolt (A) that was not significantly different from that in intermolt (P>0.05). The level of ecdysteroids in hemolymph of Y-organ donor crabs (measured by radioimmunoassay) showed an overall pattern similar to that observed for calcium fluorescence, rising from 2.9ng/mL in intermolt to 357.1ng/mL in D3 (P<0.05), and then dropping to 55.3ng/mL in D4 (P<0.05). The combined results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca++.

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y‐organs: relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5 ng/mL on D0 to 49.6 ng/mL on D6 and 87.2 ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).

Histopathology of Y-organ in Indian white shrimp Fenneropenaeus indicus, experimentally infected with white spot syndrome virus

Histopathology of Y-organ (moulting gland) in Indian white shrimp Fenneropenaeus indicus was studied after challenging the shrimp with virulent white spot syndrome virus (WSSV). Histopathological investigation on the Y-organ collected from moribund shrimps revealed the presence of intranuclear basophilic inclusions, characteristic of WSSV. More than 70% of the Y-organ cells were infected, suggesting the degenerated state of the organ. The cellular integrity of the Y-organ was completely destroyed by the WSSV. Further, Y-organ tissue samples collected from all the 16 experimental shrimp were positive by one-step polymerase chain reaction (PCR), confirming severe WSSV infection. The infected Y-organ in F. indicus with majority of the organ cells observed in the lysed condition suggests a physiological dysfunction of the organ. In uninfected and healthy controls, the lobulated Y-organ showed closely packed normal cells with prominent nuclei and sparse cytoplasm. Physiological implication of a degenerated Y-organ in the moulting and reproduction of the penaeid shrimp is discussed.

Signaling Pathways for Ecdysteroid Hormone Synthesis in Crustacean Y-organs

The Y-organs of crustaceans secrete steroid hormones (ecdysteroids) which are responsible for molting and regeneration. The Y-organs in turn are controlled (negatively) by the eyestalk peptide, molt-inhibiting hormone (MIH). We are exploring the signaling paths in Y-organ cells that lead to ecdysteroid generation when activated by the absence of MIH. The objective is to understand the connections between MIH-receptor occupancy and the depression of genes that express ecdysteroidogenic enzymes. MIH action is mediated by a rise in cyclic 5′ adenosine monophosphate (cAMP); cGMP also is involved in some species. That a cyclic nucleotide is a central regulatory component is indicated by the following selection of results: dibutyryl cAMP, activators of adenylyl cyclase or inhibitors of cyclic nucleotide phosphodiesterase each mimic the inhibitory action of MIH. Cyclic AMP inhibits the receptor-mediated uptake of cholesterol (the obligate ecdysteroid precursor), by decreasing the number of receptor sites for the lipoprotein carrier of cholesterol. MIH via cAMP also depresses de novo protein synthesis upon which ecdysteroidogenesis depends in part. A role for cellular free calcium (Ca++) is indicated by the ability of Ca++ (or a Ca++ionophore) to stimulate ecdysteroidproduction, thereby antagonizing MIH action. The mechanism involveslowering cAMP levels by enhancing phosphodiesterase activity via calmodulin, not by affecting adenylate cyclase activity. Ca++ counters the suppressive action of MIH or cAMP on protein synthesis. Consistent with the MIH-Ca++ mutual antagonism, MIH increases Ca++ efflux from 45Ca-preloaded cells. Y-Organ cells contain protein kinase C (PKC), the activation of which increases ecdysteroid production. PKC activity is not affected by MIH, but is stimulated by Ca++. These and related experiments indicate that the PKC-activated increase in ecdysteroidogenesis involves events downstream from the production of cAMP and the degradation of cAMP by Ca++. In relation to the latter, specific and non-specific inhibitors of protein tyrosine kinases (PTK) inhibit ecdysteroid synthesis dose-dependently. The relationship of PTK with MIH-cAMP and Ca++-PKC systems is under study.

Involvement of a 3 β-hydroxysteroid dehydrogenase activity in ecdysteroid biosynthesis

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas. 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5 β-ketodiol (2,22,25-trideoxyecdysone), 2,22deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3 β-OH compounds in the crab Y-organ. The enzyme involved in 3 β-dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3 β-hydroxysteroiddehydrogenase enzymes from vertebrates: it was membrane-bound and NAD +-dependent. Moreover, a NADH-dependent 3 β-reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100 000× g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3 β-hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.

Uptake of high-density lipoprotein by Y-organs of the crab Cancer antennarius: III. Evidence for adsorptive endocytosis and the absence of lysosomal processing.

Ecdysteroid hormones in crustaceans are synthesized from cholesterol in the Y-organs. Circulating cholesterol is bound to high-density lipoprotein (HDL). Experiments were conducted to find the mode of cholesterol uptake by Y-organ cells. The working hypothesis is as follows: Cholesterol is taken up by endocytosis of the entire HDL-cholesterol complex. HDL was doubly labeled in the apolipoprotein and cholesterol components with 125I and 3H, respectively. The time courses of uptake of the two labels by Y-organ segments in vitro were parallel throughout 24 hr of incubation. Chloroquine, an inhibitor of lysosomal function, had no effect on the uptake and degradation of HDL. Cycloheximide, an inhibitor of protein synthesis, depressed HDL uptake. Transmission electron microscopy of Y-organ tissue showed features characteristic of adsorptive endocytosis, including coated pits in the plasma membranes and their invagination and vesiculation. Visualization of HDL with immunogold binding showed profiles consistent with endocytosis. Quantitation of the distribution of HDL-gold particles indicated that about 75% were associated with cellular formed elements, but not with lysosomes, and significantly more particles overall were present in cells from de-eyestalked crabs than those from intact crabs. These data demonstrate that (a) cholesterol-carrying HDL is taken into Y-organ cells by adsorptive endocytosis, (b) its uptake is dependent on de novo protein synthesis, (c) its intracellular processing is independent of lysosomal enzymes, and (d) its uptake is depressed by the molt-inhibiting hormone secreted by the eyestalks.

Dissociated cell suspensions ofCarcinus maenas Y-organs as a tool to study ecdysteroid production and its regulation

In vitro ecdysteroid production by dissociated Y-organ cells of the shore crabCarcinus maenas was characterized during short-term incubations. Under optimized conditions (M199 adjusted to crab osmolality and with the addition of 10% foetal calf serum), ecdysteroid production by dispersed cells increased linearly during 4-hour incubations, with little intra-assay variation. 25-deoxyecdysone was mainly produced. PurifiedCarcinus molt inhbiting hormone (CamMIH) produced a dose-dependent inhibition of ecdysteroid production by dispersed cells. The cells were about 50 times more sensitive than whole glands to MIH. Other structurally-related peptides were tested.

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 Ca 2+ 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-methylxanthine (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) 21cAMP 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 Ca 2+ (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 Ca 2+ 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. Ca 2+ 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 45Ca 2+ entry by 45% over untreated control cells. Efflux from 45Ca 2+-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 Ca 2+ depletion, and that the effect is mediated by cAMP.

Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides

Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP>AVP>VT>>>OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3′,5′ monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y-organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto-β,β-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH 2) 5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH-suppressed steroidogensis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors.

The ultrastructural basis of steroid production in the Y-organ and the mandibular organ of the crabs Hemigrapsus nudus (Dana) and Carcinus maenas L.

The ultrastructure of the steroid producing Y-organ and the mandibular organ of the crustaceans Hemigrapsus nudus and Carcinus maenas has been studied with reference to the well investigated steroid secreting cells (SSC) of mammals. In accordance with the most important characteristic of mammalian SSC, abundant SER could be shown in the Y-organ, where it is unevenly distributed. The amount of SER seems to vary in correlation with the secretion of moulting hormone during the moult cycle. Most Y-organ cells contain a great number of mitochondria of the tubular type, another important characteristic of mammalian SSC. The ultrastructure of the mandibular organ of C. maenas differs considerably from that of the Y-organ. Some SER was found, mitochondria of unusual shape and size were conspicuous. No definite conclusion as to the function of the mandibular organ is yet to be drawn.

Some effects of eyestalk removal on the Y-organs of Cancer irroratus Say.

1. Bilateral destalking of Cancer irroratus increases the rate of RNA synthesis in the Y-organ cells during the first four days postdestalking.2. The cytoplasmic volume of the Y-organ cells also increases during the first four to five days postdestalking and then drops to a plateau that is slightly above the volume of cells from non-destalked control animals.3. Numerous areas of cytoplasmic vacuolization and cellular degeneration are evidenced within the control and destalked crabs. It is postulated that this may be evidence of holocrine activity.4. The Y-organ cells of Cancer irroratus contain numerous osmophilic granules both in control and destalked animals.