Vg Gene Expression Research Articles

Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity

Elucidation of molecular mechanisms underlying stage- and tissue-specific expression of genes activated by a blood meal is of great importance for current efforts directed towards utilizing molecular genetics to develop novel strategies of mosquito and pathogen control. Regulatory regions of such genes can be used to express anti-pathogen effector molecules in engineered vectors in a precise temporal and spatial manner, designed to maximally affect a pathogen. The fat body is a particularly important target for engineering anti-pathogen properties because in insects, it is a potent secretory tissue releasing its products to the hemolymph, an environment or a crossroad for most pathogens. Recently, we have provided proof of this concept by engineering stable transformant lines of Aedes aegypti mosquito, in which the regulatory region A. aegypti vitellogenin ( Vg) gene activates high-level fat body-specific expression of a potent anti-bacterial factor, defensin, in response to a blood meal. Further study of the Vg gene utilizing Drosophila and Aedes transformation identified cis-regulatory sites responsible for state- and fat body-specific activation of this gene via a blood-meal-triggered cascade. These analyses revealed three regulatory regions in the 2.1-kb upstream portion of the Vg gene. The proximal region, containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh, is required for the correct tissue- and stage-specific expression at a low level. The median region, carrying sites for early ecdysone response factors E74 and E75, is responsible for a stage-specific hormonal enhancement of the Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic to the Vg gene. Furthermore, our study showed that several transcription factors involved in controlling the Vg gene expression, are themselves targets of the blood meal-mediated regulatory cascade, thus greatly amplifying the effect of this cascade on the Vg gene. This research serves as the foundation for the future design of mosquito-specific expression cassettes with predicted stage- and tissue specificity at the desired levels of transgene expression.

The vitellogenin gene of the mosquito Aedes aegypti is a direct target of ecdysteroid receptor

In the female mosquito Aedes aegypti, vitellogenin (Vg), the major YPP, is activated by 20-hydroxyecdysone (20E) at the transcriptional level. We used cell transfection assays in the Drosophila S2 cells to investigate whether 20E acts directly on the Vg gene via its functional receptor, the heterodimer composed of the ecdysteroid receptor (EcR) and the ultraspiracle (USP) proteins. We demonstrated that the Vg 5′-regulatory region contains a functional ecdysteroid-responsive element (VgEcRE1) that is necessary to confer responsiveness to 20E. VgEcRE binds directly to EcR-USP produced in vitro and extracted from the vitellogenic fat body nuclei. The binding intensity of the EcR-USP-EcRE1 complex from nuclear extracts corresponds to the levels of ecdysteroids and of the Vg transcript during the vitellogenic cycle. Given the modest level of 20E-dependent activation, it is likely that the EcR-USP receptor acts synergistically with other transcription factors to bring about the high level of Vg gene expression.

Electrostatic interactions of androgens and progesterone derivatives with rainbow trout estrogen receptor

In primary cultures of immature male rainbow trout (rt) hepatocytes, vitellogenin (Vg) gene expression is regulated by E 2 via the estrogen receptor (ER). However, steroids other than estrogens can also stimulate Vg gene expression. These steroids are hardly converted into E 2 during incubation and their stimulatory activity is completely inhibited by tamoxifen implying rtER involvement. These steroids have no or a slightly positive charge on the Connolly surface. In contrast, steroids that failed to stimulate Vg gene expression had a strong positive or negative charge around rings C and D due to polarization. The amino acid sequences of the ligand binding domains (LBD) of rtER and human ERα have 57.7% homology; only one amino acid differs in the presumed steroid binding site. We modeled the three-dimensional structure of the LBD of rtER using X-ray crystallographic data for hERα in order to investigate the fit (structural and electrostatic) between steroid and rtER. Two factors are essential for binding to rtER: (i) hydroxyl or carbonyl groups near C3 and C17 of the steroids (hydrophilic regions) that can form hydrogen bonds with His(489), Arg(359), and Glu(318), (ii) a hydrophobic steroid nucleus that interacts with a hydrophobic region of the rtER LBD through van der Waals forces. If polar functional groups are present, the hydrophobic interaction between steroid and the rtER LBD is considerably weakened.

Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae)

The present paper describes the effect of juvenile hormone III (JH III) upon vitellogenin (Vg) gene expression in cardioallatectomized females of Blattella germanica. Northern blot analyses of time course studies showed that Vg mRNA can be detected 2 h after the treatment with 1 μg of JH III. Western blot analyses revealed that Vg protein is detectable 4 h after the same treatment. The study of the influence of the age showed that 48-h-old females seem more sensitive than 24-h-old females, whereas differences were less apparent between 48- and 72-h-old females. Dose–response studies indicated that 0.01 μg of JH III is ineffective, whereas the doses of 0.1, 1 and 10 μg induced the synthesis of Vg in a dose-dependent fashion. Finally, the administration of three successive doses, of 0.01 μg of JH III each, did not result in detectable Vg production, whereas two doses of 0.01 μg followed by one of 1μg of JH III induced a greater response than that resulting from a sole dose of 1μg of JH III, which suggests that sub-effective doses of JH III elicit a priming effect on Vg production.

Estrogenic properties of municipal and industrial wastewaters evaluated with a rapid and sensitive chemoluminescent in situ hybridization assay (CISH) in rainbow trout hepatocytes

The estrogenic properties of municipal and industrial wastewaters were examined using rainbow trout hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to several concentrations of industrial/municipal wastewaters for 48 h at 15°C. Estrogenicity was evaluated on the basis of the amount of vitellogenin (Vg), as determined by the alkali-labile phosphate method, excreted into the extracellular media and vitellogenin gene expression (mRNA) by a novel and very sensitive chemoluminescent in situ hybridization assay (CISH). The results show that exposure of hepatocytes to estradiol increased Vg level present in the incubation media and Vg mRNA as estimated by the CISH method. It was also found that effluents from industries that process natural products (paper recycling) and some municipal wastewaters, ultimately discharged into the St. Lawrence River, were estrogenic to hepatocytes. In addition, anti-estrogenic effects were observed in wastewater samples from the most heavily populated towns, while those from smaller towns exhibited estrogenicity to trout hepatocytes. It is suggested that the industrial component of municipal discharges is likely to contribute to anti-estrogenicity. Primary cultures of rainbow trout hepatocytes are useful means to assess the effects of complex mixtures on Vg gene expression and Vg protein secretion.

Androgen-induced vitellogenin gene expression in primary cultures of rainbow trout hepatocytes

The hormone induction of vitellogenin (Vg) gene expression was investigated in primary cultures of immature male rainbow trout hepatocytes. Vg cDNA was cloned from the liver of a female rainbow trout injected with estradiol-17 β (E 2). A Vg mRNA of about 6.6 kb was detected in the hepatocytes after stimulation with 2×10 −9 M E 2 for 24 h. The intensity of this signal increased with E 2 concentration. Several other hormones including progesterone and androgens (testosterone, androsterone, methyltestosterone) also induced Vg mRNA not only at high (2×10 −5 M) but also at low (2×10 −9 M), physiologically relevant steroid concentrations. Only cortisol failed to do so at any tested concentration but, surprisingly, like progesterone, it induced an additive effect in the presence of E 2. On the other hand, tamoxifen drastically reduced the expression of Vg gene whether stimulated by E 2 or any other tested steroid. In conclusion, our studies have highlighted the unusual specificity of Vg mRNA induction in fish.

Differential regulation of two genes implicated in fish reproduction: vitellogenin and estrogen receptor genes.

In rainbow trout as well as in other species, variability of estrogen receptor (ER) gene expression according to the cell type and the physiological state reflects a differential cell and gene sensitivity to estrogen. We previously demonstrated that expression of the rainbow trout estrogen receptor (rtER) and vitellogenin (Vg) genes were induced differently by estrogens in rainbow trout liver. Therefore, these two genes offered a suitable model to study the influence of ER concentration on gene transcriptional activities. In the present study we show that the transcription rate of rtER and Vg genes during an estrogenic treatment are affected differently by variation of cellular ER concentration. We demonstrate that rtER gene exhibits a low threshold response to loaded estrogen receptor, and increasing ER amounts do not affect the transcriptional response of this gene during an estrogenic stimulation. On the contrary, Vg gene expression requires the presence of a higher loaded estrogen receptor level to be induced, and its transcriptional response is directly proportional to the amount of synthesised ER.

Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression

Estrogen receptor (ER) and vitellogenin (Vg) gene expression are strongly up-regulated by estrogens in rainbow trout liver. In this paper, we have used primary cultured hepatocytes to examine the mechanisms implicated in estrogen regulation of ER and Vg gene expression. Treatment of hepatocytes with 1 μM estradiol (E2) led to a rapid increase in ER mRNA level (15 fold) followed by Vg mRNA induction. Transcription rate and mRNA half-life determination carried out in the presence or absence of E2, demonstrated that E2 increases both the ER and Vg gene transcriptional activity and mRNA stability (ca. 3 fold). The effect of E2 was inhibited by an excess of antiestrogens, showing that E2-stimulation of ER and Vg mRNA level is mediated by the estrogen receptor. Our data show that ER and Vg genes have different hormonal sensitivity. In fact, the Vg gene required a higher concentration of E2 to be stimulated compared to the ER gene. Examination of the mechanisms involved in post-transcriptional regulation of ER mRNA showed that the setting up and maintenance of this regulation process implies the estrogen receptor and the general translational activity within the cells, suggesting that E2-stabilization of ER mRNA depends on the synthesis of an estrogen-dependent protein. However, the cis and trans elements involved in this E2-stabilization process remain to be identified.

Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila.

The appendages of insects, like the limbs of vertebrates, grow out of the body wall after the establishment of a proximo-distal axis among a group of primordial cells. In Drosophila, the wing develops in the limbless larva from one of the imaginal discs of the thorax, which give rise to the adult epidermis. The earliest identified requirement in wing development is for the induction of vestigial (vg) gene expression at the interface between ventral cells and dorsal cells of the wing disc. It has been proposed that this event requires two reciprocal signals--one from the dorsal to the ventral cells and the other from the ventral to the dorsal cells--which trigger vg expression at the presumptive wing margin and hence initiate the development of the wing tissue. We have identified four genes--Serrate (Ser), wingless (wg), Notch and Suppressor of Hairless (Su(H))--whose activity is required during the second and early third larval instars for the expression of vg. Analysis of the functions and patterns of expression of these genes at the time of the inductive event indicates that the Ser protein acts as a dorsal signal, and the Wg protein as a ventral signal for the induction of vg expression. Furthermore, the expression of both Ser and Wg is sufficient to trigger ectopic wing development in the wing disc and leg discs. The product of the Notch gene, which encodes a receptor, is also required for this event and we suggest that its role is to integrate the inputs of Ser and Wg. We show that the induction of vg, which initiates wing development in Drosophila, requires the combined activities of Ser, wg and Notch. Based on the patterns of expression and requirements for Ser and wg in this process, we propose that Ser is a dorsal signal and that Wg is a ventral signal, and that their combination at the dorso-ventral interface activates the Notch receptor and leads to vg expression.

Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression.

Rainbow trout hepatocyte primary culture was used to test the influence of some xenobiotics on the expression of two genes implicated in reproduction, those for the estrogen implicated in reproduction, those for the estrogen receptor (ER) and vitellogenin (Vg). We showed that chlordecone, nonylphenol, a polychlorobiphenol (PCB) mixture (Aroclor 1245) and lindane were able to induce ER and Vg mRNA accumulation. Antiestrogens, 4-hydroxytamoxifen and ICI 164,384, prevented the effects of the xenobiotics, indicating that the induction of gene expression is mediated by the ER. Among these four xenobiotics, only chlordecone and nonylphenol were able to displace the binding of [3H]estradiol to ER-enriched COS-1 extracts, and to activate an estrogen-dependent reporter gene (ERE-TK-CAT) cotransfected with an expression vector containing ER cDNA. The results suggest that chlordecone and nonylphenol are direct inducers of rainbow trout ER and Vg gene expression, whereas PCBs and lindane act through their hepatic metabolites. Moreover, pentachlorophenol acts as an antagonist of the induction by estradiol of rainbow trout ER and Vg gene expression.

Cortisol-Induced Hepatic Vitellogenin mRNA in Oreochromis aureus (Steindachner)

Cortisol triggers rapid but transient transcription of the silent vitellogenin (Vg) gene in male Oreochromis aureus, producing short-lived Vg mRNA. Implantation of cocoa butter containing cortisol at 0.8, 8, 20, and 40 μg/g fish body wt induced hepatic Vg mRNA within 1 or 2 hr. Implantation of cortisol at the lowest of these dosages (0.8 μg/g body wt) produced a transient surge in serum cortisol within 1 hr. This is equivalent to a 30-fold increase over that of the resting male fish in which the endogenous cortisol levels measured 10.5 ± 2.39 and 11.4 ± 3.1 ng/mt, respectively, for fish kept communally or singly. Although sham implantation with cocoa butter also resulted in the elevation of serum cortisol, the rise was much more gradual, only reaching a peak after 6 hr. Thus, parameters like the treatment regime, sampling time intervals after implantation, and the interaction between these two factors can significantly affect the serum cortisol concentrations (P = 0.001). Although implantation of cocoa butter with or without cortisol increased serum cortisol, albeit unsynchronized. Vg mRNA was detected only in the hormone-treated group. The profile of Vg mRNA accumulation appeared to coincide with that of serum cortisol, further suggesting the activating effect of exogenous cortisol on Vg gene. Compared with an earlier study on estradiol-induced Vg gene expression, the action of cortisol was more rapid, but transient. This is reflected in the rapid clearance of the cortisol-induced Vg mRNA and, hence, the lack of plasma Vg protein during cortisol treatment.

Demethylation of the Gene Expressing a Yolk Protein Precursor in Quail

Japanese quail produce more egg-yolk protein per gram of body weight than chickens do. Because cytosine phosphoguanine (CpG) methylation has been reported to correlate inversely with gene activity, the demethylation of cytosine residues in the quail vitellogenin 2 (Vg2) gene was assessed as a possible contributing factor. Vitellogenin genes are normally transcribed in the liver of female Japanese quail that are sexually mature. Changes in the methylation pattern of CCGG sites1 of the quail Vg2 gene were studied using the methyl-sensitive restriction endonucleases Msp I and Hpa II. At least five CCGG sites were detected within the transcribed region of the Vg 2 gene; one or two of these sites were hypomethylated in all tissues examined in male and female quail, two sites underwent transcription-dependent demethylation in the liver of laying females and also in the liver of males following estradiol injection (64 μmol estradiol/mL of absolute ethanol at .25 mL/100 g of BW), and another site was demethylated in the liver of estradiol-injected males but not in laying females. Two further sites present within the 5′ flanking sequences or 5′ structural region2 underwent transcription-dependent demethylation in the liver of laying females and of estradiol-treated males; one or both of these sites appears to be demethylated in the oviduct. Another site, within the 3′ flanking sequences or the 3′ structural gene region, underwent estradiol-dependent demethylation in the liver and the oviduct. The expression-linked demethylation of the CCGG sites in the Vg2 gene of quail appears to be similar to that reported for the chicken VTGII gene. The results of this study did not explain the enhanced production of yolk protein in Japanese quail, but did suggest the presence of a fundamental, conserved mechanism that must play an important role in the expression of the avian Vg gene.

Molecular analysis of hybrid dysgenesis-induced derivatives of a P-element allele at the vg locus.

Secondary and tertiary derivatives of a P-element insertion allele at the vestigial (vg) locus were induced by hybrid dysgenesis. The derivatives were characterized by Southern analyses and, in four cases, by DNA sequencing. The alterations found were P-element internal deletions, deletions of the insert and/or adjacent vg region DNA, or novel insertions of P-element sequences into existing P-element inserts. The relatively high frequency of secondary insertions into P-element sequences observed herein is unusual, since secondary insertions have seldom been recovered in other dysgenic screens. The effects of the alleles on vg expression were determined. The results are consistent with a model in which the insertions disrupt vg gene expression by transcriptional interference.

Molecular Analysis of Hybrid Dysgenesis-Induced Derivatives of a P-Element Allele at the vg Locus

Secondary and tertiary derivatives of a P-element insertion allele at the vestigial (vg) locus were induced by hybrid dysgenesis. The derivatives were characterized by Southern analyses and, in four cases, by DNA sequencing. The alterations found were P-element internal deletions, deletions of the insert and/or adjacent vg region DNA, or novel insertions of P-element sequences into existing P-element inserts. The relatively high frequency of secondary insertions into P-element sequences observed herein is unusual, since secondary insertions have seldom been recovered in other dysgenic screens. The effects of the alleles on vg expression were determined. The results are consistent with a model in which the insertions disrupt vg gene expression by transcriptional interference.

Caractérisation de récepteurs spécifiques à l'œstradiol, induction de la vitellogénine et de son mRNA dans le foie de truite arc-en-ciel (Salmo gairdnerii)

In all egg laying vertebrates, synthesis and use of vitellogenin (Vg) are intimately bound to the active phase of reproduction. In the liver of the rainbow trout (Salmo gairdnerii), Vg synthesis is influenced by estradiol (E2) which, we believe, acts through the classical mechanism of steroid hormone action. After binding of the hormone to a soluble specific receptor protein, the estradiol-receptor complex can interact with chromatin and modulate the expression of Vg genes, leading to increased synthesis of specific mRNA and Vg. We show here: (i) the presence of specific oestrogen receptors (dissociation constant KD congruent to 1.5 X 10(-9) M for E2) in the cytosol of the male trout liver. (ii) The male liver, offering, an ideal experimental control of "zero" background, we followed-in the liver of male trout--the kinetics of induction of Vg mRNA by hybridization with Vg cDNA, after E2 stimulation, and (iii) the apparition of Vg in the serum by using an original rocket immuno-electrophoretic technique. The male trout liver vitellogenin model and the original techniques we developed will be very useful to study the influence of endogenous and exogenous factors on the different steps (receptors, transcription, translation) of vitellogenesis regulation.