Vtg Expression Research Articles

Distinctive metabolite profiles in in-migrating Sockeye salmon suggest sex-linked endocrine perturbation.

The health of Skeena River Sockeye salmon (Onchorhychus nerka) has been of increasing concern due to declining stock returns over the past decade. In the present work, in-migrating Sockeye from the 2008 run were evaluated using a mass spectrometry-based, targeted metabolomics platform. Our objectives were to (a) investigate natural changes in a subset of the hepatic metabolome arising from migration-associated changes in osmoregulation, locomotion, and gametogenesis, and (b) compare the resultant profiles with animals displaying altered hepatic vitellogenin A (vtg) expression at the spawning grounds, which was previously hypothesized as a marker of xenobiotic exposure. Of 203 metabolites monitored, 95 were consistently observed in Sockeye salmon livers and over half of these changed significantly during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health of migrating Skeena Sockeye.

Estrogen stimulates expression of chicken hepatic vitellogenin II and very low-density apolipoprotein II through ER-α

Steroid hormones and their receptors play pivotal roles throughout vertebrate reproduction and development. Egg formation in avian species is a prime example. The synthesis of egg yolk proteins by the liver is highly dependent on estrogen. Two major components of the yolk protein precursors, vitellogenin II (VTG II) and very low-density apolipoprotein II (ApoVLDL II), are synthesized in the liver of hens under estrogen stimulation and are subsequently transferred via the blood to the developing oocytes. Estrogen-inducible transcription can be mediated through estrogen receptors (ERs) (ER-α and ER-β) or through G protein–coupled receptor 30 (GPR30), but the exact participation of the individual receptor is not clear. Here, we determine the relative contribution of each transduction pathway in the synthesis of VTG II and ApoVLDL II in the hepatocytes by using selective compounds that are known to specifically interact with each of the ERs and GPR30. 17β-Estradiol and propyl pyrazole triol (PPT, ER-α agonist) induced increase in VTG II and ApoVLDL II mRNA expressions in a dose-dependent manner. A high concentration of diarylpropionitrile (DPN, which preferentially motivates ER-β) slightly stimulated the expression of VTG II and ApoVLDL II mRNAs. However, G-1 (a GPR30 agonist) failed to display any stimulating role. Methyl-piperidino-pyrazole (a highly selective ER-α antagonist) fully blocked the expression of both yolk precursors, which were upregulated by 17β-estradiol, PPT, and DPN. Considering that DPN can also provoke the action of ER-α at high concentration, this excludes the participation of ER-β and supports the role of ER-α. The aforementioned results indicate that estrogen stimulates the expression of VTG II and ApoVLDL II mRNAs predominantly through ER-α in the chicken liver.

Benzo[a]pyrene effects on reproductive endpoints in Fundulus heteroclitus.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that has been implicated in modulating aromatase enzyme function with the potential to interrupt normal reproductive function. The aim of this study was to use a fish model, Fundulus heteroclitus, to assess whether BaP exposure adversely impacts reproduction. Adult fish were exposed to waterborne BaP nominal concentrations of (0, 1, or 10 μg/l) for 28 days. Males and females were combined for the second half of the exposure (days 14-28) in order to quantitate egg production and fertilization success. Egg fertilization and subsequent hatching success of F1 embryos was significantly decreased by the high dose of BaP. In males, both gonad weight and plasma testosterone concentrations were significantly reduced compared to controls by 10 μg/l BaP. Histopathological examination of testes including spermatogonia, spermatocyte and spermatid cyst areas, percentage of cysts per phase, and area of spermatozoa per seminiferous tubule were not significantly affected. Other biomarkers, including male liver weight, liver vitellogenin (vtg) mRNA expression and sperm concentrations, were also not affected. In females, estradiol concentrations were significantly reduced after BaP exposure, but egg production, gonad weight, liver weight, vtg expression and oocyte maturation were not altered. Steroid concentrations in Fundulus larvae from exposed parents at 1 and 3 weeks posthatch were not significantly changed. BaP exposure at these environmentally relevant concentrations caused negative alterations particularly in male fish to both biochemical and phenotypic biomarkers associated with reproduction and multigenerational embryo survival.

Temporal Dynamics of Oocyte Growth and Vitellogenin Gene Expression in Zebrafish (Danio Rerio)

Little is known about how hepatic vitellogenin gene (vtg) expression relates to oogenesis in fish, especially among fractional spawners. The objective of this study was to relate hepatic vtg 1A/B expression to stage-specific oocyte development in zebrafish (Danio rerio), an asynchronous spawning fish. Liver samples were collected at seven time points postspawning (1-32 days) and fish were preserved for subsequent histological analyses. Relative vtg 1A/B expression among liver samples was quantified by reverse transcription-quantitative PCR and oogenesis was evaluated following standard hematoxylin and eosin staining of serial ovarian sections. Histological analyses indicate that a subset of previtellogenic oocytes (stages 1-2) transitioned into postvitellogenic oocytes (stages 3-4) within 4 days (96 h) postspawning. By 8 days postspawning (192 h), the majority of the ovary was occupied by mature (stage 4) oocytes, a trend that continued through 32 days postspawning. Hepatic vtg 1A/B gene expression was upregulated 3.89-fold 1-h postspawning relative to the average gene expression across all time points, but was not correlated to stage-specific oogenesis. Follicular atresia among fish sampled 32 days postspawning highlights the importance of regular spawning in zebrafish and suggests that the event of spawning itself may be integral to the regulation of oocyte development.

Sandy sediment and the bioavailability of 17β-trenbolone to adult female fathead minnows

Recent studies have detected bioavailable steroids in sediment, however, the mechanism by which these compounds become bioavailable is not completely understood. In this study, two experiments were conducted using a double aquarium system that allowed female fathead minnows to be exposed to sandy sediments without direct contact. In the first experiment, natural sediment from the Elkhorn River (Nebraska, USA) was spiked with 17β-trenbolone. Both the fish in direct contact with the sediment as well as the fish excluded from direct contact experienced significant reductions in the hepatic expression of two estrogen-responsive genes, vitellogenin and estrogen receptor α, indicating molecular defeminization. The natural sediment contained particles ranging in size from sand to clay and it was possible that the fish in experiment 1 were being exposed to trenbolone associated with the very fine particles. The sandy sediment was sieved for experiment 2, and only the particles larger than 250μm were used. In addition, the experiment was conducted at two different Tb concentrations (1× and 10×). Furthermore nuptial tubercles, a biomarker of exposure to a masculinizing androgen, were also evaluated in the females used in experiment 2. For tubercle number and vtg expression, significant results were obtained from a two-way ANOVA due to Tb concentration, but not tank location or interaction term (location vs. concentration). For ERα expression, results were found in response to Tb concentration and tank location, but not the interaction term. Overall the results from these studies suggest that the primary route of exposure of sediment-associated trenbolone to fish is through ventilation of free compound, rather than ingestion or direct contact with the sediments.

Vitellogenin is not an appropriate biomarker of feminisation in a Crustacean

The expression of the yolk protein vitellogenin (Vtg) has been used as a biomarker of feminisation in multiple fish species throughout the world. Since the late 1990s, researchers have attempted to develop similar biomarkers to address whether reproductive endocrine disruption also occurs in the males of invertebrate groups such as the Crustacea. To date, the vast majority of studies investigating Vtg induction in male Crustacea have resulted in negative or inconclusive results, leading researchers to question the utility of Vtg expression as a biomarker in this taxon. This study measured the expression of Vtg genes in two intersex phenotypes (termed internal and external) found in the male amphipod, Echinogammarus marinus, and compared them with those of normal males and females. Males presenting the external intersex phenotype are infected with known feminising parasites and display a variety of feminised traits including oviduct structures on their testes and external female brood plates (oostegites). The internal intersex male phenotype, that displays a pronounced oviduct structure on the testes without the external intersex characteristics, is not parasite infected and it is thought to be a result of environmental contamination. Given their morphology, these phenotypes might be considered highly ‘feminised’ or ‘de-masculinised’ and can be utilised to test the suitability of feminisation biomarkers. The E. marinus transcriptome was searched for genes resembling Vtg and two sequences were revealed, that we subsequently refer to as Vtg1 and Vtg2. Results from a high-throughput transcriptomic sequencing screen of gonadal cDNA libraries suggested that very low expression (in this manuscript gene transcription is taken to represent gene expression, although it is acknowledged that in addition to transcription, translation, transcript processing, mRNA stability and protein stability can regulate gene expression) of Vtg1 and Vtg2 in normal males (ESTs=1 and 0 for Vtg1 and Vtg2, respectively), internal intersex males (ESTs=0 for both Vtg sequences) and external intersex males (ESTs=5 and 0 for Vtg1 and Vtg2, respectively). In contrast, the sequencing suggested notable levels of expression of both Vtg genes in females (ESTs=1133 and 84 for Vtg1 and Vtg2, respectively). Subsequent qPCR analysis validates these expression levels, with the signal for Vtg1 and Vtg2 transcripts in all male phenotypes being indistinguishable from that caused by contamination of trace levels of genomic DNA or the low-level amplification non-target sequences. These findings suggest that Vtg expression is not notably induced in highly feminised amphipods and is therefore not an appropriate biomarker of feminisation/de-masculination in crustaceans. We discuss our findings in the context of previous attempts to measure Vtg in male crustaceans and suggest a requirement for more appropriate taxon-specific biomarkers to monitor feminisation in these groups.

Exposure to Estrogenic Chemicals Induces Ectopic Expression of vtg in the Testis of Rainbowfish, Melanotaenia fluviatilis

Vitellogenin (Vtg) is the major egg-yolk precursor protein in oviparous organisms normally synthesised only in mature females. In males and juveniles, the vtg gene, although present, is silent, but its hepatic expression may be activated by xenoestrogens. Surprisingly, its induction and potential consequences in non-hepatic tissues remain unexplored. Here we test the hepatic and testicular response of vtg expression in adult male rainbowfish Melanotaenia fluviatilis exposed to either 1, 3, 5μg/L 17β-estradiol or 100, 500μg/L 4-n-nonylphenol for 24-96h. Significant increase in the expression level of vtg mRNA in the liver and testes of exposed males was observed. The early (24h), sensitive and reliable detection of the vtg induction using qPCR demonstrates the assay's robustness to monitor xenobiotic exposure particularly in smaller fish like rainbowfish, an emerging indicator species. Whilst, the ectopic induction of vtg mRNA in testes suggests a more complex Vtg pathway.

Effects of fish oil on ovarian development in spotted scat (Scatophagus argus)

The effects of different concentrations of dietary fish oil (0, 2%, or 6%) on ovarian development in 2-year-old female Scatophagus argus were investigated. The levels of serum sex steroid hormones (estradiol-17β, E2; testosterone, T), protein phosphorus (SPP), and protein calcium (SPC), as well as vitellogenin (vtg) mRNA expression in livers and ovaries were measured. Over the eight week experimental period, oocytes did not develop further and remained at phase III in fish fed with the control diet with no supplement of fish oil. Fish fed with 2% fish oil supplement had oocytes at transition phase from III to IV. Fish fed with 6% fish oil supplement had oocytes at late phase IV. Higher gonadosmatic index, serum E2, SPP, SPC, and liver vtg expression were found in 6% fish oil group compared to that in the 2% fish oil group (except E2) and the control group (P<0.05). In addition, vtg expression in livers was 600–1000 times higher than that in the ovaries. Gonadosmatic index, E2, and SPP, as well as liver vtg expression increased during the experiment and peaked at the end of experiment. However, hepatosomatic index, serum T, and ovarian vtg expression peaked at 4 weeks, and then decreased at 8 weeks, with no significant difference among the 3 groups. In summary, we showed that 6% fish oil supplementation in S. argus could effectively promote ovarian development, with associated increases in E2 secretion and increased liver vtg mRNA expression.

Aromatic Naphthenic Acids in Oil Sands Process-Affected Water, Resolved by GCxGC-MS, Only Weakly Induce the Gene for Vitellogenin Production in Zebrafish (Danio rerio) Larvae

Process waters from oil sands industries (OSPW) have been reported to exhibit estrogenic effects. Although the compounds responsible are unknown, some aromatic naphthenic acids (NA) have been implicated. The present study was designed to investigate whether aromatic NA might cause such effects. Here we demonstrate induction of vitellogenin genes (vtg) in fish, which is a common bioassay used to indicate effects consistent with exposure to exogenous estrogens. Solutions in water of 20-2000 μg L(-1) of an extract of a total OSPW NA concentrate did not induce expression of vtg in larval zebrafish, consistent with earlier studies which showed that much higher NA concentrations of undiluted OSPW were needed. Although 20-2000 μg L(-1) of an esterifiable NA subfraction of the OSPW NA concentrate did induce expression, this was of much lower magnitude to that induced by much lower concentrations of 17α-ethynyl estradiol, indicating that the effect of the total NAs was only weak. However, given the high NA concentrations and large volumes of OSPW extant in Canada, it is important to ascertain which of these esterifiable NA in the OSPW produce the effect. Up to 1000 μg L(-1) of an OSPW subfraction containing only alicyclic NA, and considered by most authors to be NA sensu stricto, did not produce induction; but, as predicted, 10-1000 μg L(-1) of an aromatic NA fraction did. Such effects by the aromatic acids are again consistent with those of only a weak estrogenic substance. These findings may help to focus studies of the most environmentally significant OSPW-related pollutants, if reproduced in a greater range of OSPW.

Biological response of high-back crucian carp (Carassius auratus) during different life stages to wastewater treatment plant effluent

This study presents the adverse effects of endocrine disrupting chemicals (EDCs) in effluent of wastewater treatment plants (WwTPs) on fish health. A study of chronic exposure to WwTPs effluent for 10 months was undertaken in high-back crucian carp (Carassius auratus) during different life stages, covering early-life-stage (ELS), prespawning period, and postspawning period. Condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) levels were employed as indicators to assess biological effects of effluent on this gynogenesis species. Meanwhile, some high-back crucian carp were caged in Demonstration Base of Biological Purification for Filter-feeding Fish (hereinafter, Demonstration Base), as WwTPs effluent exposure controls. In the meantime, a depuration study was carried out to determine whether or not the estrogenic effects caused by effluent exposure could be reduced after moving fish into EDCs-free water. CF, HSI, GSI, and plasma Vtg levels of high-back crucian carp caged in Demonstration Base were generally in accordance with seasonal change. Effluent exposure inhibited gonadal growth, reducing GSI in ELS while increasing it around spawning, sharpened liver burdens, increasing HSI, and induced abnormal Vtg expression in juvenile high-back crucian carp, augmenting Vtg concentrations in plasma. Around spawning period, Vtg in high-back crucian carp were mainly induced by endogenous estrogens, and EDCs in effluent had less influence on them. Staying in EDCs-free water for 30 days made high-back crucian carp recover from effects of previous effluent exposure, relieving inhibition of gonadal development and hypertrophy of liver as well as reducing Vtg induced by EDCs in effluent. The results revealed that high-back crucian carp in ELS are more sensitive to WwTPs effluent exposure. Additionally, the depuration study showed a clearance of the estrogenic effects caused by effluent.

Ketamine attenuates cytochrome p450 aromatase gene expression and estradiol‐17β levels in zebrafish early life stages

Ketamine, a dissociative anesthetic, is a noncompetitive antagonist of N-methyl-D-aspartate-type glutamate receptors. In rodents and non-human primates as well as in zebrafish embryos, ketamine has been shown to be neurotoxic. In cyclic female rats, ketamine has been shown to decrease serum estradiol-17β (E2) levels. E2 plays critical roles in neurodevelopment and neuroprotection. Cytochrome p450 (CYP) aromatase catalyzes E2 synthesis from androgens. Although ketamine down-regulates a number of CYP enzymes in rodents, its effect on the CYP aromatase (CYP19) is not known. Zebrafish have been used as a model system for examining mechanisms underlying drug effects. Here, using wild-type (WT) zebrafish (Danio rerio) embryos, we demonstrate that ketamine significantly reduced E2 levels compared with the control. However, the testosterone level was elevated in ketamine-treated embryos. These results are concordant with data from mammalian studies. Ketamine also attenuated the expression of the ovary form of CYP aromatase (cyp19a1a) at the transcriptional level but not the brain form of aromatase, cyp19a1b. Exogenous E2 potently induced the expression of cyp19a1b and vtg 1, both validated biomarkers of estrogenicity and endocrine disruption, but not cyp19a1a expression. Attenuation of activated ERK/MAPK levels, reportedly responsible for reduced human cyp19 transcription, was also observed in ketamine-treated embryos. These results suggest that reduced E2 levels in ketamine-treated embryos may have resulted from the suppression of cyp19a1a transcription.

Evidence of small modulation of ethinylestradiol induced effects by concurrent exposure to trenbolone in male eelpout Zoarces viviparus

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L−1 EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L−1, TBL) or high (500 ng L−1, TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.

Hormonal effects of tetrabromobisphenol A using a combination of in vitro and in vivo assays

The aim of this study was to investigate the hormonal effects of tetrabromobisphenol A (TBBPA) in vitro on recombinant yeasts and in vivo on mosquitofish (Gambusia affinis). The in vitro bioassays for (anti-)androgenic activities showed that TBBPA had a weak androgenic activity in vitro with recombinant yeast systems carrying human androgen receptor (hAR). In the in vivo bioassays, the gene expression patterns of vitellogenin (Vtg), estrogen receptors (ERα and ERβ), and androgen receptors (ARα and ARβ) in adult males and juveniles after exposure to TBBPA for 60days were evaluated. Significant up-regulation of Vtg, ERα, and ERβ mRNAs was observed in the liver after exposure to 500nM of TBBPA. In the testis, the lowest concentration of TBBPA (50nM) markedly induced Vtg, ERβ, and ARβ mRNA expression, but the same concentration significantly inhibited ARα mRNA expression. In addition, in juveniles, 100nM of TBBPA significantly up-regulated the expression of Vtg, ERβ, and ARα mRNAs. However, TBPPA did not cause histological alterations in the liver and testis of adult male mosquitofish. The results from this present study suggest that TBBPA could display low but multiple hormonal activities despite its low toxicity to mosquitofish.

65. Development of molecular markers for stage II and stage III zebrafish ovarian follicles after in vitro culture

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but had not been successful in fish so far. Fish oocytes offers several advantages over embryos due to smaller size of the oocytes, low water content and absence of fully formed chorion. Studies in our lab have showed that early stage ovarian follicles are more suitable for cryopreservation than late stage oocytes. Hence ovarian tissue containing stage I and stage II ovarian follicles have been used in this study. Development of in vitro culture protocol for early stage ovarain follicles is important since cryopreserved early stage ovarian follicles would need to be matured in vitro. The results obtained from the in vitro culture study carried out in our lab demonstrated that the early stage ovarian fragments containing stage I and stage II follicles can be cultured in 90% L-15 medium (pH 9.0) with 20% feotal bovine serum (FBS) and 100 mIU/ml follicle stimulating hormone (FSH) for 24 h at 28 °C. The study demonstrated follicle growth in size from stage I to stage II or from stage II to stage III, respectively. Follicle viability following culture was not significantly different when compared to the cultured fresh fragments. Although follicle growth can be assessed by measuring the diameter of the follicles, molecular markers would provide more important information on follicle development. The aim of the present study was to develop a molecular marker to identify stage II and stage III ovarian follicles after in vitro culture. Studies on the levels of vtg1 and p450aromA genes in stage I, II and II ovarian follicles of zebrafish before and after culture were carried out. Ovarian follicles of zebrafish were collected from mature zebrafish females and the ovaries were placed in 90% L-15 medium. Ovarian follicles of different stages (I, II and III) were separated using syringe needles. Total RNA was extracted from ovarian follicles using trizol method. The quality of RNA of each sample was determined by the level of absorbance at 260 nm. A quantitative RT-PCR approach was used to investigate the level of gene expression. Preliminary results from the present study showed that prior to culture of the ovarian follicles the level of expression of vtg 1 was higher in stage III when compared to stage I and stage II follicles; and the level of expression in p450aromA was higher in stage II when compared to stage I and stage III ovarian follicles. Analysis on the level of expression of genes after in vitro culture of ovarian fragments are in progress. The development of molecular markers would provide a reliable method for assessing the growth of ovarian follicles in vitro.

Activation of the Ahr signalling pathway by polychlorobiphenyls causes a marked induction of cytochrome P450 only after depletion of vitellogenin in Sparus aurata

The effects of polychlorinated biphenyl congeners 126 and 153 (PCB-126 and PCB-153) on vitellogenin (Vtg) and cytochrome (CYP1A1) expression were evaluated in 60 juvenile Sparus aurata. Fish were divided into four groups and the control group (Group A) was compared to fish exposed to PCB-126 (10–8M) (Group B), PCB-153 (10–6M) (Group C) singly and also in combination (Group D) for 12, 24 and 72h. Hepatic expression of Vtg and CYP1A1 were analyzed using histological examinations and by immunochemical (Western blotting and immunohistochemistry) methods. Vtg increased in juvenile fishes of Groups B, C and D after 12h respect to Group A and decreased after 24 and 72h respect to 12h in each group. CYP1A1 increased after 12 and 24h in all groups vs control group and increased in Group B only at 72h vs in control group. The results showed that chemical interaction and endocrine disruption in fish might produce deleterious consequences not only for fish but also for human.

Creating Females? Developmental Effects of 17 -Ethynylestradiol on the Mangrove Rivulus' Ovotestis

Interest in the occurrence and fate of trace organic contaminants in the aquatic environment and their potential effects on all organisms has increased over the past two decades. Researches on contaminants have included both natural and synthetic estrogenic contaminants, neuroactive pharmaceuticals, and other endocrine disrupting chemicals that are mediated by the androgen and progesterone receptors. Exposure to very low concentrations (ng/L or parts per trillion) of compounds such as 17α-ethynylestradiol (EE(2)), a synthetic estrogen, can affect gonadal development, viability and production of eggs, fertilization rate, and sexual differentiation in fishes. Researchers and aquaculturists have used exposures to relatively higher concentrations of androgens and estrogens, for example 17α-methyltestosterone and EE(2), respectively, to direct sexual differentiation in a number of fishes. Rivulus is an androdioecious teleost that in nature exists mostly as selfing, simultaneous hermaphrodites as well as a small number of males that outcross with hermaphrodites. No one has either collected females in the wild or created functional females in the laboratory. This study had two goals: (1) to develop a reliable protocol to produce female rivulus to enable downstream technologies such as embryo injections and (2) to investigate developmental effects of EE(2) on the sexual outcome, reproductive health, and relevant gene expression in rivulus. With these goals in mind, we exposed newly hatched rivulus to nominal concentrations of 0.1, 0.5, or 1.0 parts per million (ppm) EE(2) for 4 weeks, grew them to maturity in control water, and then compared egg production; production and viability of embryos; age of reproductive maturity; and gene expression in the brain, gonad, and liver. Expression levels of seven genes with known relevance to gonadal development and function (cyp19a1b, cyp19a1a, dmrt1, figα, ERα, ERβ, and vtg) were measured using quantitative polymerase chain reaction (PCR). There was a significant decrease in cyp19a1a gene expression in the brain, corresponding to increased exposure to EE(2). Gonadal gene expression for cyp19a1a, ERα, and dmrt1 also decreased in response to EE(2). Vtg expression in the liver was unaffected. Our hypothesis that exposure to EE(2) during gonadal differentiation would direct female development was not supported by the data. However, treated fish exhibited impaired reproductive health that included reduced expression of relevant genes and, importantly, decreased fertility, increased sterility, and delay of age of reproductive maturity. The results of this study suggest that the development and maintenance of a simultaneous hermphrodite ovotestis may be particularly sensitive to its hormonal milieu.

Responses of biomarkers of a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) fish species after in situ exposure in a periurban zone of Luján river (Argentina)

The Luján River basin, which is located in the northwest area of the province of Buenos Aires, Argentina, receives different anthropogenic inputs before reaching the Río de la Plata estuary. The aim of this study was to assess the adverse impact of the river in the middle part of the basin. To this end, an in situ cage assay was conducted in two sites of the river (S1 and S2) near Luján city, and the responses of hepatic biomarkers of both a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) species were evaluated. The biomarkers studied were the condition factor and liver somatic indices (LSI), the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST), lipid peroxidation levels (thiobarbituric acid reactive substances, TBARS) and the induction of hepatic cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) proteins. After 14 days, LSI and GST activity increased, and TBARS levels decreased in both species exposed at S1 and S2. In addition, exposure at both sites promoted an increase in SOD activity and CYP1A induction in C. carpio, while Vtg expression was observed only at S1. A shorter exposure period (7 days) caused an initial response only at S2 mediated only by CAT in P. laticeps. Finally, our results demonstrate that a 14-day period of in situ exposure in Luján River could lead to antioxidant and biotransformation processes in C. carpio and to phase II biotransformation responses in P. laticeps.

Molecular cloning and characterization of estrogen receptor gene in the Scallop Chlamys farreri: Expression profiles in response to endocrine disrupting chemicals

In order to gain insights into the mechanism of sex steroid signaling in molluscs, the full-length cDNA of estrogen receptor (ER) was isolated and characterized from Chlamys farreri for the first time. The positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. Phylogenetic analysis revealed that the CfER is an ortholog of the other mollusk ERs. Tissue distribution analysis of the CfER mRNA revealed that the expression of ER mRNA was observed in various tissues, and highest in the gonad of males and females. C. farreri were exposed for 10days to endocrine disrupting chemicals including Benzo(a)pyrene (B(a)p) and polybrominated diphenyl ethers (BDE-47). B(a)p exposure at 0.4 and 2μg/L caused significant increase in mRNA expression of ER and VTG, but B(a)p at 10μg/L down-regulated CfER and VTG mRNA expression compared to control. Varying increase of ER and VTG mRNA transcripts was resulted in by BDE-47 at 0.1, 1 and 10μg/L. These results elucidate potential roles of CfER induced by xenobiotics in C. farreri and can be helpful for investigating the mechanism of sex steroid signaling in bivalve mollusks.

Evolution of yolk protein genes in the Echinodermata

Vitellogenin genes (vtg) encode large lipid transfer proteins (LLTPs) that are typically female-specific, functioning as precursors to major yolk proteins (MYPs). Within the phylum Echinodermata, however, the MYP of the Echinozoa (Echinoidea + Holothuroidea) is expressed by an unrelated transferrin-like gene that has a reproductive function in both sexes. We investigated egg proteins in the Asterozoa (Asteroidea + Ophiuroidea), a sister clade to the Echinozoa, showing that eggs of the asteroid Parvulastra exigua contain a vitellogenin protein (Vtg). vtg is expressed by P. exigua, a species with large eggs and nonfeeding larvae, and by the related asterinid Patiriella regularis which has small eggs and feeding larvae. In the Asteroidea, therefore, the reproductive function of vtg is conserved despite significant life history evolution. Like the echinozoan MYP gene, asteroid vtg is expressed in both sexes and may play a role in the development of both ovaries and testes. Phylogenetic analysis indicated that a putative Vtg from the sea urchin genome, a likely pseudogene, does not clade with asteroid Vtg. We propose the following sequence as a potential pathway for the evolution of YP genes in the Echinodermata: (1) the ancestral echinoderm produced YPs derived from Vtg, (2) bisexual vtg expression subsequently evolved in the echinoderm lineage, (3) the reproductive function of vtg was assumed by a transferrin-like gene in the ancestral echinozoan, and (4) redundant echinozoan vtg was released from stabilizing selection.

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.