Vitellogenin mRNA Levels Research Articles

Effects of exposure water volume, depuration time, and feeding status on vitellogenin mRNA induction in male medaka ( Oryzias latipes) exposed to17 β-estradiol

Bioassays measuring the induction of vitellogenin gene expression in male fish are widely used for revealing estrogenic activity in water samples. Measuring induction of vitellogenin mRNA in males by means of RT-PCR analysis is a sensitive way to detect exposure to estrogenic chemicals. To date, little work has been done to examine variations in exposure conditions for assessing estrogenic activity in water samples using this model system. Here we report the results of experiments investigating the effects of volume of treatment water, time since removal from treatment water (depuration), and short-term food deprivation on vitellogenin mRNA induction in male Japanese medaka ( Oryzias latipes). Fish exposed to a single concentration of E 2 while volume was manipulated were found to have similar levels of vitellogenin mRNA, though more E 2 was present at larger volumes. Removal of fish from E 2-treated-water to clean water after exposures reduced vitellogenin levels in as little 24 h, however, the vitellogenin levels of the fish transferred to the clean water remained above those of the control fish for at least 72 h. Depriving fish of food for up to 72 h during exposure to E 2 did not significantly reduce vitellogenin induction. Together these results support the conclusion that real time RT-PCR measurement of vitellogenin in male fish can be used as a robust indicator of exposure to estrogenic contaminants in water.

One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes

The aryl hydrocarbon receptor (AhR) and the estrogen receptor (ER) are ligand-activated transcription factors, both of which can be activated by environmental pollutants. The AhR regulates cytochrome P450 1A (CYP1A) expression and can be induced by aromatic hydrocarbons. The ER regulates vitellogenin (VTG) expression and can be induced by estrogenic substances. Both receptor responses are established biomarkers used to assess the effects of pollutants in the aquatic environment. The receptors can also be affected in situations of mixed exposure. Cross-talk between these receptor pathways has been suggested, although there are conflicting data in the literature. We investigated cross-talk between ER–VTG and AhR–CYP1A signaling pathways in primary cultures of rainbow trout hepatocytes, using quantitative PCR (qPCR) for mRNA analyses and studies of CYP1A catalytic function and protein expression. The model agonists β-naphthoflavone (BNF) and 17α-ethinylestradiol (EE 2) were used for AhR and ER activation, respectively. Combined exposure to BNF and EE 2 reduced the EE 2-mediated induction of VTG mRNA levels by about 40%, but had no effect on the BNF-mediated CYP1A mRNA levels, indicative of a one-way inhibiting AhR–ER cross-talk. However, basal levels of CYP1A mRNA were reduced 40% upon exposure to EE 2 alone, implying different cross-talk mechanism between basal and induced CYP1A mRNA levels. The mammalian ER antagonist fulvestrant (ICI) is commonly described as an absolute ER antagonist. However, ICI failed to reverse the ER activation caused by EE 2 in the present study. The CYP1A-mediated ethoxyresorufin- O-deethylase (EROD) activity was reduced by 80% in cells co-treated with BNF and EE 2, compared to cells exposed to BNF alone. In vitro inhibiting studies suggests that this reduction was a result of inhibition of the CYP1A catalyst by EE 2 since EE 2 acted as a potent inhibitor (IC 50: 4.6 μM) of the EROD activity. In addition, ICI also acted as a potent inhibitor of the EROD enzyme (IC 50: 0.6 μM). Taken together, our data supports a one-way inhibiting AhR–ER cross-talk in rainbow trout hepatocytes exposed to a mixture of BNF and EE 2.

Effects of Estradiol and Progesterone on the Reproduction of the Freshwater Crayfish Cherax albidus

In this study we have investigated the role of 17beta-estradiol and progesterone in the reproduction of the crayfish Cherax albidus by using vitellogenin (VTG) as a biomarker. Early-vitellogenic (EV), full-vitellogenic (FV), and non-vitellogenic (NV) females of Cherax albidus were treated with 17beta-estradiol, progesterone, or both for 4 weeks. Levels of VTG mRNA in the hepatopancreas were detected by RT-PCR. The PCR product was sequenced and showed 97% homology with Cherax quadricarinatus VTG. 17beta-estradiol was more effective than progesterone and 17beta-estradiol plus progesterone in increasing the vitellogenin transcript in the hepatopancreas of EV and FV females. On the contrary, progesterone was more effective than 17beta-estradiol and 17beta-estradiol plus progesterone in increasing the vitellogenin concentration in the hemolymph of EV and FV females. Hepatopancreas histology and fatty acid composition of females injected with hormones showed major modifications. No effects were registered in NV females. In conclusion, 17beta-estradiol and progesterone influence VTG synthesis, although our data indicate that they act through different pathways and are not effective until the proper hormonal environment is established, as demonstrated by their inefficacy in NV females.

Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17β-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24 h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17α-ethynylestradiol (EE2) for 24 h. Liver produced the most consistent response with up-regulation of ERα in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERβ1 by E2. ERβ2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERα and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.

Pulp and paper mill effluents induce distinct gene expression changes linked to androgenic and estrogenic responses in the fathead minnow (Pimephales promelas)

Although effluent treatment systems within pulp and paper mills remove many toxicants and improve wastewater quality, there is a need to understand and quantify the effectiveness of the treatment process. At a combined news and kraft pulp and paper mill in northwestern Ontario, Canada, fathead minnow (FHM) reproduction and physiology were examined before, during, and after a short-term (6-d) exposure to 10% (v/v) untreated kraft mill effluent (UTK), 25% (v/v) secondary treated kraft mill effluent (TK), and 100% (v/v) combined mill outfall (CMO). Although UTK exposure significantly decreased egg production, neither TK nor CMO caused any reproductive changes. The expression of six genes responsive to endocrine-disrupting compounds, stress, or metals was then examined in livers of these fish using real-time polymerase chain reaction. In female FHMs, none of the three effluents induced significant expression changes in any genes investigated. By contrast, in males there were significant increases in the mRNA levels of androgen receptor, estrogen receptor (ER) beta, and cytochrome P4501A (CYP1A) upon UTK and TK exposure but no changes in ERalpha or vitellogenin (VTG) gene expression, whereas CMO exposure significantly increased the mRNA levels of ERalpha, VTG, and CYP1A. Together, these results suggest that kraft effluent before and after biological treatment contained compounds able to induce androgenic effects in FHMs, and that combination of kraft and newsmill effluents eliminated the androgenic compounds while inducing distinct and significant patterns of gene expression changes that were likely due to estrogenic compounds produced by the newsmill.

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement

To understand the hormonal coordination of the antagonism between molting and reproduction in crustaceans, the terminally anecdysial mature female Callinectes sapidus was used as a model. The regulatory roles of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) in vitellogenesis were examined. A competitive specific RIA was used to measure the levels of MIH and CHH in the hemolymphs of mature females at pre- and mid- vitellogenic stages, and their effects on vitellogenesis at early (early 2, E2) and mid vitellogenesis (3) stages were determined in vitro. A hepatopancreas fragments incubation system was developed and the levels of vitellogenin (VtG), as well as VtG mRNA and heterogeneous nuclear (hn)VtG RNA were determined using RIA or QPCR, respectively. MIH titers were four times higher at mid-vitellogenesis than at pre-vitellogenesis, while CHH levels in the hemolymph were constant. In the in vitro incubation experiments, MIH increased both VtG mRNA levels and secretion at ovarian stage 3. At stage E2, however, MIH resulted in a mixed response: downregulation of VtG mRNA and upregulation of hnVtG RNA. CHH had no effect on any of the parameters. Actinomycin D blocked the stimulatory effects of MIH in stage 3 animals on VtG mRNA and VtG, while cycloheximide attenuated only VtG levels, confirming the MIH stimulatory effect at this stage. MIH is a key endocrine regulator in the coordination of molting and reproduction in the mature female C. sapidus, which simultaneously inhibits molt and stimulates vitellogenesis.

Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300–750mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.

Trade‐off between immune stimulation and expression of storage protein genes

Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Target of Rapamycin (TOR) Mediates the Transduction of Nutritional Signals into Juvenile Hormone Production

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Roles of ecdysteroid and juvenile hormone in vitellogenesis in an endoparasitic wasp, Pteromalus puparum (Hymenoptera: Pteromalidae)

To elucidate the endocrine regulation of vitellogenesis in an endoparastic wasp ( Pteromalus puparum), the titers of ecdysteroid and juvenile hormone (JH) from the whole bodies are measured using the method of radioimmunoassay and GC–MS, and compared with the levels of vitellogenin (Vg) mRNA in the fat bodies, hemolymph Vg and ovarian vitellin (Vt), respectively. The results show that the ecdysteroid titer and fat body Vg mRNA level have a similar dynamics tendency, and the peak titer is at adult eclosion. The titer of JH III and ovarian Vt also have a similar dynamics tendency, and the peak titer is at 48 h after eclosion. The profiles of hemolymph Vg, Vg mRNA in fat bodies and ovarian Vt, are also measured in the wasps after treated with different amounts of 20-hydroxyecdysone (20HE) or JH III in female pupa and adults. The results show that 20HE stimulates Vg synthesis in the fat bodies and its release into the hemolymph, and that JH III only accelerates Vg sequestration in the oocytes. Decapitation, which is believed to terminate synthesis of JH in insects, can not inhibit vitellogenesis and oocyte maturation in P. puparum. Furthermore, Vg gene is expressed with a lower titer of JH and depressed by a higher titer of JH III. These studies suggest that ecdysteroids play a role in Vg synthesis and believed to be the dominant hormones in regulation of vitellogenesis in P. puparum, and JHs are not the essential factors to female reproduction in this wasp.

Sensory Response System of Social Behavior Tied to Female Reproductive Traits

BackgroundHoney bees display a complex set of anatomical, physiological, and behavioral traits that correlate with the colony storage of surplus pollen (pollen hoarding). We hypothesize that the association of these traits is a result of pleiotropy in a gene signaling network that was co-opted by natural selection to function in worker division of labor and foraging specialization. By acting on the gene network, selection can change a suite of traits, including stimulus/response relationships that affect individual foraging behavior and alter the colony level trait of pollen hoarding. The ‘pollen-hoarding syndrome’ of honey bees is the best documented syndrome of insect social organization. It can be exemplified as a link between reproductive anatomy (ovary size), physiology (yolk protein level), and foraging behavior in honey bee strains selected for pollen hoarding, a colony level trait. The syndrome gave rise to the forager-Reproductive Ground Plan Hypothesis (RGPH), which proposes that the regulatory control of foraging onset and foraging preference toward nectar or pollen was derived from a reproductive signaling network. This view was recently challenged. To resolve the controversy, we tested the associations between reproductive anatomy, physiology, and stimulus/response relationships of behavior in wild-type honey bees.Methodology/Principal FindingsCentral to the stimulus/response relationships of honey bee foraging behavior and pollen hoarding is the behavioral trait of sensory sensitivity to sucrose (an important sugar in nectar). To test the linkage of reproductive traits and sensory response systems of social behavior, we measured sucrose responsiveness with the proboscis extension response (PER) assay and quantified ovary size and vitellogenin (yolk precursor) gene expression in 6–7-day-old bees by counting ovarioles (ovary filaments) and by using semiquantitative real time RT-PCR. We show that bees with larger ovaries (more ovarioles) are characterized by higher levels of vitellogenin mRNA expression and are more responsive to sucrose solutions, a trait that is central to division of labor and foraging specialization.Conclusions/SignificanceOur results establish that in wild-type honey bees, ovary size and vitellogenin mRNA level covary with the sucrose sensory response system, an important component of foraging behavior. This finding validates links between reproductive physiology and behavioral-trait associations of the pollen-hoarding syndrome of honey bees, and supports the forager-RGPH. Our data address a current evolutionary debate, and represent the first direct demonstration of the links between reproductive anatomy, physiology, and behavioral response systems that are central to the control of complex social behavior in insects.

Estrogenic Endocrine Disruption in Switzerland: Assessment of Fish Exposure and Effects

Estrogenic exposure has been reported to occur in Swiss rivers, and there is concern that reduced reproductive health, caused by disturbances of the endocrine system, may contribute to the observed decline in brown trout catch. Consequently, we aimed to determine if disturbances of the endocrine system do occur in wild brown trout (Salmo trutta) in Switzerland and, in the affirmative case, if these might affect trout population status. Our first task was to characterize the estrogenicity of Swiss midland rivers that receive effluents from sewage treatment plants (STP). Next, we performed a set of laboratory and field exposure experiments aimed at elucidating how estrogens affect sexual development and reproductive parameters as estrogen-sensitive targets in the life cycle of brown trout. Subsequently, we assessed the demographic status of brown trout populations in the field which were exposed to the cumulative impact of estrogen-active compounds and other stressors. Finally, we integrated the data into a life-cycle model to predict potential population-level consequences of the (xeno)estrogenic exposure. The estrogenicity of 18 Swiss midland rivers was characterized bioanalytically by applying the YES bioassay to water samples and by measuring plasma vitellogenin (VTG) levels in resident brown trout. Generally, estrogenic contamination of the rivers appears to occur only locally and at comparatively low levels (0.2–2 ng/l 17?-estradiol equivalents). In laboratory experiments, potential disruptive effects of estrogens on gonadal differentiation and reproduction of brown trout were investigated. The estrogen-sensitive window of brown trout gonad differentiation was found to differ from other salmonid species. Feminisation of the developing gonads occurred only after exposure to rather high estrogen concentrations. Analysis of VTG mRNA levels indicated that the yolk may accumulate environmental estrogens and act as a long-term reservoir. The experiments to study the effects of prolonged estrogen exposure on reproduction of mature brown trout showed that, while VTG was induced at low concentrations (20 ng/l estrone (E1), 2 ng/l 17?-estradiol (E2) and 400 ng/l 4-nonylphenol (NP)), effects on reproductive parameters such as fertility became evident only at the higher dose (100 ng/l estrone, 10 ng/l 17?-estradiol and 4000 ng/l 4-nonylphenol). For the field study, the river Lützelmurg (Canton Thurgau) was selected as our study site. This river is impacted by the estrogen-active effluents of one sewage treatment plant (STP). Estrogenic levels in the river were found to be highly variable over time, but showed a distinct difference between sites upstream and downstream from the STP effluent – a difference which was also reflected by the VTG levels of caged and resident brown trout. From the brown trout demographic data, it is evident that factors other than estrogen exposure, including habitat quality, strongly influence population structure. The results from the modelling supported this finding and showed that the trout population in the Lützelmurg is likely to be more sensitive to changes of survival rates in the first winter and beyond than to changes in early life stage survival or reproductive parameters. From the overall results of our project, we conclude that a significant influence of estrogenic contamination on brown trout population densities in the majority of Swiss rivers appears not to be likely.

Estrogenic effects of polychlorinated biphenyls and relation to cytochrome P4501A activity in the endangered goodeid fish Ameca splendens

The present study examines the relationships between cytochrome P4501A (CYP1A) activity and vitellogenin (VTG) induction in Ameca splendens elicited by a polychlorinated biphenyl (PCB) mixture. Ethoxyresorufin-O-deethylase (EROD) activity, mRNA levels of VTG, and VTG induction were evaluated in male and female fish exposed for 1, 2, 4, 8, and 16 d to a commercial PCB mixture. Polychlorinated biphenyls induced higher EROD in both sexes and this induction was higher in females than in males. Maximum EROD and VTG induction occurred on day 1 in females, while in males these maxima occurred on days 8 and 16. A correlation between EROD and VTG induction was found only in males (p<0.001), and VTG induction was also higher in males than in females (p<0.01). Exposure to PCBs elicited increases in VTG expression and induction over time in males, while in females these decreased at the end of the exposure period. Deficiencies in the feedback mechanisms of male A. splendens exposed in the wild to xenoestrogens such as PCBs have probably contributed to alter the sex ratio of wild populations of this species.

Effects of 2,4‐dichlorophenol on the expression of vitellogenin and estrogen receptor genes and physiology impairments in Chinese rare minnow (Gobiocypris rarus)

2,4-Dichlorophenol (2,4-DCP) is known as a toxicant, but unknown as an environmental endocrine disruptor. In the present work, Chinese rare minnow (Gobiocypris rarus) was exposed to 0.03, 0.3, and 3 mg/L 2,4-DCP for 3 d and 0.1, 0.3, and 1 mg/L 2,4-DCP for 21 d, respectively. Endpoints including somatic index, vitellogenin (VTG) mRNA and protein level, estrogen receptor (ER) mRNA and histopathology were measured. In the 3-d exposure experiment, the effect concentrations were 0.3 mg/L and above, and in 21-d exposure, the effect concentrations were 0.1 mg/L and above. When exposed to the effect concentrations, GSI was significantly reduced for both male and female, ER mRNA was upregulated in male and downregulated in female. There were no significant variations of VTG mRNA in both male and female in 3-d exposure and in male in 21-d exposure. However, VTG mRNA in female in 21-d exposure was upregulated, corresponding to an increase of VTG protein in serum. Histopathological observation showed that ovaries were degenerated in the effect concentrations, where follicular atresias were more frequently observed. Because the sex hormones related genes and toxicological endpoints were affected in the dose-dependent manner, the results suggest that 2,4-DCP could be a potential endocrine disruptor and might cause adverse effects in female sex organs through interruption of ER-mediated processes.

Molecular characterization and seasonal expression of the vitellogenin gene from Günther's walking catfish Clarias macrocephalus

The complete cDNA sequence for walking catfish vitellogenin (VTG) (4192 bp) contained 51 bases of 5′-untranslated region, an open reading frame of 4050 bp encoding 1350 amino acids, 60 bases of 3′-untranslated region, and a poly(A) tail of 31 nucleotides. The deduced amino acid sequence of walking catfish VTG shared 58.9%, 56.9%, 41.7%, 32.6% and 8.1% identity with VTGs of common carp, zebrafish, rainbow trout, Xenopus and chicken, respectively. Results agreed with previous studies which indicated that among fishes, VTG is not a highly conserved protein, but shares a common general structure. Northern blot and RT-PCR revealed that VTG was specifically expressed in the liver of female fish. To establish the relationship between gonadosomatic index (GSI) and the level of hepatic VTG transcript, monthly change in the GSI of females was monitored over a 1-year period from June 2006 to May 2007. The relative copy number of VTG mRNA was determined by quantitative real-time PCR. Significant variations of mean GSI values and VTG mRNA levels (P<0.05) were observed among months. From June to August, mean GSI values ranged from 8.60±0.35% to 13.07±0.59%. Highest mean GSI value (18.27±2.51%) and maximum VTG transcript levels were observed in September. From October through March, ovarian weight steadily declined, with the lowest mean GSI value (0.77±0.38%) in January. The expression profile of the VTG gene reflected the annual changes in reproductive cycle of female walking catfish, showing high levels during the breeding period and low levels during resting periods.

Molecular Characterization of Japanese Sillago Vitellogenin and Changes in Its Expression Levels on Exposure to 17β-Estradiol and 4-tert-Octylphenol

We cloned a full-length cDNA encoding vitellogenin (VTG) from a marine teleost, the Japanese sillago Sillago japonica. The cloned sillago VTG contained signal peptide, lipovitellin heavy chain, phosvitin, lipovitellin light chain, and beta'-component in the order from the N-terminus. An exposure to 17beta-estradiol significantly increased the levels of plasma VTG, but not hepatic VTG mRNA in males. Neither plasma VTG nor hepatic VTG mRNA levels were affected by the exposure to 4-tert-octylphenol. Hepatic VTG mRNA levels in males increased at 1 day after intraperitoneal administration of 17beta-estradiol but decreased in the subsequent 5 days. However, plasma VTG levels remained high for 5 days after administration, suggesting that the accumulation period of plasma VTG is longer than that of hepatic VTG mRNA in males. Therefore, VTG mRNA may be a suitable indicator of temporal exposure to estrogenic chemicals in the environment, whereas plasma VTG is useful to detect consecutive exposure.

Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17β-estradiol action in the neuroendocrine axis of male goldfish

Auto-regulation of the three goldfish estrogen receptor (ER) subtypes was examined simultaneously in multiple tissues, in relation to mRNA levels of liver vitellogenin (VTG) and brain transcripts. Male goldfish were implanted with a silastic implant containing either no steroid or 17beta-estradiol (E2) (100 microg/g body mass) for one and seven days. Liver transcript levels of ERalpha were the most highly up-regulated of the ERs, and a parallel induction of liver VTG was observed. In the testes (7d) and telencephalon (7d), E2 induced ERalpha. In the liver (1d) and hypothalamus (7d) ERbeta1 was down-regulated, while ERbeta2 remained unchanged under all conditions. Although aromatase B levels increased in the brain, the majority of candidate genes identified by microarray in the hypothalamus (1d) decreased. These results demonstrate that ER subtypes are differentially regulated by E2, and several brain transcripts decrease upon short-term elevation of circulating E2 levels.

Estrogen‐like properties of perfluorooctanoic acid as revealed by expressing hepatic estrogen‐responsive genes in rare minnows (Gobiocypris rarus)

Perfluorooctanoic acid (PFOA) is an important perfluorinated compound (PFC) with various applications and has been widely disseminated in the environment, wildlife, and humans. The present study investigated the effects of waterborne PFOA on the expression of hepatic estrogen-responsive genes, vitellogenin (VTG), and estrogen receptor beta (ERbeta) and on the gonadal development in a freshwater rare minnow (Gobiocypris rarus). The mRNA levels of VTG and ERbeta were determined using reverse transcription polymerase chain reaction (RT-PCR) techniques, and VTG protein levels were identified using enzyme-linked immunosorbent assay. A significant increase of VTG expression in the livers of both mature males and females was observed after 14 and 28 d of exposure to 3, 10, and 30 mg/L PFOA, indicating that PFOA could induce VTG synthesis. The expression of ERbeta increased significantly in livers of both mature males and females after a 14-d exposure, although no difference was observed after a 28-d exposure. The development of oocytes in testes exposed to PFOA also provided evidence of estrogenic activity in males. The ovaries of PFOA-exposed females underwent degeneration, as reported in other fish species exposed to environmental estrogens. This preliminary study indicates that PFOA can disturb the activity of estrogen in mature male rare minnows by inducing hepatic estrogen-responsive genes, VTG and ERbeta, and barrier female reproduction.

Influence of municipal effluents on the expression of connexins in the brook trout ( Salvelinus fontinalis) testis

Gap junctions are essential for spermatogenesis. Exposure to municipal wastewater effluent can modify spermatogenesis in fish. The present aim was to determine if municipal wastewater effluent could alter the expression of testicular connexins (Cxs) in brook trout. Trout were exposed for 4 and 12 weeks to various concentrations of municipal effluent (0, 1, 10 and 20%, v/v). Hepatic vitellogenin ( vtg) mRNA levels were not different between exposure groups after 4 weeks. At this time, testicular cx43 and cx31 mRNA levels increased in the 1% group, but cx30 and cx43.4 levels were not different at any concentration. Immunolocalization of each Cx did not differ between groups after 4 weeks. After 12 weeks, spermatogenesis in the 1% group was more advanced than in other groups, and hepatic vtg mRNA levels were significantly increased at the higher exposure concentrations. Testicular cx43 mRNA levels were higher than controls at all doses, while cx43.4 levels increased in a dose-dependent manner but remained lower than controls. cx31 mRNA levels were significantly lower in the 1 and 10% groups than in control and 20% group, while cx30 levels did not vary. Immunolocalization of Cxs did not differ between groups except for Cx43.4, which was expressed between spermatocytes in the 1% group. Furthermore, the Cx31 immunoreaction appeared to decrease in testicular blood vessels of fish exposed to the highest dose. Furthermore, vegf mRNA levels were unaltered by treatment at both time points. Thus, long-term exposure to environmental concentrations of wastewater effluent can alter the expression of testicular Cxs.