Vitellogenin mRNA Levels Research Articles

Insulin-like peptides regulate vitellogenesis and oviposition in the green lacewing, Chrysopa septempunctata.

Insulin-like peptides (ILPs) act through a conserved insulin signaling pathway and play crucial roles in insect metabolism, growth, reproduction, and aging. Application of bovine insulin is able to increase vitellogenin (Vg) mRNA and protein levels in female insects. Here, we first show that injection of bovine insulin into previtellogenic Chrysopa septempunctata female adults promoted ovarian growth, increased Vg protein abundance, elevated reproductive performance, and enhanced protease activity. These data suggested that ILPs play crucial roles in reproductive regulation of the green lacewing, C. septempunctata.

Mechanistic study of chlordecone-induced endocrine disruption: Based on an adverse outcome pathway network

The adverse outcome pathway (AOP) framework could be helpful for chemical risk assessment and mechanistic research. The aim of the present study was to unravel the mechanism of chlordecone-induced endocrine disruption by illustrating the main molecular initiating event (MIE)/perturbations responsible for the observed effects. In silico simulations were performed to predict the MIE(s), and the results pointed to agonistic interaction with estrogen receptors (ERα, ERβ), androgen receptor (AR), cytochrome P450 (CYP19A) by chlordecone. In vivo endocrine disruptions were evaluated in rare minnow (Gobiocypris rarus) exposed to 0.01, 0.1, 1 and 10 μg L−1 chlordecone from 2 h post-fertilization until sexually mature. In the females, increases of vitellogenin (vtg) mRNA levels in liver and gonad, plasma estradiol (E2), testosterone (T) and E2/T, and renalsomatic index confirmed the role of agonism of ER and CYP19A as MIEs, but the decreased gonadosomatic index, degenerated ovaries as well as the feed-forward response pointed to other potential but important MIEs and corresponding AOPs. In the males, increased E2/T ratio, increased testis vtg mRNA levels and occurrence of intersex confirmed the roles of agonism of ERα and CYP19A as main MIEs in chlordecone-induced endocrine disruptions. Our results also fetches out the limit of AOPs in predicting the adverse outcomes and explaining the mechanism of chemicals at present, thus reflected a critical need for expanding AOPs and AOP network before using it in chemical risk assessment.

S6 protein kinase activates Juvenile Hormone and vitellogenin production in the cockroach Blattella germanica

AbstractNutritional status regulates different processes, such as growth and development, through TOR (target of rapamycin) and insulin receptor signalling pathways. The ribosomal S6 protein kinase (S6K) is a downstream element of both pathways. Using cDNA from the German cockroach Blattella germanica (L.), two S6K isoform sequences (BgS6K) are cloned. The long isoform differs from the short one by the insertion of a 22‐amino acid duplication, involving the key phosphorylation position Thr390. As a result of this, the long isoform presents a new, potentially regulatory phosphorylation site. RNA interference knockdown of both BgS6K isoforms induces an increase in the length of the last nymphal instar, together with a reduction in the mRNA levels of a number of enzymes of the Juvenile Hormone biosynthetic pathway in the corpora allata, vitellogenin mRNA levels in the fat body and basal oocyte length. Thus, BgS6K is important for nymphal development and is necessary for the full induction of Juvenile Hormone synthesis and vitellogenin production in adult females.

Thyroid endocrine status of wild European eels (Anguilla anguilla) in the Loire (France). Relationships with organic contaminant body burdens

In teleost fish, thyroid function is involved in various critical physiological processes. Given the complexity of the hypothalamo–pituitary–thyroid (HPT) axis, a large number of genes and proteins can be the potential target of endocrine-disrupting compounds (EDCs). The aim of this study was to evaluate, in yellow and silver European eels (Anguilla anguilla), potential effects of EDCs on thyroid status by analyzing the associations between EDC body burdens and thyroid hormones (THs). In yellow individuals, greater free T3/T4 ratios (FT3/FT4) and lower plasma FT4 levels were associated with greater concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), as highlighted by significant correlations with many congeners. Few positive relationships with alkylphenols were noticed. In contrast, silver eels usually exhibited less significant correlations between THs and contaminant loads. Expression of a series of genes involved in the HPT axis was also investigated in the silver individuals. Concerning mRNA expression in silver females, some main correlations were noticed: thyroid-stimulating hormone (TSHβ) gene expression was significantly correlated to numerous PCBs, and hepatic mRNA levels of deiodinase 2 (Deio 2) were negatively correlated to 2-hydroxyfluorene (2-OHF) and 2-naphtol (2-OHNa). Thyroid receptor (TRα and TRβ) mRNA levels exhibited weak negative correlations with some PBDEs in silver females and males. Hepatic vitellogenin (Vtg) mRNA levels were detected in all silver males but at lower levels than in silver females. In males, Vtg mRNA levels were positively associated to FT4/TT4. In silver females, strong positive correlations were found between congeners of PCBs, PBDEs and PFAS suggesting potential estrogenic effects. Overall, the observed results indicate that several organic contaminants, mainly dl-, ndl-PCBs and PBDEs, could be associated with changes in thyroid homeostasis in these fish, via direct or indirect interactions with peripheral deiodination, metabolism of T4 and mechanisms involved in TSHβ, Deio 2 and Vtg gene transcription.

Intersex and alterations in reproductive development of a cichlid, Tilapia guineensis, from a municipal domestic water supply lake (Eleyele) in Southwestern Nigeria

The objectives of this study were to develop and validate biomarker techniques for aquatic environmental monitoring of endocrine disrupting chemicals (EDCs) in Nigeria aquatic ecosystems, using the Eleyele Lake, which is a major source of domestic water supply to Ibadan and its surrounding towns, as a model aquatic environment and Tilapia guineensis, as a model organism. A total of 55 male and 28 female fish were used for this study. No significant difference in condition factor was observed between the sexes. Evaluation of gross gonadal morphology of the sampled fish showed 33% intersex prevalence in the sampled population, of which respective 71 and 29% were males and females, with visible testis and ovary developing alongside phenotypic females and males. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) were performed, showing that male fish had significantly higher plasma LH and E2 concentrations, compared to females. Vitellogenin (Vtg) and zona radiata proteins (Zrp) mRNA levels were significantly higher in males, compared to female fish. Contaminant analysis revealed that PCB 81, 123, 138 and 196 were the only PCB congeners detected in sediment and fish muscle (PCB153 in sediment), while dieldrin was the only organochlorine compound (OC) detected in Eleyele sediment. These responses were used in a multivariate analysis, showing that two principal components were extracted and accounted for 74% of total variation in the dataset. The principal component analysis (PCA) showed that male fish variables were positively correlated with PCB congeners 18 and 123, while female fish showed positive correlations with congener 81, 138, 189, 196, indicating sex-specific pattern of association between PCBs concentrations and biomarker expression. In addition, strong positive correlation between male fish and LH, E2, FSH and Vtg was observed, while female fish positively correlated with 11-KT and GSI. These relationships suggest feminization and masculinization of male and female fish, respectively.

Expression of RXR, EcR, E75 and VtG mRNA levels in the hepatopancreas and ovary of the freshwater edible crab, Oziothelphusa senex senex (Fabricius, 1798) during different vitellogenic stages.

The objective of the present study was to investigate the expression profile of retinoid X receptor (RXR), ecdysone receptor (EcR) and ecdysone inducible gene (E75) in the hepatopancreas and ovary of Oziothelphusa senex senex during different vitellogenic stages. RXR, EcR and E75 complementary DNAs (cDNAs) were isolated from the ovaries, while vitellogenin (VtG) cDNA was isolated from the hepatopancreas of vitellogenic female crab. Deduced amino acid sequence of the messenger RNAs (mRNAs) of RXR, EcR and E75 showed more than 80% identity with their respective mRNAs of other brachyurans. VtG mRNA was not detected in the ovary throughout vitellogenic stages. RXR and EcR were significantly increased in the ovaries during vitellogenic stage I. The levels of EcR, E75 and VtG in the hepatopancreas elevated significantly during vitellogenic stages I and II, whereas the levels of RXR elevated only in vitellogenic stage I. During vitellogenic stage III, the levels of RXR, EcR and VtG in the hepatopancreas were significantly decreased. Immunoprecipitation analysis revealed the presence of VtG in the haemolymph, hepatopancreas and ovary extracts from the females but absent in haemolymph and hepatopancreas extract of males. It can be inferred that RXR, EcR and E75 are involved in the regulation of synthesis of VtG in hepatopancreas, whereas in ovary, it is hypothesized that they play an important role in the uptake of VtG from the haemolymph, probably by regulating the levels of vitellogenin receptor. These are the first data showing an association between the expression levels of RXR, EcR and E75 and vitellogenesis and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation to induce vitellogenesis and ovarian maturation in crustaceans.

Isolation of crustacean hyperglycemic hormone-family peptides with vitellogenesis-inhibiting activity from the shiba shrimp Metapenaeus joyneri

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. In order to characterize multiple VIH molecules in the shiba shrimp Metapenaeus joyneri, four CHH-family peptides designated as Mej-SGP-I, -II, -III, and -IV were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The vitellogenesis-inhibiting activities of these peptides were examined using ex vivo incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. All of the four peptides significantly reduced vitellogenin mRNA levels in the ovarian fragments. Among them, the inhibitory activity of Mej-SGP-III which was the most abundant CHH-family peptide in the sinus gland was apparent. Therefore, a cDNA encoding the Mej-SGP-III precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE. The mature Mej-SGP-III was found to consist of 72 amino acid residues containing six conserved cysteine residues and possess an amidated C-terminus. The mature Mej-SGP-III showed the highest sequence identity to Pej-SGP-V (one of six CHH/VIHs in M. japonicus) and Liv-SGP-G (one of six VIHs in Litopenaeus vannamei). These results indicate that Mej-SGP-III is one of the hormones regulating vitellogenesis in M. joyneri.

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

Functional characterization of recombinant gonad-inhibiting hormone (GIH) and implication of antibody neutralization on induction of ovarian maturation in marine shrimp

Ovarian maturation in crustacean is controlled by an eyestalk neuropeptide called gonad-inhibiting hormone (GIH) that is presumed to inhibit vitellogenin synthesis. In this study, a recombinant protein of GIH of Penaeus monodon (rPem-GIH) was expressed in Pichia pastoris expression system and purified to homogeneity by reversed-phase SPE (C18). The purified rPem-GIH significantly reduced vitellogenin mRNA level in primary tissue culture derived from previtellogenic ovary of P. monodon broodstock by 45.7% compared with the untreated group. This effect was similar to that of partially purified optic lobe extract, and demonstrated that the rPem-GIH possessed gonad-inhibiting activity. A monoclonal antibody specific to Pem-GIH (anti-GIH mAb) was able to neutralize the activity of GIH from the partially purified optic lobe extract to about 58% by in vitro assay in primary tissue culture of the ovary. Peptide mapping revealed that amino acids 16 to 20 (MYNKV) of the mature Pem-GIH may serve as an epitope of anti-GIH mAb. A single injection of anti-GIH mAb into wild previtellogenic P. monodon was able to induce ovarian maturation and spawning at a comparable rate to the eyestalk ablation. Our results demonstrate neutralization effect of anti-GIH mAb on vitellogenesis inhibitory action of GIH, and thus provide a potential alternative to induce ovarian maturation in P. monodon broodstock.

RNAi Silencing of the HaHMG-CoA Reductase Gene Inhibits Oviposition in the Helicoverpa armigera Cotton Bollworm

RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests.

Induction and Recovery of Estrogenic Effects After Short-Term 17β-Estradiol Exposure in Juvenile Rainbow Trout (Oncorhynchus mykiss)

Estrogenic compounds found in the aquatic environment include natural and synthetic estrogen hormones as well as other less potent estrogenic xenobiotics. In this study, a comprehensive approach was used to examine effects on fish endocrine system endpoints during a short-term xenoestrogen exposure as well as after post-exposure recovery. Rainbow trout (Oncorhynchus mykiss) were exposed to an aqueous 17β-estradiol (E2) concentration of 0.473 μg l(-1) for 2 and 7 days (d) followed by a 14-d recovery period. At d2 and d7, plasma E2 concentrations in treated fish were 458- and 205-fold higher than in control fish and 23- and 16-fold higher than the exposure water concentration. E2 treatment resulted in significant increases in hepatosomatic index (HSI), plasma vitellogenin (VTG) protein concentrations, and liver VTG and estrogen receptor alpha mRNA levels. All of these parameters, with the exception of plasma VTG protein, returned to baseline values during the recovery period. Plasma cortisol concentrations were unaffected by treatment. This research shows varied time frames of the estrogen-responsive molecular-, biochemical-, and tissue-level alterations, as well as their persistence, in juvenile rainbow trout treated with aqueous E2. These results have implications for feral rainbow trout exposed to xenoestrogens and indicate the importance of evaluating a comprehensive suite of endpoints in assessing the impact of this type of environmental contaminant.

Endocrine disruption in thicklip grey mullet (Chelon labrosus) from the Urdaibai Biosphere Reserve (Bay of Biscay, Southwestern Europe)

Endocrine disruptors (EDs) interfere with the development and functioning of the endocrine system, causing reproductive disturbance in aquatic wildlife. The aim of the present work was to determine the presence of EDs in sediments and to investigate possible exposure and effects of EDs in the estuary of the Urdaibai Biosphere Reserve (Gernika) in comparison with the Arriluze marina. For this, gonad histology, plasma vitellogenin (VTG) protein levels and mRNA levels of vitellogenin (vtg), cyp19 aromatases, estrogen receptor (er) and retinoid X receptor (rxr) were studied in Chelon labrosus. The presence of alkylphenols (APs) in fish bile was also assessed. In sediments, estrogenic hormones were below the detection limit and levels of bisphenol A were very low. In Gernika organotin compounds were low but in Arriluze levels of up to 12μg/g were found. Moderate levels of APs and phthalate levels of up to 8μg/g were found in sediments. In fish, a high prevalence up to 33% of intersex gonads was found in Gernika, whereas only one intersex was found in Arriluze. Accordingly, mullets from Gernika showed higher concentrations of APs in bile. VTG protein levels were detected not only in females but also in some undifferentiated, male and intersex fish. mRNA of vtg was detected in one male from Gernika. mRNA of er and rxr showed significant differences between seasons. In conclusion, the present study demonstrated that C. labrosus from the Urdaibai estuary were exposed to EDs and showed clear signs of endocrine disruption.

Pathogenic infection confounds induction of the estrogenic biomarker vitellogenin in rainbow trout

To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17β-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA levels than healthy fish. Transcriptome data suggest that this was due to energetic constraints. Reduced responsiveness of the VTG biomarker in parasitized fish might obscure detection of low-level field exposure.

Co-expression and regulation of ovarian vitellogenins in the Pacific oysterCrassostrea gigas

In addition to the reported single cDNA sequence encoding vitellogenin in the Pacific oyster Crassostrea gigas (referred as Cg-Vtg1), we isolated and characterized the 3′ end of a novel vitellogenin (Vtg) in C. gigas, named Cg-Vtg2. The transcript size is up to 6 kb long and includes a D-type von Willebrand domain, characteristic of most vitellogenins, but not occurring in Cg-Vtg1. The expression of Cg-Vtg2 was found restricted to follicular cells in maturing and ripe female gonads. Quantitative expression analysis of both vitellogenins showed a closely correlated transcription pattern during the female gametogenic cycle, with the highest expression during the ripe (mature) stage, and a declining expression, to almost undetectable levels, in post-spawning (resting) stage. Levels of vitellogenin mRNA associates with the quantity of ingested food. Mature female oysters fed either a 2% or a 12% algal biomass per female biomass, showed a 2.9-fold increase in expression of Cg-Vtg1 and a 3.7-fold increase of Cg-Vtg2 expression levels in those females fed the higher level of algae, suggesting that Vtg's could be used as quantitative indicator of vitellogenesis. Our results suggest that vitellogenins are synthesized during the late period of vitellogenesis, when most of the yolk has already been incorporated into maturing oocytes.

Induction of Ovarian Maturation in Penaeus monodon by Molecular Signal Interventional Approach

Vitellogenin (VTG) synthesis in the hepatopancreas and ovary is negatively regulated by vitellogenesis-inhibiting hormone (VIH) produced in the neurosecretory cell of X-organ/sinus gland complex of the eyestalks of penaeid shrimp. Eyestalk ablation is used commercially to induce ovarian maturation in shrimps which leads to an eventual loss of the spawner. The aim of the present study was to understand the molecular mechanism of VIH regulation in ovarian development and its inhibition of VTG gene expression by using a MEK-specific inhibitor (U0126). The real-time quantitative PCR results showed VTG mRNA level was progressively increased in the ovary and hepatopancreas of unilateral eyestalk-ablated and inhibitor-treated shrimps. Western blot analysis also showed that phosphoMEK was detected only in the unilateral eyestalk-ablated and control shrimp, whereas phospho-MEK was not detected in inhibitor-treated shrimp. DAX-1, SF-1, and StAR expression correlated with changes in VIH mRNA and altered phospho-ERK levels. This is consistent with the hypothesis that suppression of DAX-1 results in SF-1-mediated StAR protein upregulation of estradiol that is implicated in vitellogenesis. This is the first report that demonstrates the molecular mechanism of VIH suppression via MEK pathway to induce ovarian maturation in female Penaeus monodon by molecular signal intervention, a less-invasive method than traditional eyestalk ablation.

Differential expression of immune genes of adult honey bee (Apis mellifera) after inoculated by Nosema ceranae

Nosema ceranae is a microsporidium parasite infecting adult honey bees (Apis mellifera) and is known to affects at both the individual and colony level. In this study, the expression levels were measured for four antimicrobial peptide encoding genes that are associated with bee humoral immunity (defensin, abaecin, apidaecin, and hymenoptaecin), eater gene which is a transmembrane protein involved cellular immunity and gene encoding female-specific protein (vitellogenin) in honey bees when inoculated by N. ceranae. The results showed that four of these genes, defensin, abaecin, apidaecin and hymenoptaecin were significantly down-regulated 3 and 6days after inoculations. Additionally, antimicrobial peptide expressions did not significantly differ between control and inoculated bees after 12days post inoculation. Moreover, our results revealed that the mRNA levels of eater and vitellogenin did not differ significantly following N. ceranae inoculation. Therefore, in this study we reaffirmed that N. ceranae infection induces host immunosuppression.

FoxO inhibits juvenile hormone biosynthesis and vitellogenin production in the German cockroach

The transcription factor Forkhead-box O (FoxO) is the main transcriptional effector of the Insulin Receptor/Phosphatidylinositol 3-kinase (InR/PI3K) pathway. In a situation of nutrient restriction, the pathway is inactive and FoxO translocates to the nucleus to exert its transcriptional action. In starved females of the cockroach Blattella germanica, the reproductive processes, and in particular the synthesis of juvenile hormone in the corpora allata and that of vitellogenin in the fat body, are arrested. In the present report we examine the possible role of FoxO in the transduction of the nutritional signals to these reproductive events. We first cloned FoxO cDNA from B. germanica (BgFoxO), and showed that its expression is not nutritionally regulated. BgFoxO knockdown using systemic RNAi in vivo in starved females elicited an increase of juvenile hormone biosynthesis, although without modifying mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, HMG-CoA reductase or methyl farnesoate epoxidase (CYP15A1) in corpora allata. In addition, BgFoxO RNAi treatment produced a remarkable increase of vitellogenin mRNA levels in fat body and of vitellogenin protein in the haemolymph. Our results indicate that BgFoxO plays an inhibitory role on juvenile hormone biosynthesis and vitellogenin production in a situation of nutrient shortage.

Molecular cloning and sequence of retinoid X receptor in the green crab Carcinus maenas: a possible role in female reproduction

Retinoid X receptor (RXR) belongs to an ancient superfamily of nuclear hormone receptors, and plays an important role in reproduction of vertebrates. However, the reproductive role of RXR has not been clarified in crustaceans. In this investigation, we first report the cloning of two alternative splice variants of RXR cDNA from green crab ovarian RNA. RXR mRNA levels were quantified in different vitellogenic stages of the crab hepatopancreas (HP) and ovary. The expression of RXR mRNA relative to the arginine kinase mRNA was significantly increased in the HP of vitellogenic crabs in a stage-dependent manner. The relative levels of RXR mRNA in the ovary were significantly lower in vitellogenic stage III crabs than in crabs in the other three stages. These data indicate that the HP and ovary of the crab are capable of expressing RXR, which may regulate, in part, vitellogenesis in the crab. We also examined the effects of methyl farnesoate (MF) and RXR-dsRNA treatments on vitellogenin and RXR gene expression. Vitellogenin and RXR mRNA levels in HP and ovarian fragments incubated in MF were significantly (P<0.001) higher than in control tissue fragments prepared from the same animal. Treatment of crabs with RXR-dsRNA significantly (P<0.001) reduced mRNA levels for RXR and for vitellogenin as well as MF levels in hemolymph. These results indicate that, MF and RXR form a complex (MF-RXR) directly and together stimulate ovarian development in these green crabs. This interaction of RXR, MF, and ovary development axis is a novel finding and is the first report to the best of our knowledge.

17Beta-estradiol affects the response of complement components and survival of rainbow trout ( Oncorhynchus mykiss) challenged by bacterial infection

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17β-estradiol (E2) using rainbow trout ( Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 μg 17β-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria ( Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids ( Daphnia magna)

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids ( Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.