Vitellogenin Concentrations Research Articles

Effects of Sublethal Concentrations of Insecticides on the Fecundity of Sogatella furcifera (Hemiptera: Delphacidae) via the Regulation of Vitellogenin and Its Receptor.

White-backed planthopper (Sogatella furcifera, Hemiptera: Delphacidae) is an important migratory pest of rice. It causes severe economic losses by reducing crop production. Vg and VgR are important proteins that help in the successful reproduction of insects and have been studied in many insects. To understand the molecular mechanisms underlying the effects of insecticides on white-backed planthopper reproduction, we studied the expression profiles of SfVg, SfVg-like, and SfVgR in white-backed planthopper exposed to insecticides. SfVg and SfVgR silencing inhibited the ovarian development, number of eggs laid by, and hatching rate of white-backed planthopper. Thiamethoxam LC10 significantly inhibited SfVg-like and SfVgR expression. In contrast, triazophos LC25 significantly promoted SfVg, SfVg-like, and SfVgR expression and increased vitellogenin content in white-backed planthopper. These results demonstrate that insecticides can regulate the reproduction of white-backed planthopper by altering the expression of SfVg and SfVgR, thereby affecting the population density of white-backed planthopper. These findings build a foundation for improving our understanding of the molecular mechanisms underlying the effects of insecticides on the reproduction and resurgence of pests.

Proteomics and lipidomics analyses reveal modulation of lipid metabolism by perfluoroalkyl substances in liver of Atlantic cod (Gadus morhua)

The aim of the present study was to investigate effects of defined mixtures of polycyclic aromatic hydrocarbons (PAHs) and perfluoroalkyl substances (PFASs), at low, environmentally relevant (1× = L), or high (20× = H) doses, on biological responses in Atlantic cod (Gadus morhua). To this end, farmed juvenile cod were exposed at day 0 and day 7 via intraperitoneal (i.p.) injections, in a two-week in vivo experiment. In total, there were 10 groups of fish (n = 21-22): two control groups, four separate exposure groups of PAH and PFAS mixtures (L, H), and four groups combining PAH and PFAS mixtures (L/L, H/L, L/H, H/H). Body burden analyses confirmed a dose-dependent accumulation of PFASs in cod liver and PAH metabolites in bile. The hepatosomatic index (HSI) was significantly reduced for three of the combined PAH/PFAS exposure groups (L-PAH/H-PFAS, H-PAH/L-PFAS, H-PAH/H-PFAS). Analysis of the hepatic proteome identified that pathways related to lipid degradation were significantly affected by PFAS exposure, including upregulation of enzymes in fatty acid degradation pathways, such as fatty acid β-oxidation. The increased abundances of enzymes in lipid catabolic pathways paralleled with decreasing levels of triacylglycerols (TGs) in the H-PFAS exposure group, suggest that PFAS increase lipid catabolism in Atlantic cod. Markers of oxidative stress, including catalase and glutathione S-transferase activities were also induced by PFAS exposure. Only minor and non-significant differences between exposure groups and control were found for cyp1a and acox1 gene expressions, vitellogenin concentrations in plasma, Cyp1a protein synthesis and DNA fragmentation. In summary, our combined proteomics and lipidomics analyses indicate that PFAS may disrupt lipid homeostasis in Atlantic cod.

Impact of herbicide pretilachlor on reproductive physiology of walking catfish, Clarias batrachus (Linnaeus).

Herbicide pretilachlor is widely used in paddy fields to control annual weeds. The present study has been carried out in walking catfish, Clarias batrachus, to evaluate the impact of herbicide pretilachlor on reproductive physiology after chronic exposure. Based on the median lethal concentration value (96 h), fish were exposed to three nominal test concentrations of pretilachlor ((SL-I (1/20th LC50), SLII (1/15th LC50), and SL-III (1/10th LC50)) for 30, 45, and 60 days after which plasma sex steroid profile, plasma vitellogenin concentration, and gonadal aromatase activity were analyzed in both sexes. Plasma concentration of testosterone decreases in herbicide-exposed male fish. Significant increase in plasma 17β-estradiol, plasma vitellogenin concentration, and gonadal aromatase activity were observed in herbicide-exposed male fish. All these alterations in reproductive parameters in male fish are dependent on concentration and exposure duration of herbicide. On the other hand, significant decrease in plasma concentration of testosterone was observed in female fish which was also dependent on concentration and exposure duration of herbicide. No significant changes in plasma 17β-estradiol concentrations, plasma vitellogenin concentration, and gonadal aromatase activity were observed in female fish. Above findings clearly suggested that herbicide pretilachlor acts as endocrine disruptor in fish and affects overall reproductive physiology of fish, but its ability to induce reproductive toxicity in male and female differs considerably.

Reproductive Performances and Egg Qualities in African Catfish (<i>Clarias gariepinus</i>) Broodstocks Supplemented with Curcumin and Thyroxine Hormone

An experiment was designed to study the effects of curcumin and thyroxine hormone supplementation in the diet to improve the reproductive performance of catfish broodstock by improving egg quality that eventually increases the production of seedlings. Catfish used in this experiment were supplemented with curcumin and thyroxine hormone through their feeds for 12 weeks. The results showed that there was no significant difference (p > 0.05) in the hepatosomatic index (HSI), gonadosomatic index (GSI), percentage of gonad maturity, total cholesterol concentration in the spawned eggs, high density lipoprotein (HDL) concentration in the spawned eggs, fertilization rate of spawned eggs, and hatching rate of fertilized eggs. However, there were significant differences (p < 0.05) in the concentration of vitellogenin in the spawned eggs, egg diameter of the spawned eggs, and the triglycerides contents of spawned eggs. It was concluded that curcumin and thyroxine supplementations of African catfish increased vitellogenin concentrations and diameters of spawned eggs that have great potential to improve the reproductive performance.

Multi-approach assessment for the evaluation of spatio-temporal estrogenicity in fish from effluent-dominated surface waters under low instream flow

Current practices employed by most wastewater treatment plants (WWTP) are unable to completely remove endocrine disrupting compounds (EDCs) from reclaimed waters, and consistently discharge these substances to receiving systems. Effluent-dominated and dependent surface waters, especially during low instream flows, can increase exposure and risks to aquatic organisms due to adverse biological effects associated with EDCs. Given the ecological implications that may arise from exposure to such compounds, the present a multi-approach study examined spatio-temporal estrogenic potential of wastewater effluent to fish in East Canyon Creek (ECC), Utah, USA, a unique urban river with instream flows seasonally influenced by snowmelt. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at different upstream and downstream sites from an effluent discharge during the summer and fall seasons. In the summer, where approximately 50% of the streamflow was dominated by effluent, fish from the upstream and a downstream site, located 13 miles away from the effluent discharge, presented significantly elevated concentrations of plasma vitellogenin (VTG). Similarly, significantly high 17β-estradiol to 11-ketotestosterone ratios were measured in the summer across all sites and time points, compared to the fall. In the laboratory, juvenile fish and primary hepatocytes were exposed to concentrated effluent and surface water samples. Quantification of VTG, although in significantly lower levels, resembled response patterns observed in fish from the field study. Furthermore, analytical quantification of common EDCs in wastewater revealed the presence of estriol and estrone, though these did not appear to be related to the observed biological responses, as these were more significant in sites were no EDCs were detected. These combined observations suggest potential estrogenicity for fish in ECC under continuous exposures and highlight the advantages of following weight-of-evidence (WoE) approaches for environmental monitoring, as targeted analytically-based assessments may or may not support the identification of causative contaminants for adverse biological effects.

Impact of Endocrine Disruptors on Vitellogenin Concentrations in Wild Brown Trout (Salmo trutta trutta).

The adverse effects of endocrine disruptors (EDs) on aquatic wildlife and human health represent a current issue of high public concern.Substantial knowledge of the level of estrogenic EDs in fish has accumulated from field surveys.For this purpose, a survey of wild brown trout (Salmo trutta trutta) was carried out to assess the incidence of EDs in the feral fish population living in the Liri river (Abruzzi, Italy). The results of this study show that this aquatic environment possesses an estrogenic potency that triggered the increase of vitellogenin levels in both female and male trouts. Fish exposed to different pesticides and urban waste in downstream river showed higher vitellogenin levels in comparison to the headwater site. Furthermore, some trouts coming from the downstream reported the presence of several pesticides and fungicides, some of these banned several years ago.

Effect of dietary essential fatty acids on level of oestradiol‐17β and vitellogenin, reproductive performance and larval quality of striped catfish(Pangasianodon hypophthalmus)in out‐of‐spawning season

A study was conducted to determine the effect of essential fatty acid in striped catfish (Pangasianodon hypophthalmus) broodstock diets on a level of oestradiol-17β and vitellogenin, reproductive performance and larval quality in out-of-spawning season. Five female and five male broodstock were stocked into a net cage (3 × 5 × 1.5 m3) and fed with pelleted diets containing 1.5% fish oil and different amounts of corn oil (CO) in diet formulation, that is 0%, 1%, 2% and 3% respectively. Gonadal development, oestradiol-17ß and vitellogenin concentrations were examined every 2 weeks. Oestradiol-17ß and vitellogenin concentrations showed an increase during the maturation process. The significantly highest percentage of female mature, fecundity, hatching rate, larval production and larval survival rate were obtained from fish fed with 2% corn oil in the diet. This result revealed that the administration of 2% corn oil in the diet as a source of n-6 (linoleic acid) and 1.5% fish oil as a source of n-3 (linolenic acid) can improve the reproductive performance of striped catfish broodstock in out-of-spawning season.

Dietary Supplementation With Icariin Affects Estrogen Synthesis, Vitellogenesis, and Oocyte Development in the Chinese Mitten Crab, Eriocheir sinensis

In Eriocheir sinensis aquaculture, crabs feeding an artificial diet without steroid hormones available in ice-fresh fish or snails show delayed ovarian maturation and a delay in listing. Icariin (ICA), a safe monomer compound derived from plant, can effectively promote oestradiol (E2) synthesis and ovarian development in mammals, but its use on crustaceans has not been reported. We investigated the effects of ICA on oestrogen synthesis, vitellogenesis, and oocyte development in Chinese mitten crabs fed one of four diets containing 0, 50, 100, and 200 mg/kg ICA, respectively, for eight weeks. Results showed that vitellogenin contents, gonadosomatic indexes, and oocyte volumes in ICA50 and ICA 100 crabs were significantly higher than those in the control. The highest up-regulated expression of Vitellogenin (VTG) in hepatopancreases and ovaries were detected in ICA100 crabs. Haemolymph E2 contents were also significantly increased in ICA100 crabs. Enzyme-linked immunosorbent assay and western blot analysis indicated that the increase in E2 concentrations was attributable to an elevation in aromatase protein levels in ovaries through the cyclic adenosine monophosphate/ protein kinase A/ cAMP response element binding protein pathway. These findings suggest that ICA could serve as a natural additive to resolve the problem of delayed ovarian maturation of crabs raised in ponds.

Xenobiotics Result in Hormonal and Enzymatic Dysregulations in the Red Mussel Mytilus galloprovincialis (Lamarck, 1819) (Bivalvia, Mytilidae)

<b>Background and Objective:</b> The contaminants in a marine ecosystem like mercury and synthetic hormones can disrupt the regulation of natural endocrine and reproductive systems of most organisms. This study aims to study the effect of organic and inorganic mercury on the viscera of <i>Mytilus galloprovincialis</i> after intracoelomic injection of 17α-ethinylestradiol, 17β-estradiol and Dichlorodiphenyltrichloroethane (DDT) and check the histological changes in the gonads. <b>Materials and Methods:</b> Mussels are collected during June-August, 2018 from Ras el tin beach of the Mediterranean Sea of Alexandria, Egypt. This study aims to: test the effect of 17α-ethinylestradiol, 17β-estradiol and DDT on vitellogenin (VTG) synthesis, enzymes dysfunction through intracoelomic injection of methyl mercury in a 0.75 μg/0.1 mL and mercury chloride to a 75 μg/0.1 mL. Gonads are studied histologically in control and treated mussels. Water-administered E2 and EE2 at 120 μL dose induced VTG expression in males 14 days exposure. <b>Results:</b> The relative concentration of VTG in the induced groups increases significantly as compared to the control. Alterations in the gonadal tissues and the maturation stages of the mussels are observed. The imposex mussels are characterized by concomitant secondary male sexual characteristics and the female gonad shows testicular structure. Superoxide Dismutase (SOD) activity in mussel digestive glands differed significantly (p = 0.002) after 72 hrs of MeHg exposure. <b>Conclusion:</b> Significant correlation can be observed between the activities of Glutathione S-Transferases (GST) and Glutathione Reductase (GR) in the digestive glands of mussels treated with MeHg, the enzyme activities of digestive glands treated with HgCl<sub>2</sub> and between Superoxide Dismutase<i>-</i>Catalase (SOD-CAT), SOD-GR and GST-GR.

Dietary genistein disrupts sex steroid and vitellogenic response in female common carp, Cyprinus carpio L.

A 60-day feeding trial was conducted to evaluate the impact of dietary genistein on steroidogenesis and vitellogenesis pattern in female common carp, Cyprinus carpio L. Adult female C. carpio were fed with graded levels of genistein in different groups is 0 (Control), 1 (Gen-1), 3 (Gen-3), 6 (Gen-6) and 9 (Gen-9) mg% of feed. As a result, inclusion of genistein significantly reduced the number and size of vitellogenic oocyte in the ovary of C. carpio. Serum vitellogenin (VTG) concentration as well as the expression of erβ and vtgb2 in liver were significantly reduced in genistein fed female C. carpio. Serum testosterone and 17β-estradiol (E2) concentration in female C. carpio were increased with increasing dose of genistein, whereas the expression of cyp19a from the ovary was downregulated in genistein fed C. carpio. The study concludes that structural resemblance of genistein to endogenous estrogen may have led to competing with E2 for binding to estrogen receptors of the liver, which further impaired vitellogenin synthesis and hence created steroid hormonal imbalance. These endocrine disrupting events caused an impairment in ovarian growth of C. carpio.

Mul-tiomics analysis of cadmium stress on the ovarian function of the wolf spider Pardosa pseudoannulata

Cadmium (Cd) pollution is widespread in paddy filed soil in China. In this study, the toxicity of Cd with regard to the female reproductive system of paddy spider Pardosa pseudoannulata was investigated by means of multi-omics analyses (transcriptome, proteome, and miRNAs). Decreased activities of detoxifying enzymes including peroxidase (POD), Glutathione S-transferases (GST), and superoxide dismutase were detected in the ovary of P. pseudoannulata. Of these, GST and POD were consistently down-regulated at the transcriptional and translational levels. Vitellogenin content and fecundity of the spider were also reduced by Cd burden. Five vitellogenin encodes genes were down-regulated while only vitellogenin-6 protein was up-regulated. But protein lipovitellin-1, the main composition of vitellin, was down-regulated. In addition, the correlation between the mitogen-activated protein kinase (MAPK) signaling pathway and Cd stress was identified. A down-regulated gene that encoding connector of kinase to AP-1 in the MAPK signaling pathway was regulated by the up-regulated miRNA (miRNA id: miRNA dan-miR- 318>der-miR-318>dgr-miR-318>dme-miR-318–3p > dmo-miR-318>dpe-miR-318>dps-miR-318>dse-miR-318>dsi-miR-318>dvi-miR-318>dwi-miR-318>dya-miR-318). In conclusion, Cd stress possesses distinct female reproductive toxicity on P. pseudoannulata through impairing antioxidant system and synthesis of vitellin.

Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

Plastic originated bisphenol A: A potential endocrine disruptor for fish reproduction

Bisphenol A (BPA) is an organic synthetic compound, abundantly used worldwide for the production of polycarbonate plastic and epoxy resins. It is an endocrine disruptor that can mimic estradiol by binding to and activating the same estrogen receptor as the natural hormone. It is not only hazardous to human population but also found to be acutely toxic to aquatic organisms in the range 1000–10,000μg/L for freshwater and marine species. In 2010, the U.S. Environmental Protection Agency reported that over one million pounds of BPA are released into the environment annually. BPA can enter the environment either directly from chemical, plastics, coat and staining manufacturers, from paper or material recycling companies, or indirectly leaching from plastic, paper and metal waste in landfills or ocean-borne plastic trash. BPA in anaerobic or semi aerobic sediment environments can persist for a prolonged period of time, leading to higher BPA levels in sediments than in surface water. Interestingly BPA can persist longer in seawater than in fresh water without any degradation (about 30 day) and the possibility of BPA contamination is higher marine than freshwater organisms. BPA possess endocrine disruption in different types of fishes by vitellogenin induction, upregulation of brain aromatase isoform mRNA, reduction of total sperm counts and induction of testis ova and poor somatic growth of male. It was also observed that environmentally relevant low level of BPA increased the expression of genes related to reproduction axis such as kiss1, kiss1r, Gnrh3, LH β, FSHβ, and ERα and dmrt1. BPA is also assumed to involve THR, in increasing the rate of early embryonic development in several fish species. Further, BPA exhibit very high estrogenic activity on the cyp19a1b gene, and increase concentration of vitellogenin in swim-up fry of freshwater fish species. Keeping in view of these, the present study is aimed to elucidate a baseline information about contamination routes of BPA in the aquatic environment and its endocrine-disruptive effects on aquatic organisms.

Histology and design-based estimation of hepatocellularity and volumes of hepatocytes in control and ethynylestradiol exposed males of platyfish (Xiphophorus maculatus)

The liver hosts numerous vital functions, such as biotransformation and excretion of xenobiotics. Synthetic oestrogens influence liver structure and function, leading to adaptations or to dysfunctions/injury. They are often stated to induce increases in fish liver weight, but there is controversy regarding how: if by changes in hepatocyte size (hypertrophy) and/or number (hyperplasia). Using platyfish as the experimental model, our primary aim was to assess if/how hepatocytes reacted to a sub-acute oestrogenic exposure. A complementary aim was to generate fundamental structural data for the liver of that model organism. Adult males were injected intramuscularly with 17α-ethinylestradiol (EE2) (25 μg/g), every 72 h for two weeks. Control fish were given solvent only. Body and liver morphometry were registered, and hepatocytes examined through histology and stereology at light microscopy. Immunohistochemistry evaluated hepatocytic vitellogenin (VTG) content. Treated and control fish did not differ as to quantitative parameters. Nevertheless, exposed fish were sensitive to EE2. VTG tagging was positive in their hepatocytes and these tended to be more basophilic, though not fully oestrogenized. We hypothesise that the platyfish liver is not particularly sensitive to the disrupting action of EE2 because of its reproductive mode; with no production peaks of VTG and no huge changes in endogenous sex-steroids. The fish may have had no evolutionary pressure for hepatocytes to be particularly reactive to oestradiol (E2). In the end, this study offers the first unbiased estimation of the liver cellularity in the platyfish, as well of the hepatocytic volume, serving now as a baseline reference.

Difference in biochemical markers in the gibel carp (Carassius auratus gibelio) upstream and downstream of the hydropower plant

However the physiological stress in aquatic organisms associated with hydropower plants (HPP) ecosystems has been previously investigated, no studies have so far assessed it on biochemical level. Therefore this study evaluated an oxidative stress and toxicity in the gibel carp Carassius auratus gibelio associated with a small-scale HPP in the West Ukraine. A battery of liver, brain and blood markers was evaluated individuals inhabiting upstream and downstream of the dam of the small-scale Kasperivtci HPP (KHPP; an installed capacity of 7.5 MW), and from a reference site. Number of alterations were noted in fish from the KHPP impoundment facility including signs of oxidative stress (a decrease in superoxide dismutase (SOD) activity and an increase in protein carbonyls) and cytotoxicity (an increase in micronucleated erythrocytes and caspase-3 activity). No changes in DNA fragmentation in hepatocytes or brain cholinesterase activity were detected. As demonstrated by the integral stress index, fish associated with downstream of the dam revealed the greatest alterations reflected by the combined oppression of antioxidant system (SOD, catalase) and pro-oxidants (thiobarbituric acid reactive substances and oxyradicals), low concentration of metallothioneins, but high cathepsin D activity (as markers of lysosomal dysfunction and autophagy) and increased vitellogenin concentration in males (indicating an endocrine disruption). The study highlights that fish inhabiting ecosystems associated with HPP, particularly downstream of the dam, may face additional stresses with long-term effects yet to be evaluated.

Bioaccumulation of TBT and Its Cellular Toxic Effects on the Freshwater Prawn Macrobrachium rosenbergii.

To test the toxic effects of tributyltin (TBT), Macrobrachium rosenbergii were exposed to three concentrations of TBT viz. 10ng/L, 100ng/L and 1000ng/L for 90days. The bioaccumulation of TBT level varied in hepatopancreas based upon dose dependent manner. Histopathological results revealed the reduction in basement membrane thickness, disruption of the hepatopancreatic tubules and abnormal lumen in hepatopancreas of TBT treated prawns. The ultrastructure of the control prawn showed normal architecture of cellular organelles with prominent nuclei in hepatocytes. On the other hand, many vacuoles, irregular arrangements of microvilli, swollen mitochondria, distorted rough endoplasmic reticulum cisternaes and abnormal nucleus were seen in the TBT treated group. Further, the biochemical and vitellogenin content were altered remarkably due to TBT exposure. It directly indicated that TBT had conspicuously inhibited the vitellogenesis. Therefore, it was inferred that the administration of TBT has considerably affected the hepatopancreatic functions in M. rosenbergii.

Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei

The regulation of female reproduction in crustaceans is controlled by a variety of hormones. Previous studies of hormone-initiated cellular mechanisms controlling ovarian maturation focused mainly on those initiated by inhibitory hormones. In order to facilitate research on ovarian development, it is necessary to gain a better understanding of factors that promote ovarian growth on the cellular level. Here, we used eyestalk ablation to firstly induce a state of ovarian maturation in Litopenaeus vannamei. Gonadosomatic index, hemolymph vitellogenin (Vg) concentrations, and Vg gene transcript levels in the ovaries were significantly elevated at 1 and 2 weeks after eyestalk ablation (P < 0.05). Correspondingly, immunoblot analysis revealed a remarkable decrease in anti-PKC-α immunoreactivity in both cytosol and membrane fractions of ovarian tissue homogenates: it was strongly apparent in intact animals, but decreased with time after eyestalk ablation, showing a stronger tendency to do so in the membrane fraction than in the cytosol fraction. Considered overall, the data presented strongly suggest that PKC-α isoform plays a role in the regulation of ovarian growth in L. vannamei through a negative-based regulating mechanism.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 μg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17β-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17βhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.

Effects of Water Samples in Polyethylene Terephthalate Bottles Stored at Different Conditions on Zebrafish Embryos with Relevance to Endocrine Disrupting Chemical Migration and Adenomatous Polyposis Coli Tumor Suppressor Gene

Objective: Polyethylene terephthalate (PET) is a material that is most commonly used for production of clear plastic bottles. Adenomatous polyposis coli (APC) and β-catenin have been related with cancer. Aim was to investigate the effects of PET bottled water samples that were exposed to sunlight and hot water on zebrafish embryos. Moreover the effects of these water samples on APC knockdown zebrafish embryos were also evaluated.Methods: Phthalate concentrations in water samples were determined using ELISA. Immunohistochemical method and RT-PCR were used to analyse the expressions of proliferative cell nuclear antigen (PCNA), β catenin, Wnt 3a and Gsk3β. Biochemical parameters were measured using spectrophotometric methods. Vitellogenin concentrations were measured using ELISA and apoptotic cells were evaluated by Acridine Orange staining.Results: Increased PCNA, β-catenin, Wnt 3a, Gsk3β expressions, vitellogenin, nitric oxide, apoptosis and impaired oxidant-antioxidant balance were observed in the exposure groups with these increases being more profound in APC knockdown groups.Conclusion: APC knockdown embryos were more prone to the deleterious effects of water samples used in this study.

A combined FSTRA-shotgun proteomics approach to identify molecular changes in zebrafish upon chemical exposure

The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.