Alterations in neurotransmitters, steroid hormones, vitellogenin, and antioxidant system induced by di-n-butyl phthalate and di-isopentyl phthalate on catfish Rhamdia quelen.

Phthalates, such as di-n-butyl phthalate (DBP) and di-isopentyl phthalate (DiPeP), are pollutants with a high potential for endocrine disruption. This study aimed to evaluate parameters of endocrine disruption in specimens of the Neotropical fish Rhamdia quelen exposed to DBP and DiPeP through their food. After 30 days of exposure, the fish were anesthetized and then euthanized, and blood, hypothalamus, liver, and gonads were collected. DBP caused statistically significant alterations in the serotoninergic system of males (5 and 25 ng/g) and females (5 ng/g) of R. quelen and it increased testosterone levels in females (25 ng/g). DiPeP significantly altered the dopaminergic system in females, reduced plasma estradiol levels (125 ng/g) and hepatic vitellogenin expression (25 ng/g), and changed the antioxidant system in gonads (125 ng/g). The results suggest that DBP and DiPeP may have different response patterns in females, with the former being androgenic and the latter being anti-estrogenic. These findings provide additional evidence regarding the molecular events involving DBP and DiPeP in the endocrine disruption potential in juvenile specimens of Rhamdia quelen.

Effects of trophic 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure in Oreochromis niloticus: A multiple biomarkers analysis.

Polybrominated diphenyl esters are emerging environmental contaminants with few toxicological data, being a concern for the scientific community. This study evaluated the effects of BDE-47 on the health of Oreochromis niloticus fish. The animals were exposed to three doses of BDE-47 (0, 0.253, 2.53, 25.3 ng g-1) every 10 days, for 80 days. The BDE-47 affected the hepatosomatic and gonadosomatic index in female and the condition factor by intermediate dose in both sexes. The levels of estradiol decreased and the T4 are increased, but the vitellogenin production was not modulated in male individuals. Changes in AChE, GST, LPO and histopathology were observed while the integrated biomarker response index suggests that the lowest dose of BDE-47 compromised the activity of antioxidant enzymes. The oral exposure to BDE-47 in environmental concentrations is toxic to O. niloticus and the use of multiple biomarkers is an attribution in ecotoxicology studies and biomonitoring programs.

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 17ß-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 17ß-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.

Deleterious impacts of heat stress on steroidogenesis markers, immunity status and ovarian tissue of Nile tilapia (Oreochromis niloticus).

The water temperature of aquacultures is a primary factor of fish welfare, reproductive patterns, and immunity. To elucidate the molecular and biological processes of the temperature modulation of reproduction and immunity, female Nile tilapia (190 ± 10g) were allocated into five groups following acclimatization (150 females, three replicates, each n = 10). Each group was subjected to various temperatures (28 °C, 30 °C, 32 °C, 34 °C, and 37 °C), the group at 28 °C representing the control. Their serum levels of estradiol, cortisol, and vitellogenin were measured as well as serum triiodothyronine (T3) hormone, thyroxine (T4) hormone, and non-specific immunity (phagocytic and lysozyme activity). In addition, steroidogenic acute regulatory protein (STAR), vitellogenin gene receptor, and heat shock protein 70 (HSP70) gene expression were evaluated. The serum levels of estradiol, cortisol, and vitellogenin markedly declined (P < 0.05) in fish group at higher temperatures. In addition to T3, T4 was significantly affected (P < 0.05) in the control group. The expressions of the STAR gene (steroidogenesis) and vitellogenin receptors were also considerably down-regulated. The histopathological photomicrograph of fish subjected to high water temperature revealed injuries in ovary tissues, demonstrating its harmful effects. The experimental results verified the possible role of water temperature as a main stressor on Nile tilapia' physiology through modulation of steroidogenesis-related gene expression and immunity.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

Trade-off between aerobic performance and egg production in migratory macaroni penguins.

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E2) to support vitellogenesis and egg production, but E2 secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E2-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 µg Zn ml-1, and 9.70 ± 0.19 mmol l-1, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens.

Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

A Preliminary Survey of Estrogenic Effects in Cultured Adult Male Nile Tilapia (Oreochromis niloticus) in the Seven Lakes of San Pablo City, Philippines.

This study aimed to investigate the impact of estrogenic endocrine disruptors in adult male Nile Tilapia (Oreochromis niloticus) from the Seven Lakes (Bunot, Calibato, Mohicap, Palakpakin, Pandin, Sampaloc and Yambo) of San Pablo City in Laguna, Philippines. Fish were collected from aquaculture operations in each lake from July to September 2017 and examined for plasma vitellogenin (VTG) levels, hepatosomatic index (HSI) and gonadosomatic index (GSI). In comparison to the reference group (Pandin Lake), fish from Mohicap, Sampaloc and Yambo had higher levels of plasma VTG. HSI was increased in fish from Bunot Lake while it was markedly reduced in fish from Sampaloc Lake. Comparable GSI were obtained in all samples. While most examined fish are in active spermatogenesis, fish from Sampaloc Lake had immature gonads with no spermatozoa, and with minimal to moderate Leydig cell vacuolation and hyperplasia. The results suggest that cultured Nile tilapia from Sampaloc Lake is most adversely affected by endocrine-active compounds. The identification and quantification of these chemicals are recommended in future studies.

Chronic exposure to environmental concentrations of phenanthrene impairs zebrafish reproduction.

Phenanthrene (PHE) is a tricyclic polycyclic aromatic hydrocarbon which distributed extensively in the aquatic environment. However, the knowledge about its impact on fish reproduction is still limited, particularly under a chronic exposure regime. In this study, we exposed zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 µg/L) of PHE for 4 months and assessed the impact on reproduction. The results demonstrated that egg production was decreased in fish exposed to PHE, with a significant reduction at 5.0 µg/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or E2 in male fish. In addition, plasma vitellogenin levels were significantly inhibited after PHE exposure in female fish. The transcription of hypothalamic-pituitary-gonadal (HPG) axis related genes (GnRH2, FSHß, LHß, 17ß-HSD, CYP11A1, and CYP19a) were significantly altered in a sex-specific manner. In addition, embryos derived from exposed parents exhibited increased malformation and decreased hatching success in the F1 generation. Taken together, these results demonstrate that chronic exposure to environmentally relevant concentration of PHE could cause adverse effects on reproduction and impair the development of offspring, ultimately leading to fish population decline in aquatic environment.

Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17ß (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ±â€¯28.9 µg/mL (VtgAa), 57.9 ±â€¯30.7 µg/mL (VtgAb) and 12.6 ±â€¯4.8 µg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 µg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ±â€¯733.93 µg/mL) was significantly higher than for VtgAa (150.33 ±â€¯22.35 µg/mL) or VtgC (57.08 ±â€¯6.00 µg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.

Long-term exposure to waterborne nonylphenol alters reproductive physiological parameters in economically important marine fish.

Low concentrations of nonylphenol (NP) in aquatic environment can induce drastic effects on the endocrine system in animals. In this study, we examined the modulatory effects of NP on reproductive and physiological parameters in juveniles of the red seabream and black rockfish following waterborne NP exposure (0, 1, 10, and 50 µg L-1) for 60 days. In red seabream exposed to 50 µg L-1 NP, plasma levels of 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) were significantly lower at 30 and 60 days, while E2 levels were slightly higher in 10 µg L-1-exposed individuals at day 30. Similarly, significantly lower levels of E2 and 11-KT were observed in 10 and 50 µg L-1-exposed black rockfish at 60 days, whereas the E2 level was higher in 1 µg L-1-exposed individuals at day 30. After exposure to NP, plasma and mRNA levels of vitellogenin (VTG) were significantly higher in both species at 30 and 60 days, similar to the inducible effects from synthetic estrogen. Plasma cortisol levels were significantly elevated by relatively higher concentrations of NP (10 and 50 µg L-1) at 30 and 60 days. Finally, 60 days of exposure of 50 µg L-1 NP significantly decreased the gonadosomatic index (GSI) and increased the hepatosomatic index (HSI) in both species. The results obtained from this study provide an evidence of the endocrine disrupting potential of waterborne NP on early stages of economically important marine fish. The NP-triggered endocrine modulation can induce effects on the development of reproductive and metabolic organs in fish species.

Joint effects of chronic exposure to environmentally relevant levels of nonylphenol and cadmium on the reproductive functions in male rockfish Sebastiscus marmoratus.

Nonylphenol (NP) and Cadmium (Cd) are two common contaminants that can be detected in aquatic environments. Nevertheless, the combined toxicity of NP and Cd at environmentally relevant concentrations in aquatic organisms has not been thoroughly characterized to date. In the present study, the interactions between NP and Cd on male Sebastiscus marmoratus were studied. After 21 days of exposure, the brain aromatase activity was observed to be significantly induced by 100 ng/L NP and 40 µg/L Cd, whereas all of the concentrations of co-treatment resulted in an increase in brain aromatase activity. Additionally, NP could also reduce plasma testosterone concentration, while NP, Cd and their mixture could induce plasma 17ß-estradiol (E2) concentration and VTG concentration. The interactions between NP and Cd on the reproductive physiology were antagonism. Our results also support the notion of using these indicators as biomarkers for exposure to EDCs and further extend the boundary of biomonitoring to environmental levels.

Two-generational reproductive toxicity assessment of 6:2 chlorinated polyfluorinated ether sulfonate (F-53B, a novel alternative to perfluorooctane sulfonate) in zebrafish.

As an alternative to perfluorooctane sulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used in the Chinese chrome plating industry for over four decades. It has been increasingly detected in environmental matrices in recent years, causing great concern regarding its potential health risks to humans and wildlife. However, its adverse effects on biota remain largely unknown. To explore the chronic toxicity of F-53B on reproduction, a two-generational study was conducted using zebrafish (Danio rerio). Adult zebrafish (F0 generation) were chronically exposed to different concentrations of F-53B (0, 5, 50, and 500 µg/L) for 180 d using a flow-through exposure system, with F1 and F2 generations reared without exposure. The reproductive toxicity endpoints were assessed in F0 and F1 adult fish. Results showed that F-53B accumulated in the F0 gonads and transferred to the F1 generation via maternal eggs, and even remained in F1 adult fish and their eggs (F2) after 180 d depuration. In the F0 generation, F-53B exposure significantly inhibited growth and induced reproductive toxicity, including decreased gonadosomatic index and egg production/female, changes in the histological structure of the gonads, and increased serum testosterone levels. In particular, serum estradiol and vitellogenin levels were significantly increased in 5 µg/L F-53B-exposed adult males. The transcriptional levels of several genes along the hypothalamic-pituitary-gonadal axis were altered in F0 generation fish. Testis transcriptome analysis revealed that F-53B exposure disrupted spermatogenesis in F0 male zebrafish. Maternal transfer of F-53B also induced adverse effects on growth and reproduction in the F1 generation. Furthermore, the higher occurrence of malformation and lower survival in F1 and F2 embryos indicated that parental exposure to F-53B could impair the embryonic development of offspring. Taken together, this study demonstrated that F-53B could induce reproductive toxicity in zebrafish similar to that induced by legacy PFOS, and its potential adverse effects on offspring deserve further investigation.

Indicators of exposure to estrogenic compounds at Great Lakes Areas of Concern: species and site comparisons.

Adverse effects resulting from potential exposure of wild fishes to estrogenic endocrine disruptors were assessed at seven United States Great Lakes Areas of Concern using biomarkers ranging from organismal (gonadosomatic indices) to tissue/plasma (histology, plasma vitellogenin) and molecular (hepatic gene transcripts) levels. Biomonitoring was conducted on pelagic, top predator species, largemouth Micropterus salmoides and smallmouth M. dolomieu bass and benthic, omnivorous white sucker Catostomus commersonii. Seasonal (spring and fall) comparisons were conducted at select sites. Intersex (testicular oocytes), plasma vitellogenin, and hepatic vitellogenin transcripts were commonly observed in bass species. Testicular oocyte severity was positively, although weakly, correlated with plasma vitellogenin, hepatic transcripts of vitellogenin, estrogen receptor α, and estrogen receptor ß2, while negatively correlated with androgen receptor ß and phosphoenolpyruvate carboxykinase. No testicular oocytes were observed in white sucker; however, plasma vitellogenin and hepatic vitellogenin transcripts were commonly detected in the males. The results demonstrate the importance of utilizing multiple endpoints to assess exposure to estrogenic compounds as well as the importance of choosing sensitive species.

Plasma biomarkers in juvenile marine fish provide evidence for endocrine modulation potential of organotin compounds.

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used to control marine fouling. Here, we show that organotin stimulation reduces the hormone levels in the plasma of two economically important aquaculture fish. Blood plasma samples were collected from juvenile red seabream and black rockfish exposed to environmentally realistic concentrations of TBT and TPT for 14 days. The levels of two plasma biomarkers, namely the yolk protein precursor vitellogenin (VTG) and the sex steroid 17ß-estradiol (E2), were measured to determine the endocrine disrupting potential of the organotin compounds. Both organotin compounds were dose-dependently accumulated in the blood of two fish. Exposure to waterborne TBT and TBT significantly decreased the plasma VTG levels in both the juvenile fish in a dose-dependent manner. In contrast, the treatment with E2, a well-known VTG inducer, significantly increased the plasma VTG levels in both the fish. In addition, the mRNA levels of vtg were also downregulated in the liver tissues of both the fish at 100 and/or 1000 ng L-1 of TBT or TPT exposure. The plasma E2 titers were significantly suppressed at 100 and/or 1000 ng L-1 of TBT or TPT exposure for 14 days compared to their titer in the control. Since estrogen directly regulates vtg gene expression and VTG synthesis, our results reveal the endocrine disrupting potential of organotin compounds, and subsequently the endocrine modulation at early stage of fish can trigger further fluctuations in sexual differentiation, maturation, sex ration or egg production. In addition, the results demonstrate their effects on non-target organisms, particularly on animals reared in aquaculture and fisheries.

Comparison of proteomic profiles in the ovary of Sterlet sturgeon (Acipenser ruthenus) during vitellogenic stages.

One of the challenges of sturgeon aquaculture is that sturgeon takes an extended amount of time to reach sexual maturity. The pattern of the protein expression in relation to the late maturity of sturgeon can help to better understand changes in sexual maturity. 17ß-estradiol (E2), testosterone (T) and vitellogenin (Vtg) levels were examined at all stages of sexual maturation in Sterlet sturgeon (Acipenser ruthenus). Two-dimensional gel electrophoresis and mass spectrometry analysis were used to show the pattern of the ovarian proteins. The T levels increased from the previtellogenic to the postvitellogenic stages (P < 0.05) and Vtg showed a decremental pattern in pre- and postvitellogenic, and atresia (not significantly). The analysis showed 900 protein spots, 19 of which were successfully identified and had significant differences between the previtellogenic and the vitellogenic groups (P < 0.05). Among the identified proteins, 40% involved in cell defense (heat shock protein, Glutathione peroxidase, natural killer enhancing factor, peroxiredoxin-2), 30% in transcription and translation (constitutive photomorphogenesis 9 and Ybx2), 20% in metabolism and energy production (triose-phosphate isomerase (TPI)) and 10% in transport (glycolipid transfer protein). In the vitellogenic stage, the proteins were related to metabolism and energy production (TPI, ES1, creatin kinase, enolase, nucleoside diphosphate kinase, 50%), cell defense (thioredoxin and dislophid isomerase, 20%) and transport (fatty acid binding protein, 10%). Our findings show changes in protein expression pattern from cell defense to metabolism during egg development.

Standardization of plasma vitellogenin level for surveying environmental estrogen pollution using the Japanese common goby Acanthogobius flavimanus.

Field surveys of the impact of environmental estrogen (EE) pollution in aquatic wildlife have been conducted using vitellogenin (VTG) as a biomarker to evaluate the influence of EE. However, a standard baseline of VTG level that can be used to evaluate EE pollution has not been fully determined. To the best of our knowledge, the present study is the first to determine the standard baseline VTG level for evaluating the biological effects of EE pollution using the Japanese common goby (Acanthogobius flavimanus) as the target model fish. Plasma VTG and estradiol-17ß (E2 ) levels associated with the reproductive cycle of wild goby inhabiting an unpolluted environment were measured. Mean plasma VTG and E2 levels exhibited similar changes, increasing in the yolk vesicle stage and peaking in the tertiary yolk stage in females. However, plasma VTG and E2 levels showed no significant changes in males, remaining at low levels throughout the reproductive cycle. The highest VTG levels in females and males were 1.6 mg ml-1 and 124.87 ng ml-1 , respectively. These results indicate that the baseline level (normal level) in males was approximately 130 ng ml-1 at most. We concluded that the threshold between normal and abnormal levels with a 10% risk rate was 150 ng ml-1 in the wild male goby. Plasma VTG levels in males captured from Nagasaki Harbor were higher than the threshold in each reproductive developmental stage, indicating the possibility of EE pollution at this site. The biological standard baseline for VTG established in this study is useful for assessing EE pollution in natural waters.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

Feminization of male and juvenile fish because of exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTPs) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust biomarkers for estrogenic exposure, and feminized fish have been observed downstream of WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream of central WWTP outlets. However, between 2000 and 2004, investigations of Danish streams not receiving sewage from central WWTPs revealed a high variation in vitellogenin concentrations of male juvenile brown trout (Salmo trutta); some individuals had high concentrations, probably as a result of estrogenic point sources, and the plasma concentration was >50 ng mL-1 in 79% of the juvenile males. The streams were reinvestigated in 2010 to 2016, and the average male level had decreased to a hitherto unseen baseline level; in 2010 only 0.7% (one individual) of the males had a vitellogenin concentration >50 ng mL-1 , which could indicate that the estrogenicity of the streams decreased after 2004. We examined possible estrogenic sources in streams unaffected by central WWTP effluents, and found that the reduced vitellogenin levels are most likely explained by a national effort to improve on-site wastewater treatment in scattered houses not connected to central WWTPs. Environ Toxicol Chem 2018;37:839-845. © 2017 SETAC.

Chronic nitrate exposure alters reproductive physiology in fathead minnows.

Nitrate is a ubiquitous aquatic pollutant that is commonly associated with eutrophication and dead zones in estuaries around the world. At high concentrations nitrate is toxic to aquatic life but at environmental concentrations it has also been purported as an endocrine disruptor in fish. To investigate the potential for nitrate to cause endocrine disruption in fish, we conducted a lifecycle study with fathead minnows (Pimephales promelas) exposed to nitrate (0, 11.3, and 56.5 mg/L (total nitrate-nitrogen (NO3-N)) from <24 h post hatch to sexual maturity (209 days). Body mass, condition factor, gonadal somatic index (GSI), incidence of intersex, and vitellogenin induction were determined in mature male and female fish and plasma 11-keto testosterone (11-KT) was measured in males only. In nitrate-exposed males both 11-KT and vitellogenin were significantly induced when compared with controls. No significant differences occurred for body mass, condition factor, or GSI among males and intersex was not observed in any of the nitrate treatments. Nitrate-exposed females also had significant increases in vitellogenin compared to controls but no significant differences for mass, condition factor, or GSI were observed in nitrate exposed groups. Estradiol was used as a positive control for vitellogenin induction. Our findings suggest that environmentally relevant nitrate levels may disrupt steroid hormone synthesis and/or metabolism in male and female fish and may have implications for fish reproduction, watershed management, and regulation of nutrient pollution.