Molting cycle-dependent expression of CYP4C15, a cytochrome P450 enzyme putatively involved in ecdysteroidogenesis in the crayfish, Orconectes limosus (original) (raw)
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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.
Endocrinology, 2007
Cytochrome P450c17 is the single enzyme that mediates the 17␣-hydroxylase and 17, 20 lyase activities during the biosynthesis of steroid hormones in the gonads and adrenal gland. However, the mechanism underlying its dual action continues to be a controversy in the field of steroidogenesis in fish. In an attempt to resolve this issue, we identified a novel type of P450c17 (P450c17-II) by an in silico analysis from the genomes of six fish species. We cloned P450c17-II from tilapia and medaka, and comparison with the conventional P450c17-I revealed that they differ in gene structure and enzymatic activity. Enzymatic assays by thin-layer chromatography revealed that P450c17-II possesses only the 17␣-hydroxylase activity without any 17, 20 lyase activity, unlike P450c17-I, which has both these activities. In testis, both P450c17-I and -II express in the interstitial cells. Remarkable differences, revealed by in situ hybridization, in the expression patterns of the P450c17-I and -II in the ovary and head kidney of tilapia during various stages of development strongly suggest that P450c17-I is responsible for the synthesis of estradiol-17 in the ovary, whereas P450c17-II is required for the production of C21 steroids such as cortisol in the head kidney. More interestingly, a temporally controlled switching is observable in the expression of these two genes during the steroidogenic shift from estradiol-17 to the C21 steroid, 17␣, 20-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of fish oocytes) in the fish ovary, revealing a role for P450c17-II in the production of hormones that induce oocyte maturation in fish.
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.
General and Comparative Endocrinology, 2008
The marine copepod Calanus finmarchicus is the most abundant zooplankton species in the northern regions of the Atlantic Ocean and the Barents Sea. Very little is known about molecular regulation of hormone metabolism, moulting and reproduction in copepods. To investigate these processes, we sampled adult male and female copepods (females at three distinct reproductive stages) and copepodites stage five (CV) from the culture at SINTEF/NTNU Sealab. Copepods were individually photographed, analyzed biometrically (body size, length and lipid storage size) and for ecdysteroid concentrations. In addition, we analyzed copepods for gene expression of three putative cytochrome P450 enzymes possibly involved in ecdysteroid regulation: CYP301A1, CYP305A1 and CYP330A1. The CV group exhibited the highest ecdysteroid concentrations and the largest lipid storage size, and a significant positive correlation was found between these parameters. Also, two of the P450 enzymes (CYP305A1 and CYP330A1) were more highly expressed at CV than at the adult stage, suggesting that these P450 enzymes are involved in ecdysteroid synthesis and lipid storage regulation. The expression of CYP330A1 was higher in newly moulted females than in females that had produced eggs. In addition, we observed that ecdysteroid concentrations were higher in females with large egg sacs, suggesting that ecdysteroids may be involved in egg maturation and reproduction. The CYP301A1 was more highly expressed in males and post-spawning females, and may be involved in ecdysteroid degradation since these groups also exhibited the lowest ecdysteroid concentrations.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1997
Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b 5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/ kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β-and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.
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.
Cell Tissue Res, 1998
We investigated the distribution of 3 types of steroidogenic enzymes, P450scc, 3βHSD, and P450c17, in wild raccoon dog ovaries by immunohistochemistry. Six pairs of ovaries were obtained from wild raccoon dogs between 2001 and 2003, with 3 of the 6 pairs of ovaries containing corpora lutea. P450scc, 3βHSD, and P450c17 were localized in the granulosa and theca cells of these raccoon dogs. Furthermore, lutein cells were stained positively for P450scc and 3βHSD in the pregnant and non-pregnant raccoon dogs. These results suggest that granulosa and theca cells may synthesize progesterone and androgens, which may play an important role in follicular development, and that lutein cells are a major source of progesterone in wild raccoon dogs.
Journal of Experimental Zoology, 2001
Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17α-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the “extra-gonadal” form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475–483, 2001. © 2001 Wiley-Liss, Inc.
General and comparative endocrinology, 2015
Estradiol-17β (E2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17α,20β-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17α-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cyp17a2-which encodes P450c17-II-was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I-V) encoding P450c17-...