Vitamin D3 promotes fish oocyte development by directly regulating gonadal steroid hormone synthesis†.

Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis.

Extra-Fortification of Zinc Upsets Vitellogenin Gene Expression and Antioxidant Status in Female of Clarias magur brooders.

The present experiment was designed to evaluate the effect of graded level of zinc on Vitellogenin gene (Vtg) expression and antioxidant enzymes in threatened catfish, Clarias magur (C. magur). One hundred and eighty female C. magur with an average weight of 145 ± 5 g were allocated in twelve cemented tanks with dimension 4.5 × 2 × 1 m for a period of 60 days. Fish were distributed in four groups with three replicates following the completely randomised design. The first group treated as control (C) fed with basal diet contained normal zinc level, and remaining groups were fed with basal diets having 50, 200 and 300 mg/kg zinc acetate and treated as T1, T2 and T3 respectively. To evaluate the effect of dietary zinc supplementation on Vtg gene expression, three sampling were carried out, I sampling (April, before starting the experimental trail), II sampling (May, after 1 month of feeding trail) and III sampling (June before breeding season). In the present study, a dose-dependent relationship between Vtg gene expression and zinc inclusion in the diet of threatened catfish, C. magur, was reported. Vtg gene expression increased in all groups from I sampling to II sampling but the highest Vtg gene expression was found in T1 group and the lowest in T3 group at II sampling. Vtg gene expression among the treatments differs significantly (P < 0.05) in each sampling. Accumulation of zinc was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) in C. magur and it was reported that the significantly higher (P < 0.05) zinc was accumulated in the liver and ovary of T3 group as compared to other groups. The antioxidant enzyme activities (superoxide dismutase, SOD, catalase and GST) were also measured in different tissues (liver, gill and ovary) to evaluate the effect of extra-supplementation of zinc on the antioxidant status. In T3 group, SOD, catalase and GST activities were significantly higher than those in other groups. In the current study, serum glucose level was also measured and it was found in increasing trend with inclusion of zinc in the diet of C. magur. In the present study, it can be concluded that the zinc exhibits beneficial effect only up to 50 mg/kg. Thus, it is concluded that supplementation of zinc at 200 mg/kg or more disrupts Vtg gene expression and antioxidant status.

Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent.

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin (vtg) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish's developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures.

Reproductive performance and vitellogenin mRNA transcript abundance in the hepatopancreas of female Litopenaeus vannamei fed diets with different soy lecithin content.

A 30-day nutritional study was conducted for determining the effects of supplementing soy lecithin (SL) in the diet of female Litopenaeus vannamei. Four isonitrogenous and isoenergetic diets were supplemented with graded amounts of SL including 0 (control), 20, 40 and 60 g Kg-1. The brooding specimens fed the 40 g SL/kg diet had the greatest gonadosomatic index (3.4 ±â€¯0.2 %) and the shortest latency period from eyestalk ablation to the first spawning (5 days). In addition, there was a larger content of long chain polyunsaturated fatty acids (LC-PUFA) in the hepatopancreas of brooding specimens fed with the 20 and 40 SL/kg diets compared to the other treatments. Furthermore, brooding specimens fed with the 40 g SL/kg diet had the greatest hemolymph cholesterol and high density lipoprotein concentrations. The abundance of vitellogenin mRNA transcript was greater in the hepatopancreas of brooding specimens fed with the 40 and 60 g SL/kg diets compared with the other groups. Results indicate that supplementing diets with 40 g SL/kg can improve growth and reproductive performance in L. vannamei female brooding specimens probably by enhancing LC-PUFA deposition and enhancing vitellogenin gene expression, as indicated by a greater abundance of mRNA transcript for vitellogenin, in the hepatopancreas.

Probiotic Intake Increases the Expression of Vitellogenin Genes in Laying Hens.

A promising approach for slowing down the rate of reproductive aging is the use of probiotic bacteria as a feed additive. In the current study was investigated the influence of the intake of a potential probiotic on the follicle content and expression of vitellogenin genes (vtg1, vtg2, vtg3) in aged hens. RNA was isolated from liver samples collected from 570-day-old laying hens and gene expression levels were measured using RT-PCR. Bacillus subtilis KATMIRA1933 supplementation had a positive effect on the number of formed follicles in hens and also triggered a significant increase in the relative expression levels of vtg1, vtg2, and vtg3. A Bacillus amyloliquefaciens B-1895 enriched diet or a combination of the two strains had a modest effect on both the number of follicles and the expression of vitellogenin genes. Additionally, the study demonstrates that vitellogenin mRNA expression levels can be considered as a biomarker in a convenient approach for analyzing the hen's egg-laying ability.

Advancing the Zebrafish embryo test for endocrine disruptor screening using micro-injection: Ethinyl estradiol as a case study.

Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro-injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single-dose micro-injection was compared with continuous aquatic exposure in terms of compound accumulation and biological responses. 17α-Ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor activation was concentration/injected dose dependent, increased daily, and was related to esr2b transcription. Transcription of vitellogenin 1 (vtg1) and brain aromatase (cyp19a1b) was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase in cyp19a1b transcripts from 48 hours post fertilization (hpf) onward, whereas after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, whereas others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro-injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1and cyp19a1b transcription and estrogen receptor activation are suitable biomarkers for endocrine disruptor screening in both scenarios. Environ Toxicol Chem 2019;38:533-547. © 2018 SETAC.

Nutritional and reproductive signaling revealed by comparative gene expression analysis in Chrysopa pallens (Rambur) at different nutritional statuses.

BACKGROUND: The green lacewing, Chrysopa pallens Rambur, is one of the most important natural predators because of its extensive spectrum of prey and wide distribution. However, what we know about the nutritional and reproductive physiology of this species is very scarce. RESULTS: By cDNA amplification and Illumina short-read sequencing, we analyzed transcriptomes of C. pallens female adult under starved and fed conditions. In total, 71236 unigenes were obtained with an average length of 833 bp. Four vitellogenins, three insulin-like peptides and two insulin receptors were annotated. Comparison of gene expression profiles suggested that totally 1501 genes were differentially expressed between the two nutritional statuses. KEGG orthology classification showed that these differentially expression genes (DEGs) were mapped to 241 pathways. In turn, the top 4 are ribosome, protein processing in endoplasmic reticulum, biosynthesis of amino acids and carbon metabolism, indicating a distinct difference in nutritional and reproductive signaling between the two feeding conditions. CONCLUSIONS: Our study yielded large-scale molecular information relevant to C. pallens nutritional and reproductive signaling, which will contribute to mass rearing and commercial use of this predaceous insect species.

Alteration in molecular markers of oocyte development and intersex condition in mullets impacted by wastewater treatment plant effluents.

Wastewater Treatment Plant (WWTP) discharges are an important source of endocrine disrupting chemicals (EDCs) into the aquatic environment. Fish populations inhabiting downstream of WWTP effluents show alterations in gonad and gamete development such as intersex condition, together with xenoestrogenic effects such as vitellogenin up-regulation. However, the molecular mechanisms participating in the development of intersex condition in fish are not elucidated. The aim of this study was to assess the impact of two WWTPs effluents (Gernika and Bilbao-Galindo situated in the South East Bay of Biscay) with different contaminant loads, in thicklip grey mullet (Chelon labrosus) populations inhabiting downstream, examining the presence and severity of intersex condition, during two seasons. Molecular markers of xenoestrogenicity and oocyte differentiation and development (vtgAa, cyp19a1a, cyp19a1b, cyp11b, foxl2, dmrt1 and gtf3a) were also studied. Intersex mullets were identified downstream of both WWTPs and vtgAa was upregulated in intersex and non intersex males. Sex dependent differential transcription levels of target genes were detected in mullets from Galindo. However, no such pattern was observed in mullets from Gernika, suggesting an attenuating effect over studied genes caused by a higher presence of EDCs in this site, as indicated by the elevated prevalence of intersex mullets in this population. In conclusion, no direct association between xenoestrogenic responses and intersex condition was established. Mullets from Gernika showed signs of severe EDC exposure compared to those from Galindo, as demonstrated by the higher prevalence of intersex males and the reduction in transcription profile differences between sexes of gametogenic gene markers.

Transcriptome Profile Reveals that Pu-Erh Tea Represses the Expression of Vitellogenin Family to Reduce Fat Accumulation in Caenorhabditis elegans.

Due to misbalanced energy surplus and expenditure, obesity has become a common chronic disorder that is highly associated with many metabolic diseases. Pu-erh tea, a traditional Chinese beverage, has been believed to have numerous health benefits, such as anti-obesity. However, the underlying mechanisms of its anti-obesity effect are yet to be understood. Here, we take the advantages of transcriptional profile by RNA sequencing (RNA-Seq) to view the global gene expression of Pu-erh tea. The model organism Caenorhabditis elegans was treated with different concentrations of Pu-erh tea water extract (PTE, 0 g/mL, 0.025 g/mL, and 0.05 g/mL). Compared with the control, PTE indeed decreases lipid droplets size and fat accumulation. The high-throughput RNA-Sequence technique detected 18073 and 18105 genes expressed in 0.025 g/mL and 0.05 g/mL PTE treated groups, respectively. Interestingly, the expression of the vitellogenin family (vit-1, vit-2, vit-3, vit-4 and vit-5) was significantly decreased by PTE, which was validated by qPCR analysis. Furthermore, vit-1(ok2616), vit-3(ok2348) and vit-5(ok3239) mutants are insensitive to PTE triggered fat reduction. In conclusion, our transcriptional profile by RNA-Sequence suggests that Pu-erh tea lowers the fat accumulation primarily through repression of the expression of vit(vitellogenin) family, in addition to our previously reported (sterol regulatory element binding protein) SREBP-SCD (stearoyl-CoA desaturase) axis.

Effects of the Dietary ω3:ω6 Fatty Acid Ratio on Body Fat and Inflammation in Zebrafish (Danio rerio).

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT-PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.

Analysis of lifespan-promoting effect of garlic extract by an integrated metabolo-proteomics approach.

The beneficial effects of garlic (Allium sativum) consumption in treating human diseases have been reported worldwide over a long period of human history. The strong antioxidant effect of garlic extract (GE) has also recently been claimed to prevent cancer, thrombus formation, cardiovascular disease and some age-related maladies. Using Caenorhabditis elegans as a model organism, aqueous GE was herein shown to increase the expression of longevity-related FOXO transcription factor daf-16 and extend lifespan by 20%. By employing microarray and proteomics analysis on C. elegans treated with aqueous GE, we have systematically mapped 229 genes and 46 proteins with differential expression profiles, which included many metabolic enzymes and yolky egg vitellogenins. To investigate the garlic components functionally involved in longevity, an integrated metabolo-proteomics approach was employed to identify metabolites and protein components associated with treatment of aqueous GE. Among potential lifespan-promoting substances, mannose-binding lectin and N-acetylcysteine were found to increase daf-16 expression. Our study points to the fact that the lifespan-promoting effect of aqueous GE may entail the DAF-16-mediated signaling pathway. The result also highlights the utility of metabolo-proteomics for unraveling the complexity and intricacy involved in the metabolism of natural products in vivo.

Developmental exposure to the organophosphorus flame retardant tris(1,3-dichloro-2-propyl) phosphate: estrogenic activity, endocrine disruption and reproductive effects on zebrafish.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant that is detectable in the environment and biota, prompting concern over its risk to wildlife and human health. Our objective was to investigate whether long-term exposure to low concentrations of TDCPP can affect fish reproduction. Zebrafish embryos were exposed to low concentrations (0, 4, 20 and 100µg/L) of TDCPP from 2h post-fertilization until sexual maturation. Exposure to TDCPP significantly increased plasma estradiol and testosterone levels in females, but had no effect in males. TDCPP exposure also caused a significant reduction in fecundity as indicated by decreased egg production. Real-time PCR was performed to examine selected genes in the hypothalamic-pituitary-gonadal (HPG) axis and liver. Principle component analysis (PCA) showed that sex hormone levels and fecundity were related to the mRNA level of several genes in the HPG axis. Furthermore, hepatic vitellogenin (vtg1 and vtg3) expression was upregulated in both females and males, suggesting TDCPP has estrogenic activity. Histological examination revealed promotion of oocyte maturation in the females, but retardation of spermiation in males. Reduced egg quality (e.g., egg diameter) and increased malformation rates were observed in the F1 generation. Chemical analysis showed significant levels of TDCPP and its metabolite bis(1,3-dichloro-2-propyl) phosphate in the gonads of males and females. In conclusion, long-term exposure to low concentrations of TDCPP impairs fish reproduction.

Dose-dependent compensation responses of the hypothalamic-pituitary-gonadal-liver axis of zebrafish exposed to the fungicide prochloraz.

Compensation responses and adaptability of hypothalamic-pituitary-gonadal (HPG) axis have been reported in fish exposed to model chemicals, however due to its importance in predictive toxicology further study was needed to elucidate details of the integrated responses to model chemicals. Transcriptional profiles of the hypothalamic-pituitary-gonadal (HPG) axis and concentrations of 17ß-estradiol (E2) in plasma were measured in male and female zebrafish that had been exposed to one of seven concentrations of the fungicide, prochloraz: low (1, 3 or 10µg/L), medium (30 or 100µg/L) or high concentrations (300 or 1000µg/L) for 4 days. In zebrafish exposed to the low and medium concentrations of prochloraz, compensation responses of the HPG axis through transcription, occurred in brain (up-regulation of gnrh, gnrhr and lhß) and both brain and gonad (up-regulation of steroidogenic genes), respectively. Concentrations of E2 in plasma and expression of estrogen receptor 1 (er1) and vitellogenins (vtgs) in liver did not change. This result suggested that compensatory responses were successful in maintaining homeostasis. In zebrafish exposed to the two greatest concentrations, compensatory responses occurred in brain, gonad and liver through up-regulation of er2ß, but it failed to maintain concentration of E2 in blood plasma and expression of er1 and vtgs in liver. Collectedly, the results observed in this study allowed characterization of dose-dependent compensatory responses along the HPG axis and liver and identified key linkages between compensatory responses occurring in brain, gonad and liver after exposure to prochloraz.

Isolation of estrogen receptor subtypes and vitellogenin genes: expression in female Chalcalburnus tarichi.

Reproductively arrested gonadal development has been previously described in the teleost pearl mullet (Chalcalburnus tarichi, Cyprinidae) from Van Edremit Gulf of Lake Van, Turkey. Oocyte development in some females was arrested at the previtellogenic stage, while gonadosomatic index (GSI) and plasma 17ß-estradiol (E2) level were low. A subset of the females was found to have normal ovaries and relatively higher plasma E2 and GSI. These two groups were termed reproductively arrested (RA) and reproductively non-arrested (RN) females. In this study, we cloned estrogen receptor (ER) isoforms (ERα, ERß1 and ERß2) and vitellogenin (Vtg), and their mRNA levels were measured in RA and RN fish tissues. C. tarichi ERs fell in the same clade with other fish ERs and ERα and ERß1 had 97% and 98% identity with the roach (Rutilus rutilus) ERs, respectively. Both Vtg and ER isoforms' mRNA abundance were higher in the liver than in the ovary and hypothalamus (liver>ovary>hypothalamus). The level of ERα mRNA was significantly lower in the liver, ovary and brain of RA fish than in the RN fish tissues. ERß1 mRNA levels were not different in the liver and ovary from RA and RN fish while ERß2 expression significantly increased in the liver and ovary from RA fish. All ER subtype expression was found to be lower in the brain from RA fish than RN fish. The level of Vtg mRNA was significantly lower in the liver and ovary from RA fish than RN fish tissue. These results suggest that ER subtypes are differentially regulated by E2, and their functions are also different in vitellogenesis. Analysis of organic contaminants in sediments revealed that C. tarichi living in Van Edremit Gulf of Lake Van are exposed to the contaminants bis(2-ethylhexyl) phthalate and 4,4(') DDT. We suggest that the RA fish represent a segment of the population that is more sensitive to exposure to endocrine disrupting compounds.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Genetics of reproduction and regulation of honeybee (Apis mellifera L.) social behavior.

Honeybees form complex societies with a division of labor for reproduction, nutrition, nest construction and maintenance, and defense. How does it evolve? Tasks performed by worker honeybees are distributed in time and space. There is no central control over behavior and there is no central genome on which selection can act and effect adaptive change. For 22 years, we have been addressing these questions by selecting on a single social trait associated with nutrition: the amount of surplus pollen (a source of protein) that is stored in the combs of the nest. Forty-two generations of selection have revealed changes at biological levels extending from the society down to the level of the gene. We show how we constructed this vertical understanding of social evolution using behavioral and anatomical analyses, physiology, genetic mapping, and gene knockdowns. We map out the phenotypic and genetic architectures of food storage and foraging behavior and show how they are linked through broad epistasis and pleiotropy affecting a reproductive regulatory network that influences foraging behavior. This is remarkable because worker honeybees have reduced reproductive organs and are normally sterile; however, the reproductive regulatory network has been co-opted for behavioral division of labor.

Toxicity of silver nanoparticles to rainbow trout: a toxicogenomic approach.

Silver (Ag) nanoparticles are used as antimicrobial adjuvant in various products such as clothes and medical devices where the release of nano-Ag could contaminate the environment and harm wildlife. The purpose of this study was to examine the sublethal effects of nano-Ag and dissolved Ag on Oncorhynchus mykiss rainbow trout. Hepatic Ag contents and changes in gene expression were monitored to provide insights on bioavailability and mode of action of both forms of silver. Fish were exposed to increasing concentrations (0.06, 0.6 and 6 µg L(-1)) of nano-Ag (20 nm) and silver nitrate (AgNO(3)) for 96 h at 15°C. A gene expression analysis was performed in the liver using a DNA microarray of 207 stress-related genes followed by a quantitative polymerase chain reaction on a selection of genes for validation. The biochemical markers consisted of the determination of labile zinc, metallothioneins, DNA strand breaks, lipid peroxidation (LPO) and vitellogenin-like proteins. The analysis of total Ag in the aquarium water revealed that nano-Ag was mostly aggregated, with 1% of the total Ag being dissolved. Nevertheless, hepatic Ag content was significantly increased in exposed fish. Indeed, dissolved Ag was significantly more bioavailable than nano-Ag only at the highest concentration with 38 ± 10 and 11 ± 3 ng Ag mg(-1) proteins for dissolved and nano-Ag respectively. Exposure to both forms of Ag led to significant changes in gene expression for 13% of tested gene targets. About 12% of genes responded specifically to nano-Ag, while 10% of total gene targets responded specifically to dissolved Ag. The levels of vitellogenin-like proteins and DNA strand breaks were significantly reduced by both forms of Ag, but DNA break levels were lower with nano-Ag and could not be explained by the presence of ionic Ag. Labile zinc and the oxidized fraction of metallothioneins were increased by both forms of Ag, but LPO was significantly induced by nano-Ag only. A discriminant function analysis revealed that the responses obtained by biochemical markers and a selection of ten target genes were able to discriminate completely (100%) the effects of both forms of Ag. Exposure to nano-Ag involved genes in inflammation and dissolved Ag involved oxidative stress and protein stability. Hence, the toxicity of Ag will differ depending on the presence of Ag nanoparticles and aggregates.

Phytoestrogens genistein and daidzein affect immunity in the nematode Caenorhabditis elegans via alterations of vitellogenin expression.

SCOPE: Phytoestrogens, such as the soy isoflavones genistein and daidzein, are suggested to beneficially affect lipid metabolism in humans and thereby contribute to healthy ageing. New evidences show that phytoestrogens might slow ageing processes also by affecting immune processes. METHODS AND RESULTS: We tested in the nematode Caenorhabditis elegans the effects of 17ß-estradiol, genistein, and daidzein on resistance versus the nematode pathogen Photorhabdus luminescens with focus on vitellogenins, which are invertebrate estrogen-responsive genes that encode homologues to ApoB100 with impact on immune functions. Here, we show that the estrogen 17ß-estradiol increases the resistance of C. elegans versus P. luminescens by enhancing vitellogenin-expression at the mRNA and protein level. Knockdown of single out of five functional vits by RNA-interference blunted the life-extending effects under heat-stress of 17ß-estradiol, demonstrating a lack of redundancy for the vitellogenins. RNAi for nhr-14, a suggested nuclear hormone receptor for estrogens, displayed no influence on 17ß-estradiol effects. The soy isoflavone genistein reduced vitellogenin-expression and also resistance versus P. luminescens whereas daidzein increased resistance versus the pathogen in a vitellogenin-dependent manner. CONCLUSION: Our studies show that induction of estrogen-responsive vitellogenin(s) by the phytoestrogen daidzein potently increases resistance of C. elegans versus pathogenic bacteria and heat whereas genistein acts in an antiestrogenic manner.

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.

Dual effects of daidzein on chicken hepatic vitellogenin II expression and estrogen receptor-mediated transactivation in vitro.

Two in vitro systems were employed to delineate the estrogenic activity of daidzein (Da), alone or in combination with high or low concentrations of estrogen in two cell types possessing different estrogen-receptor (ER) isoforms, ERalpha and/or ERbeta: (1) vitellogenin II (VTG), the egg yolk precursor protein and the endpoint biomarker for estrogenicity, in chicken primary hepatocytes, and (2) CHO-K1 cells transiently co-transfected with ERalpha or ERbeta and estrogen-response elements (ERE) linked to a luciferase reporter gene. Da (100 microM) alone induced VTG mRNA expression in chicken hepatocytes, albeit with much less potency compared to estradiol (E(2)). Da exhibited different effects in the presence of 1 microM and 10 microM E(2). At a concentration of 100 microM, Da enhanced 1 microM E(2)-induced VTG transcription by 2.4-fold, but significantly inhibited 10 microM E(2)-induced VTG mRNA expression in a dose-dependent fashion from 1 to 100 microM. Tamoxifen completely blocked the estrogenic effect of daidzein, alone or in combination with 1 microM of E(2), but did not influence its anti-estrogenic effect on 10 microM E(2)-induced VTG mRNA expression. Furthermore, neither E(2) nor daidzein, alone or in combination, affected ERalpha mRNA expression, yet all the treatments significantly up-regulated ERbeta mRNA expression in chicken hepatocytes. E(2) effectively triggered estrogen-response elements (ERE)-driven reporter gene transactivation in CHO-K1 cells expressing ERalpha or ERbeta and showed much greater potency with ERalpha than with ERbeta. In contrast, daidzein was 1000 times more powerful in stimulating ERbeta- over ERalpha-mediated transactivation. Daidzein, in concentrations ranging from 5 nM to 50 microM, did not affect ERbeta-mediated transactivation induced by 1 nM E(2), but it significantly inhibited ERbeta-mediated transactivation induced by 10 nM E(2) at 500 nM. Despite the tremendous difference in sensitivity between the two in vitro systems, daidzein exhibited greater potency as an estrogen-antagonist for ERbeta-mediated activity.