The clearance in vivo and metabolism of ecdysone and 3-dehydroecdysone in tissues of the crabCancer antennarius

Abstract

The Y‐organs of Cancer antennarius secrete the hormones ecdysone (E) and much greater amounts of 3‐dehydroecdysone (3DE), but the dominant derivative in the circulation is 20‐hydroxyecdysone (20E). The uptake and clearance of E, 3DE, and metabolites were compared in selected tissues in vivo, and the capacity of each to generate 20E was assessed in vitro. [3H]‐labeled E or 3DE, injected into intermolt crabs, rapidly cleared from hemolymph, while being taken up and then rapidly cleared from tissues (muscle, heart, epidermis, ovary) within 1 h. Labeled 20E from E or 3DE appeared in all tissues in the first hour, cleared to varying degrees from tissues, and then reaccumulated over 24 h. Among these tissues, ovary accumulated the most labeled 20E. Generally by 24 h more labeled 20E accumulated from 3DE than from E. Excising the eyestalks (inducing premolt) did not affect the outcomes except that deeyestalking caused much greater concentrations of 3DE than E to appear in all tissues initially. Tissue extracellular volumes ([14C]inulin spaces) were not altered by deeyestalking, except transiently in epidermis, and otherwise did not account for the observed ecdysteroid dynamics. A parallel study with eyestalk neural tissue from crabs receiving [3H]3DE showed extraordinary accumulation of 3DE and labeled metabolites. The normalized tissue/hemolymph ratio was 72 at 8 h, compared with 0.6–2.6 for other tissues. The apparent extracellular volume of eyes was high (80%) but did not account for the high ecdysteroid accumulation. Tissues were incubated 18 h with [3H]3DE. Activities representing 3β‐reductase and 20‐hydroxylase generally were present, evidenced by finding labeled E and 20E. Tissue blanks, hemolymph, and muscle were devoid of activity, hepatopancreas and epidermis were intermediate, ovary, testis, and hindgut were high, and eyestalk tissue was highest in activities. Low amounts of labeled high‐polar material were usually present, and a labeled derivative tentatively identified as 3‐dehydro‐20‐hydroxyecdysone (3D20E) also appeared in ovary, hepatopancreas, hindgut, and eyes, indicating some direct conversion of 3DE to 3D20E. In composite, these data indicate that 3DE secreted by Y‐organs is converted to E, thence to 20E by several tissues, accounting for the preponderance of 20E in crustacean hemolymph. The findings support the hypotheses that 20E is the definitive molting hormone (receptor ligand) and that an ecdysteroid feedback mechanism controls release of the molt‐inhibiting hormone from the eyestalks. J. Exp. Zool. 279:609–619, 1997. © 1997 Wiley‐Liss, Inc.

A crustacean nitric oxide synthase expressed in nerve ganglia, Y-organ,gill and gonad of the tropical land crab,Gecarcinus lateralis

“…In blowfly, for example, steroid synthesis is inhibited by NO and cGMP. However, crustacean ovary accumulates ecdysteroid, but there is little evidence that it is a significant site for ecdysteroid synthesis (Gunamalai et al, 2003;Spaziani et al, 1997;Subramoniam, 2000;Suzuki et al, 1996;Warrier et al, 2001). The localization of NOS in connective tissue and epithelia in Y-organ and gill suggests that it functions in an immune response to pathogens.…”

Ecdysteroid metabolism in crustaceans

“…YOs cannot convert 3DE to ecdysone or ecdysone to 20E [31,43,61]. In peripheral tissues, ecdysone is hydroxylated at C20, probably by Shd, to produce 20E [38,40,41,[62][63][64]. A 20-hydroxylase activity in the hepatopancreas of the blackback land crab (Gecarcinus lateralis) requires NADPH, is inhibited by metyrapone (an inhibitor of cytochrome P450 enzymes), and is associated with the mitochondrial fraction [62].…”

“…[ 3 H]25-hydroxycholesterol (25C) is converted to 7-dehydro-25C by the YOs of green crab (Carcinus maenas), shrimp (Penaeus vannamei), and crayfish (Orconectes limosus); the 7-dehydro-25C is converted to 3DE in C. maenas or to ecdysone and 3DE in P. vannamei and O. limosus [37]. The YOs from Cancer antennarius can also synthesize 3DE from 25C [38]. Although it is not a normal intermediate in the pathway, these results show that 25C can be a substrate for 7,8-dehydrogenase.…”

“…Although it is not a normal intermediate in the pathway, these results show that 25C can be a substrate for 7,8-dehydrogenase. The uptake of 25C is much faster than cholesterol, which makes it a useful tool for the study of the regulation of ecdysteroidogenesis [37,38].…”

“…However, YO cells convert a 3-oxo-4 intermediate hydroxylated at C25 to 3DE and ecdysone [36]. This is consistent with studies showing that these ecdysteroids are synthesized from 25C as the precursor [32,37,38].…”