Vitellogenin Gene Expression Research Articles

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP ( BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.

Effects of eutrophication on vitellogenin gene expression in male fathead minnows ( Pimephales promelas) exposed to 17α-ethynylestradiol in field mesocosms

This study evaluated the effect of aquatic secondary nutrient supply levels (nitrogen and phosphorus) on the subcellular response of adult male fathead minnows ( Pimephales promelas) exposed to a single nominal concentration of 17α-ethynylestradiol (EE2), a potent synthetic estrogen, under quasi-natural field conditions. Outdoor mesocosms were maintained under low, medium, and high nutrient supply conditions as categorized by total phosphorus (TP) level (nominal 0.012, 0.025, and 0.045 mg TP/L, respectively), and treated with EE2 with and without a carrier solvent. Using reverse transcription-polymerase chain reaction methods, vitellogenin gene ( Vg) expression was determined in the fish collected at 0 h, 8 h, 24 h, 4 d, 7 d, and 14 d post-exposure. Induction of Vg was detected as early as 8 h post-exposure, with and without the carrier solvent, and persisted through Day 14. Results showed Vg to be significantly greater at low nutrient levels ( p < 0.05), suggesting that EE2 bioavailability to the fish was likely greater under less-turbid water conditions.

Equal contribution of hepatopancreas and ovary to the production of vitellogenin (PmVg1) transcripts in the tiger shrimp, Penaeus monodon

Although the gene fragment and partial cDNA for the vitellogenin (Vg) of Penaeus monodon have been reported for several years, the complete gene sequence and full-length cDNA of this Vg has not been reported. Here we report the gene organization and cloning of the full-length cDNA of P. monodon Vg. Similar to the MeVg1 of M. ensis, the Vg gene of P. monodon (PmVg1) is confirmed to consist of 14 introns and 15 exons. Additionally, genomic DNAs that show high sequence identity to PmVg1 were isolated from a single shrimp. The result confirmed the existence of multiple Vg genes in P. monodon. PmVg cDNA is 7.8 kb in size and the deduced precursor is most similar to the Vg of Penaeus merguenesis (86% identity). Contrary to the previous report, similar levels of PmVg1 transcripts can be detected from the hepatopancreas and ovary suggesting that the ovary also plays a major role in the contribution of PmVg1 transcript during vitellogenesis. In shrimp at early vitellogenesis (i.e., GSI < 3%), the expression of PmVg1 in the ovary and hepatopancreas is low; the expression reached the maximum level in shrimp with GSI 4–8% and decreased in shrimp with GSI > 9%. Farnesoic acid and 20-hydroecdysone can cause a significant stimulation of PmVg1 expression in an in vitro hepatopancreas explant culture. In conclusion, vitellogenesis in P. monodon is most likely the result of the expression of multiple vitellogenin genes and the hepatopancreas and ovary contribute equally to the production of PmVg1 transcripts.

Transcriptional modulation of brain and hepatic estrogen receptor and P450arom isotypes in juvenile Atlantic salmon ( Salmo salar) after waterborne exposure to the xenoestrogen, 4-nonylphenol

The molecular basis for vitellogenin (Vtg) and Zr-protein gene (and protein) expression shows that the Vtg and Zr-protein gene activations are receptor-mediated responses that are ligand structure-dependent interactions with ER, probably involving all isoforms, in addition to other co-activators. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of the male and female reproductive tracts. In vertebrates, the cytochrome P450 aromatase (P450arom) is a crucial steroidogenic enzyme catalyzing the final step in the conversion of androgens to estrogens. The present study was undertaken to investigate the effects of nonylphenol on brain and liver ER and P450arom isotypes gene expressions as sensitive biomarker of effect. Fish were exposed to static and continuous waterborne nonylphenol at 5, 15 and 50 μg/L. Blood, brain and liver samples were collected after 0 (control), 3 and 7 days post-exposure. Plasma levels of Vtg and Zr-proteins were analyzed using immunoblotting and enzyme linked immunosorbent assay (ELISA) methods. A quantitative RT-PCR method was used to analyse gene expression patterns using cloned plasmid containing amplicons of interest as standards. Our data demonstrate that both ER and P450arom gene isotypes showed differential organ-specific, nonylphenol concentration- and time-dependent expression patterns after exposure to environmental relevant concentrations of nonylphenol. Liver and plasma Vtg and Zr-protein gene and protein expression showed a concentration- and time-dependent induction at day 3 and 7 post-exposure. This is the first study evaluating, in parallel, the ERs, P450aroms and protein xenoestrogen biomarkers of effect in any fish species or lower vertebrate. Therefore, the present study shows that the brain and hepatic ER and P450arom isotypes evaluated using state-of-the-art molecular approaches are sensitive xenoestrogen biomarkers of effect. These responses should be evaluated in parallel with plasma protein biomarkers such as Vtg and Zr-proteins.

Vitellogenin gene expression as a biomarker of endocrine disruption in the invertebrate, Mytilus edulis

Vitellogenin levels are often used as a biomarker of endocrine disruption in fish. For invertebrates, there is a general lack of knowledge regarding endocrine regulation and, consequently, there are few direct biomarkers of endocrine disruption in such species. This study focuses on the marine mussel Mytilus edulis, which is often employed in biomonitoring studies. A partial vitellogenin mRNA has been isolated and gene expression quantified using real-time PCR.

The effects of crustacean hyperglycemic hormone-family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus

In crustaceans, eyestalk ablation induces gonadal maturation of which vitellogenin gene expression is an essential step. However, the molecular mechanisms by which the hormones produced by the X-organ/sinus gland complex in the eyestalk regulate vitellogenesis remain poorly understood. We therefore investigated the effects of sinus gland extracts and certain sinus gland peptides belonging to the crustacean hyperglycemic hormone peptide family on vitellogenin gene expression in ovarian fragments of immature kuruma prawn, Marsupenaeus japonicus. Vitellogenin mRNA levels in incubated ovarian fragments were significantly higher than those in unincubated ovarian fragments prepared from the same animal. Sinus gland extracts and sinus gland peptide-III (type I peptide) both reduced vitellogenin mRNA levels in a dose-dependent manner. In contrast, neither molt-inhibiting hormone (sinus gland peptide-IV) nor molt-inhibiting hormone B, both of which are type II peptides, exerted significant effects on vitellogenin mRNA levels. These results suggest that, in the immature ovary, sinus gland peptide-III is involved in the suppression of vitellogenin gene expression. The existence of such a peptide in the X-organ/sinus gland complex provides a rationale for the significant increase in vitellogenin mRNA levels in the ovaries of eyestalk-ablated prawns.

Induction of vitellogenin mRNA in juvenile chinese sturgeon (acipenser sinensis gray) treated with 17β‐estradiol and 4‐nonylphenol

It has been demonstrated that 4-nonylphenol (4-NP) exerts estrogenic effects in diverse fishes. The present study investigated the effects of 4-NP on Chinese sturgeon (Acipenser sinensis Gray) vitellogenin (VTG) gene expression. By reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers, a 462-base pair fragment of Chinese sturgeon VTG, corresponding to a 154-amino acid sequence, was amplified and sequenced. This sequence exhibited 152/154 identity to the amino acid sequence of white sturgeon (A. transmontano) VTG. Conventional RT-PCR and quantitative real-time RT-PCR were established and used to study the VTG and beta-actin gene expression in the liver of juvenile Chinese sturgeon injected three times with 17beta-estradiol (5 mg/kg body wt/week) or 4-NP (10 or 100 mg/kg/week) in the course of three weeks. Significant induction of VTG gene expression was detected in all treated groups, and no VTG mRNA was detected in the control group. The ratio of VTG to beta-actin analyzed from the results of quantitative real-time RT-PCR reached 0.041 +/- 0.024 (mean +/- SD) in the group receiving 10 mg/kg/week of 4-NP and 4.51 +/- 1.68 in the group receiving 100 mg/kg/week of 4-NP. Chemical analysis of 4-NP showed that the concentrations of 4-NP in the 10 mg/kg/week group and the 100 mg/kg/week group were 2.78 +/- 2.41 and 31.38 +/- 0.26 mcirog/ g wet weight, respectively. Compared with the 4-NP concentrations (0.8-1.92 microg/g wet wt) in fish from the Yangtze River, China, a potential hazard exists regarding 4-NP in Chinese sturgeon. These results represent the first indication of the risk of endocrine-disrupting chemicals for Chinese sturgeon.

The dynamics of vitellogenin gene expression differs between intact and eyestalk ablated kuruma prawn Penaeus (Marsupenaeus) japonicus

In order to compare the dynamics of vitellogenin gene expression between naturally maturing prawns and prawns induced to mature artificially by eyestalk ablation, a cDNA encoding vitellogenin was cloned from a cDNA library prepared from the hepatopancreas of the kuruma prawn Penaeus (Marsupenaeus) japonicus, and a quantitative real-time reverse transcription — polymerase chain reaction (RT-PCR) system was developed. Sequence analysis revealed the likely possibility that vitellogenin cDNA from the hepatopancreas was identical to that from the ovary which had been isolated in a previous study. Based on this information, a quantitative real-time RT-PCR system was established and the dynamics of vitellogenin mRNA levels were examined. In naturally maturing prawns, vitellogenin mRNA levels were maintained at low levels during the previtellogenic stage, and thereafter, levels increased as vitellogenesis progressed but decreased during the latter stages of maturation in the hepatopancreas and ovary. In contrast, in eyestalk-ablated prawns, changes in mRNA levels differed in both tissues; an obvious increment of mRNA levels was revealed in the ovary, whereas mRNA levels were negligible in the hepatopancreas. This suggests that eyestalk ablation cannot be used to accurately simulate the natural process of vitellogenin gene expression during vitellogenesis, and that vitellogenin gene expression is regulated in a tissue-specific manner.

Expression of the Reproductive Female-Specific Vitellogenin Gene in Endocrinologically Induced Male and Intersex Cherax quadricarinatus Crayfish1

In oviparous females, the synthesis of the yolk precursor vitellogenin is an important step in ovarian maturation and oocyte development. In decapod Crustacea, including the red-claw crayfish (Cherax quadricarinatus), this reproductive process is regulated by inhibitory neurohormones secreted by the endocrine X-organ-sinus gland (XO-SG) complex. In males, the C. quadricarinatus vitellogenin gene (CqVg), although present, is not expressed under normal conditions. We show here that endocrine manipulation by removal of the XO-SG complex from male animals induced CqVg transcription. The CqVg gene was expressed differentially during the molt cycle in these induced males: no expression was seen in the intermolt stages, but expression was occasionally detected in the premolt stages and always detected in the early postmolt stages. Relative quantitation with a real-time reverse transcriptase-polymerase chain reaction showed that expression of CqVg in induced early postmolt males was an order of magnitude lower than that in reproductive females, a finding that was consistent with RNA in situ hybridization results. The SDS-PAGE of high-density lipoproteins from the hemolymph of endocrinologically induced early postmolt males did not show the typical vitellogenin-related polypeptide profile found in reproductive females. On the other hand, removal of the XO-SG complex from intersex individuals, which are chromosomally female but functionally male and possess an arrested female reproductive system, induced the expression, translation, and release of CqVg products into the hemolymph, as was the case for vitellogenic females. The expression of CqVg in endocrinologically manipulated molting males and intersex animals provides an inducible model for the investigation and understanding of the endocrine regulation of CqVg expression and translation in Crustacea as well as the relationship between the endocrine axes regulating molt and reproduction.

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.

Responses of molecular indicators of exposure in mesocosms: Common carp (Cyprinus carpio) exposed to the herbicides alachlor and atrazine

Common carp (Cyprinus carpio) were treated in aquatic mesocosms with a single pulse of the herbicides atrazine or alachlor to study the bioavailability and biological activity of these herbicides using molecular indicators: Liver vitellogenin gene expression in male fish for estrogenic activity, liver cytochrome P4501A1 gene expression, and DNA damage in blood cells using the single-cell gel electrophoresis method. Both alachlor and atrazine showed dose-related increases in DNA strand breaks at environmentally relevant concentrations (<100 ppb). Gene expression indicators showed that neither herbicide had estrogenic activity in the carp, whereas atrazine at concentrations as low as 7 ppb induced cytochrome P4501A1. These results support the study of molecular indicators for exposure in surrogate ecosystems to gauge relevant environmental changes following herbicide treatments.

Assessment of Estrogenic Endocrine-Disrupting Chemical Actions in the Brain Using in Vivo Somatic Gene Transfer

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

The aryl hydrocarbon receptor-mediated disruption of vitellogenin synthesis in the fish liver: Cross-talk between AHR- and ERα-signalling pathways

BackgroundIn the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERα). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERα genes, in primary cultured salmon hepatocytes, have been investigated.ResultsThe expression of the genes encoding VTG and ERα were strongly induced by 17β-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor α-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERα mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERα genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERα genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERα mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERα. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERα or that activated AHR blocked the ability of ERα to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA.ConclusionIn fish hepatocytes E2 induces ERα and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERα. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERα- and AHR-signalling pathways is unidirectional.

Estradiol-induced gene expression in largemouth bass ( Micropterus salmoides)

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E 2). Single injections of E 2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ERα sequences. After acute E 2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36–42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E 2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E 2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen γ was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E 2 involves a complex network of responses over time.

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7–10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40–50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.

Measurement of vitellogenin gene expression by RT-PCR as a tool to identify endocrine disruption in Japanese medaka (Oryzias latipes)

In order to monitor vitellogenin gene expression in the Japanese medaka (Oryzias latipes), a reverse transcription-polymerase chain reaction (RT-PCR) system was developed. To date cDNA for medaka vitellogenin has not been published; therefore, initially a sequence fragment had to be obtained and compared with other known vertebrate vitellogenins. For this, a 1.2 kb cDNA of medaka vitellogenin (M-Vg1.2) was amplified by RT-PCR and cloned into a pCR® II-TOPO bacterial vector. On Northern blot analysis, the antisense cRNA of M-Vg1.2 stained a 5.5 kb gene product found exclusively in female fish, but not in males. Additionally, the 5'-end of medaka vitellogenin cDNA was amplified by 5'-RACE-PCR. The analysed nucleotide sequence of 1.6 kb shared significant similarities with vitellogenins known from other fish species: approximately 72% similarity with mummichog (Fundulus heteroclitus) vitellogenin I and approximately 62% with fathead minnow (Pimephales promelas) vitellogenin. To develop a semiquantitative RT-PCR for the measurement of vitellogenin gene expression, primers specific to a 500 bp sequence of the vitellogenin cDNA (M-Vg0.5) were constructed using the gene product of elongation factor 1α as internal standard. Induction of vitellogenin gene expression was measured in male medaka exposed to 0, 2, 20 and 50 μg l-1 nonylphenol and 0, 2.5, 25 and 100ng l-1 17α -ethinyloestradiol for 7 days. The LOECs for vitellogenin induction in male medaka were 20 μg l-1 and 25ng l-1 for nonylphenol and 17α -ethinyloestradiol, respectively.

Induction of vitellogenin gene transcription in vitro by juvenile hormone in Blattella germanica

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10 −7 or 10 −8 M), partially inhibited the response to JH III.

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.

Temperature-dependent vitellogenin-mRNA expression in primary cultures of rainbow trout ( Oncorhynchus mykiss) hepatocytes at 14 and 18°C

In order to study the influence of temperature on vitellogenin gene and estrogen receptor gene expression in primary hepatocytes from rainbow trout ( Oncorhynchus mykiss), cells were exposed to 17β-estradiol, bisphenol-A and nonylphenol for 48 and 96 hr. Induction of vitellogenin-mRNA expression was detected in a non-radioactive dot blot/RNAse protection assay and by RT-PCR. In the dot blot/RNAse protection assay, the estrogenic potentials of bisphenol-A and nonylphenol were about 10 4- to 10 5-fold and 10 5-fold lower than that of 17β-estradiol, respectively. The relative estrogenic potential did not show any difference between 14 and 18°C. In contrast, at 18°C, RT-PCR analysis revealed increased amounts of vitellogenin- and estrogen receptor-mRNA after 12 and 24 hr of exposure to 17β-estradiol, if compared to 14°C. Owing to increased vitellogenin gene expression at 18°C, the sensitivity of primary hepatocytes to 17β-estradiol and bisphenol-A could be increased.

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.