Expression of ecdysteroids and cytochrome P450 enzymes during lipid turnover and reproduction in Calanus finmarchicus (Crustacea: Copepoda) (original) (raw)
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Insect Biochemistry and Molecular Biology, 2002
A cytochrome P450 enzyme cDNA (CYP4C15) has been previously cloned from a cDNA library of crayfish steroidogenic glands (Y-organs). The conceptual translation of the CYP4C15 cDNA sequence was analyzed for regions of putative high antigenicity and a mixture of two synthetic peptides was chosen for the production of a specific polyclonal antibody. Western blot analysis on Y-organ subcellular fractions indicated an endoplasmic reticulum location of CYP4C15, in agreement with the structural feature of the predicted protein, i.e. the presence of a hydrophobic N-terminal segment.
Marine Ecology Progress Series, 2004
Ecdysteroid levels were investigated by HPLC-MS over the moult cycle and in relation to reproduction in male shore crabs Carcinus maenas. Ecdysone (E), 20-hydroxyecdysone (20E) and Ponasterone A (PoA) were quantified in the haemolymph, hepatopancreas and testis. Also, the expression of 2 recently discovered Cytochrome p 450 genes (CYP330A1 and CYP4C39) inducible by ecdysteroids was studied in the hepatopancreas by Northern blot hybridisation analysis. In the haemolymph and hepatopancreas, all 3 ecdysteroids varied over the moult cycle with high levels in premoult and low levels in postmoult and intermoult. In the testis, 20E and E were present at high levels except in Postmoult Stage A, where low levels were observed. PoA was never observed in the testis. Ecdysteroids were quantified in the red and green colour forms of late intermoult C 4 crabs. In both phenotypes, 20E was the dominating ecdysteroid in late intermoult. In the haemolymph, 20E levels did not vary between the 2 phenotypes, but haemolymph 20E levels were negatively related to size. Also, haemolymph 20E levels varied with season in late-intermoult crabs, with higher levels during spring and autumn than during summer. Green crabs had significantly higher testicular E levels than red crabs. Ecdysteroid levels were negatively related to CYP330A1 and CYP4C39 gene expression. CYP330A1 and CYP4C39 mRNA levels were low during intermoult and premoult but high during postmoult. The results suggest that E and 20E are involved in both growth and reproduction whereas PoA is involved in moulting but not in reproduction and that the testis of male shore crab may be a possible source of ecdysone production in addition to the Y-organ. The results also support the concept that the probability of male shore crabs entering a new moult cycle decreases with increasing size.
Frontiers in Zoology, 2014
Introduction: Daphnia magna exhibits a parthenogenetic reproductive cycle linked to a moulting cycle, but regulatory mechanisms of neither moulting nor reproductive cycle are understood in daphnids. Moulting is regulated by ecdysteroids in insects. A previous study showed that a titre of ecdysteroids changed during the reproductive cycle in D. magna; however, no clear correlation among titre, moulting and reproductive cycles has been proved in daphnids. To understand endocrine mechanisms underlying the coordinated reproductive cycle, we analysed the expression of genes coding for enzymes in ecdysteroids synthesis or inactivation pathways, and the effects of 20-hydroxyecdysone (20E) on moulting and ovulation in D. magna. Results: We cloned orthologues of neverland (nvd) and shade (shd) in the ecdysteroids synthesis pathway, and Cyp18a1 in the ecdysteroids inactivation pathway previously identified in insects. Gene expression of Cyp18a1 changed conversely with the fluctuation in ecdysteroids titre during the intermoulting period. Tissue-specific expression analysis of nvd showed a prominent expression in the gut. Furthermore, treatment of adult female D. magna with 20E inhibited moulting and/or ovulation. Conclusions: Our cloning and phylogenetic analyses showed that nvd and shd as well as Cyp18a1 are evolutionary conserved in D. magna, suggesting that these genes appeared in arthropods before the radiation of insects. The gene expression analysis during the reproductive cycle indicated that Cyp18a1 possibly regulates the decline of ecdysteroid titre before moulting and ovulation. Furthermore, the expression of nvd in the gut suggested that ecdysone might be synthesised in the gut. Exogenous 20E-treatment resulted in the failure of not only moulting, but also ovulation, suggesting that a low level of ecdysteroids before moulting is required for moulting and ovulation in D. magna.
Aquatic Toxicology, 2008
Anthropogenic chemicals released into the environment may have so-called endocrine disruptive effects. For instance, innumerous observations on subtle alterations of fish reproductive systems have been published in the scientific literature during the last decades. At the same time, the evidence is scarce regarding similar effects in crustaceans, which is probably related to the limited understanding of basic crustacean endocrine systems and pathways, rather than absence of endocrine disruption within the crustacean subphylum. This knowledge gap is particularly evident in micro-crustacean species, which are frequently used in environmental risk assessment of chemicals, and adequate tools for identification of potential endocrine disrupters are missing in current chemicals regulation. The main objective of the current study was therefore to utilize an enzyme immunoassay to establish a stable protocol for analysis of individual ecdysteroid levels in the benthic harpacticoid copepod Nitocra spinipes, a species which has been used in ecotoxicological investigations for more than 30 years. Further, to assess the usefulness of the individual ecdysteroid level as a stressor endpoint, it was integrated with a growth-related stressor endpoint battery, i.e. individual RNA content, mean development times, and growth rate, by exposing individual N. spinipes to the insecticide and known ecdysteroid antagonist lindane at 25-400 g L −1 . The results showed that the ecdysteroid levels were significantly different from the control (71 pg individual −1 ) in the 100 g L −1 treatment (124 pg individual −1 ). Although the ecdysteroid levels were not significantly different from the control in the 25-50 g L −1 treatments (83-93 pg individual −1 ), these results still show a clear concentration-related trend. In the 200 g L −1 treatment, the ecdysteroid content was the highest (165 pg individual −1 ), however not significantly different from the control due to high variation. The 400 g L −1 treatment resulted in lowered ecdysteroid levels (107 pg individual −1 ), indicating a profound lindane-induced stress, which was confirmed by high mortality in the same treatment (79%). For all other endpoints there were clear concentration-related effects of lindane, with development time and growth rate as the most sensitive endpoints. In conclusion, this study presents for the first time a tool for identification of endocrine alterations in N. spinipes. By using the established enzyme immunoassay protocol, we obtained individual ecdysteroid levels that integrated well with the growth-related stressor endpoints previously used on this species.
Journal of experimental zoology. Part A, Comparative experimental biology, 2004
Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined e...
2014
Title of dissertation: The functional importance and significance of ecdysteroids in molt-cycle regulation of the blue crab, Callinectes sapidus Sirinart Techa, Doctorate of Philosophy, 2014 Dissertation directed by Associate Professor J. Sook Chung Marine Estuarine Environmental Science This study aims to expand our understanding of how ecdysteroids and neuropeptide hormones (MIH/CHH) regulate molting in crustaceans using the blue crab Callinectes sapidus as a model animal. The hypothesis of this study is that ecdysteroids have a stimulatory effect on MIH/CHH production in eyestalks while generating both positive and negative feedback on ecdysteroidogenesis in Y-organs. Since ecdysteroids exert their signals through an ecdysteroid receptor complex, composed of an ecdysone receptor (EcR) and its partner, the retinoid-X receptor (RXR), the functional activity of ecdysteroids on tissues of interest is examined through EcR expression. Endogenous levels of ecdysteroids as well as expres...
Archives of Biochemistry and Biophysics, 1984
Experiments were performed to determine the ability of the cytochrome P-450 present in hepatopancreas microsomes from the spiny lobster, Panulirus argus, to catalyze oxidation of progesterone, testosterone, and ecdysone. Preparations of hepatopancreas microsomes fortified with NADPH cytochrome P-450 reductase from pig liver efficiently catalyzed NADPH-dependent 16a-and 6@-hydroxylation of progesterone and testosterone, and 21-hydroxylation of progesterone. These products were also found if NADPH and NADPH cytochrome P-450 reductase were replaced by cumene hydroperoxide. Cytochrome P-450 purified from hepatopancreas microsomes catalyzed NADPH-and reductase-dependent 16a-hydroxylation of progesterone and testosterone 10 times more rapidly than the original microsomal preparation. While ecdysone was not a substrate for the hepatopancreas microsomal cytochrome P-450, low ecdysone 20-monooxygenase activity was found in hepatopancreas mitochondria. Further studies showed that homogenates of green gland, ovaries, and testes had higher ecdysone monooxygenase activities than hepatopancreas, and that the activity in green gland was localized in mitochondria.
Biochemical and Biophysical Research Communications, 1999
Biosynthesis of ecdysteroids, arthropod steroid molting hormones, proceeds from dietary cholesterol through a complex and still incompletely elucidated pathway. Most of the known steps are catalyzed by cytochrome P450 enzymes (CYPs) but none of their genes has yet been identified. We have established a cDNA library of crayfish steroidogenic glands (Y organs). A full length CYP-cDNA was characterized containing a 1539 bp open reading frame encoding a predicted protein of 513 amino acid residues. This novel CYP was assigned to the CYP4 family and designated CYP4C15. Northern blots demonstrated predominant expression of this gene in the active molting glands, suggesting a role in ecdysteroid biosynthesis rather than detoxification.
Journal of Biological Chemistry, 2004
Ecdysteroids mediate a wide variety of developmental and physiological events in insects. In the postembryonic development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although many studies have revealed the biochemical and physiological properties of the enzymes for ecdysteroid biosynthesis, most of the molecular identities of these enzymes have not been elucidated. Here we describe an uncharacterized cytochrome P450 gene, designated Cyp306a1, that is essential for ecdysteroid biosynthesis in the PGs of the silkworm Bombyx mori and fruit fly Drosophila melanogaster. Using the microarray technique for analyzing gene expression profiles in PG cells during Bombyx development, we identified two PG-specific P450 genes whose temporal expression patterns are correlated with changes in ecdysteroid titer during development. Amino acid sequence analysis showed that one of the Bombyx P450 genes belongs to the CYP306A1 subfamily. The temporal and spatial expression pattern of the Drosophila Cyp306a1 homolog is essentially the same as that of Bombyx Cyp306a1. We also found that Drosophila Cyp306a1 is disrupted in the phantom (phm) mutant, known also as the Halloween mutant. The morphological defects and decreased expression of ecdysone-inducible genes in phm suggest that this mutant cannot produce a high titer of ecdysone. Finally we demonstrate that S2 cells transfected with Cyp306a1 convert ketodiol to ketotriol via carbon 25 hydroxylation. These results strongly suggest that CYP306A1 functions as a carbon 25 hydroxylase and has an essential role in ecdysteroid biosynthesis during insect development.