Regulation of ecdysteroidogenesis in blue crab, Callinectes sapidus, Y -organs by molt -inhibiting hormone (MIH)

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide produced and released by the X-organ/sinus gland complex of the eyestalks. In the present studies, cellular signaling pathways involved in the regulation of Y-organ function by MIH were investigated in the blue crab, Callinectes sapidus. By using ADP-ribosylation and Western blot analysis, the presence of G _iα- and G_sα-proteins in Y-organs and thoracic ganglia was confirmed. A569 (anti-Gα) detected proteins of calculated molecular weight ∼57 kD, ∼50 kD, and ∼40 kD in Y-organs. Proteins of molecular weight, ∼50 kD and ∼40 kD, are likely to be G_sα- and G_i/oα-proteins, respectively, based on their molecular weights and immunospecificity. In Y-organ incubation studies, 8-Br-cAMP (a cAMP analog) suppressed incorporation of [³⁵S]-methionine into Y-organ proteins; the effect was concentration-dependant. Addition of sinus gland extract (a MIH source), cholera toxin (a G_sα protein activator), IBMX (a phosphodiesterase inhibitor), or forskolin (an adenylyl cyclase activator) likewise suppressed the incorporation of [³⁵S]-methionine into Y-organ proteins. Surprisingly, these reagents failed to suppress ecdysteroid synthesis in cultured Y-organs. Two cAMP analogs, 8-Br-cAMP and db-cAMP, were without effect at any concentrations used. In addition, forskolin and cholera toxin also showed no effects. By contrast, 8-Br-cGMP (a cGMP analog) significantly suppressed ecdysteroidogenesis. Finally, recombinant MIH increased the cGMP concentration in Y-organs but was without effect on cAMP concentration. The combined results suggest that, although a G_sα-protein- and cAMP-mediated regulatory mechanism is present in Y-organs, it is unlikely that they are actively involved in MIH signaling. By contrast, our data strongly support the possibility of cGMP being a primary second messenger for MIH-mediated suppression of ecdysteroid secretion by the Y-organs of C. sapidus .