Ecdysteroid-phosphate Phosphatase Research Articles

Expressions of sugar transporter genes during Bombyx mori embryonic development.

Sugar transporters (Sts) play important roles in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. Few studies have been conducted on expressions of Sts during insect embryonic development. In the present study, we investigated temporal expressions of St genes during the embryonic diapause process in Bombyx mori. We found that in HCl-treated developing eggs, high gene expressions of trehalose transporter 1 (Tret1) were detected during middle and later embryonic development. St4 and St3 gene expressions gradually increased during the early stages, reached a small peak on Day 3, and large peaks were again detected on Day 7. However, in diapause eggs, expression levels of the Tret1, St4, and St3 genes all remained at low levels. Differential temporal changes in expressions of the Tret1, St4, and St3 genes found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited similar changing patterns as those of HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development. In addition, high gene expressions of Tret1 were also detected when dechorionated eggs were incubated in the medium. The addition of LY294002 (a specific phosphatidylinositol 3-kinase [PI3K] inhibitor) and U0126 (a mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase [MEK] inhibitor) partially inhibited Tret1 gene expression in dechorionated eggs, but did not affect either ecdysteroid-phosphate phosphatase gene expression or ecdysteroid biosynthesis, clearly indicating that both PI3K and ERK are involved in increased gene expression of Tret1 that was independent of ecdysteroid levels. To our knowledge, this is the first comprehensive report to demonstrate the transcriptional regulation of St genes during embryonic development, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

Changes in expressions of ecdysteroidogenic enzyme and ecdysteroid signaling genes in relation to Bombyx embryonic development.

Although the role of ecdysteroids in regulating egg diapause process in Bombyx mori is well documented, temporal changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling are less well understood. In the present study, we studied changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling during embryonic development of B. mori. Results showed that in diapause eggs, the expression of ecdysteroid-phosphate phosphatase (EPPase) gene and Halloween genes (Spook [Spo] and Shade [Shd]) remained at very low levels. However, in eggs whose diapause initiation was prevented by HCl, significant increases in the messenger RNA (mRNA) levels of EPPase, Spo, and Shd were detected during embryonic development. Other Halloween genes (Neverland [Nvd] and Phantom [Phm]) also showed different changes between diapause and HCl-treated eggs. However, genes of Disembodied (Dib) and Shadow (Sad) showed similar changes in both diapause and HCl-treated eggs. We further investigated changes in expression levels of ecdysone receptor genes (EcRA, EcRB1, and USP) and downstream signaling genes (E75A, E75B, E74A, E74B, Br-C, HR3, HR4, KR-H1, and FTZ-F1). Results showed that genes of EcRA and the other nuclear receptors (E75A, E75B, E74A, HR3, HR4, KR-H1, and FTZ-F1) exhibited significant differential patterns between diapause and HCl-treated eggs, with increased levels being detected during later stages of embryonic development in HCl-treated eggs. Differential temporal changes in expressions of genes involved ecdysteroid biosynthesis and its downstream signaling found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited the same changing patterns as those in HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development in B. mori. To our knowledge, this is the first comprehensive report to study the transcriptional regulation of ecdysteroidogenic and ecdysteroid signaling genes, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori

Steroid hormones, represented by estrogen and testosterone, act as sex hormones that play an essential role in the sexual differentiation of vertebrates. However, it remains unclear whether ecdysteroids, typical steroid hormones in insects, function as sex hormones. In this study, we investigated whether ecdysteroids or ecdysone signals are involved in the sexual differentiation of the silkworm (Bombyx mori) embryo. Quantitative analysis using LC-MS/MS demonstrated that there was no significant difference in the 20-hydroxyecdysone (20E) titer between sexes during embryonic development. Consistent with this result, expression levels of 2 genes encoding ecdysteroid-phosphate phosphatase (EPPase) and ecdysone 20-hydroxylase (E20OHase), which are essential for the biosynthesis of ecdysone and 20E in eggs, did not show a significant difference between male and female embryos. Expression levels of ecdysone receptor (EcR) and E75, which is one of a small set of genes induced directly by 20E, were also similar between the 2 sexes. However, knockdown of EPPase and one isoform of EcR (EcR-A) resulted in decreased expression of Bombyx doublesex (Bmdsx), a master regulatory gene for sexual differentiation of the silkworm in both male and female embryos. In vitro analysis with cultured testes revealed that expression levels of Bmdsx were increased in a dose-dependent manner of the ecdysone analog, ponasterone A. These results suggest that ecdysone signaling may play a role in indirectly regulating the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx in the silkworm.

Timing of embryonic quiescence determines viability of embryos from the calanoid copepod, Acartia tonsa (Dana)

Like 41 other calanoid copepods, Acartia tonsa, are capable of inducing embryonic quiescence when experiencing unfavorable environmental conditions. The ecdysone-signaling cascade is known to have a key function in developmental processes like embryogenesis and molting of arthropods, including copepods. We examined the role of ecdysteroid-phosphate phosphatase (EPPase), ecdysone receptor (EcR), ß fushi tarazu transcription factor 1 (ßFTZ-F1), and the ecdysteroid-regulated early gene E74 (E74), which represent different levels of the ecdysone-signaling cascade in our calanoid model organism. Progression of embryogenesis was monitored and hatching success determined to evaluate viability. Embryos that were induced quiescence before the gastrulation stage would stay in gastrulation during the rest of quiescence and exhibited a slower pace of hatching as compared to subitaneous embryos. In contrast, embryos developed further than gastrulation would stay in gastrulation or later stages during quiescence and showed a rapid pace in hatching after quiescence termination. Expression patterns suggested two peaks of the biological active ecdysteroids, 20-hydroxyecdysone (20E). The first peak of 20E was expressed in concert with the beginning of embryogenesis originating from yolk-conjugated ecdysteroids, based on EPPase expression. The second peak is suggested to originate from de novo synthesized 20E around the limb bud stage. During quiescence, the expression patterns of EPPase, EcR, ßFTZ-F1, and E74 were either decreasing or not changing over time. This suggests that the ecdysone-signaling pathway play a key role in the subitaneous development of A. tonsa embryogenesis, but not during quiescence. The observation is of profound ecological and practical relevance for the dynamics of egg banks.

Early embryonic expression of a putative ecdysteroid-phosphate phosphatase in the water flea, Daphnia magna (Cladocera: Daphniidae).

Ecdysteroids, known as molting hormones, play central roles in the onset of molting, metamorphosis, and reproduction in arthropods. The ecdysteroids stored in eggs also play an important role in embryogenesis. In insects, ecdysteroids are stored as phosphate esters, which are converted to an active form by ecdysteroid-phosphate phosphatase (EPPase). Although EPPase is believed to be widely conserved in the Ecdysozoa, little is known about its expression in clades other than Insecta. In this study, we cloned a putative EPPase gene from a small fresh water crustacean known as a water flea, Daphnia magna Straus (Cladocera: Daphniidae), and examined its expression during embryogenesis. The amino acid sequence of the putative crustacean EPPase cDNA showed high similarity to insect EPPase and human suppressor of T-cell receptor signaling-1. We also found that the D. magna EPPase was highly expressed during early embryogenesis; its expression rapidly decreased 6 h after oviposition. This timing corresponds to the onset of organogenesis in D. magna . The expression of EPPase could not be detected in diapaused eggs. This is the first report of an EPPase from crustaceans, and the results suggest that the function of EPPase is conserved between insects and crustaceans.

Synthesis and Phosphorylation of Ecdysteroids During Ovarian Development in the Silkworm, Bombyx mori

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.

Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori

In various insects, 20-hydroxyecdysone (20E) is indispensable for embryonic development. In eggs of the silkworm Bombyx mori, 20E has been demonstrated to be produced by two metabolic pathways: de novo synthesis from cholesterol and dephosphorylation of ovary-derived physiologically inactive ecdysteroid phosphates. In the former, ecdysone 20-hydroxylase (E20OHase) has been suggested to be a key enzyme. In the latter, it has been demonstrated that the dephosphorylation of ecdysteroid phosphates is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (EPPase). In this study, a cDNA encoding E20OHase was cloned from 3-day-old nondiapause eggs of B. mori and sequenced using PCR techniques. The protein exhibited the signature sequences characteristic of P450 enzymes, and mediated the conversion of ecdysone to 20E using the baculovirus expression system. Semi-quantitative analysis revealed that the E20OHase mRNA is expressed predominantly during gastrulation and organogenesis in nondiapause eggs, but is scarcely detected in diapause eggs whose development is arrested at the late gastrula stage. The developmental changes in the expression patterns of E20OHase and EPPase suggest that both enzyme activities are regulated at the transcription level, and both enzymes contribute cooperatively to 20E formation during embryonic development.

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme, Ecdysteroid 22-Kinase

This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365-26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase.

ERK/MAPK regulates ecdysteroid and sorbitol metabolism for embryonic diapause termination in the silkworm, Bombyx mori

The eggs of the silkworm Bombyx mori undergo a state of suspended overt development and diminished metabolism called ‘diapause’ to escape adverse environmental conditions. Termination of Bombyx embryonic diapause requires 2–3 months of low temperature (5 °C), but the molecular mechanisms underlying diapause termination are unknown. Diapause termination requires a decrease in the sorbitol concentration, which arrests embryonic development, and the secretion factors from yolk cells that promote embryonic development. In the present study, we report that 20-hydroxyecdysone promoted the development of denuded embryos and that ecdysteroid-phosphate phosphatase (EPPase), which is a key enzyme for active ecdysteroid production, was induced by incubation of diapausing eggs at 5 °C. In dechorionated egg cultures, extracellular signal-regulated kinase (ERK), which is activated by incubating diapausing eggs at 5 °C, regulated sorbitol–glycogen conversion, ecdysteroid secretion via gene transcription of key enzymes, sorbitol dehydrogenase-2, and EPPase, suggesting that ERK has a key role in diapause termination.

Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions.

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3'- and 5'-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.