Masculinization and reproductive effects in western mosquitofish (Gambusia affinis) after long-term exposure to androstenedione.

Androstenedione (AED) is a naturally occurring steroid hormone. It is metabolized to potent androgens, which may induce androgenic effects in fish. However, little is known whether and how the androgens interfere with the fish gonadal development and reproduction. This study aimed at demonstrating the effects of long-term AED exposure on reproduction and development in mosquitofish (Gambusia affinis). The growth, development and several morphological endpoints, including the segment number and length of anal fin, histological changes of gonads and liver, were evaluated in mosquitofish during development from fertilized embryo to adulthood (180 days) after exposure of AED at environmentally relevant concentrations. We found that the growth (length, body weight and condition factor) of fish was negatively correlated with AED concentration in females, but not in males. The significant elongation of the ray and increment of segment numbers in the anal fin, were detected in all mosquitofish after exposure. Moreover, AED exposure (0.4gµ/L) caused damages in gonads and reduced the number of pregnant females. These findings indicate that AED has adverse effects on the growth and development of the western mosquitofish after long-term exposure (180d). Long-term exposure (180d) to AED, including environmentally relevant concentration (0.4µg/L and 4µg/L), induced masculinization in female mosquitofish under the experimental conditions.

Modulation of transcription of genes related to the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes in zebrafish (Danio rerio) embryos/larvae by androstenedione.

This study aimed to determine the effects of Androstenodione (AED) on the transcriptional expression of genes involved in the hypothalamic-pituitary-gonadal (HPG) and the hypothalamic-pituitary-adrenal (HPA) axes in the zebrafish embryos/larvae. Zebrafish embryos were exposed to 0, 4.0, 45.0, 487.0, and 980.0 ng/L of AED from the day of fertilization to 144 h post fertilization (hpf), during which the transcriptional profiles of key genes related to the HPG and HPA axes were examined daily using quantitative real-time PCR. The AED exposure significantly up-regulated several receptor signaling pathways and the key genes involved in various steps of the steroidogenic pathways were also affected. In addition, the AED exposure could significantly modulate the transcriptional profiles of the other target genes related to hypothalamic and pituitary hormones. The findings of this study suggest that AED, at environmentally relevant concentrations, affects the adrenal endocrine systems and the reproduction of zebrafish by interrupting the HPG and HPA axes.

Changes in Histopathology, Enzyme Activities, and the Expression of Relevant Genes in Zebrafish (Danio rerio) Following Long-Term Exposure to Environmental Levels of Thallium.

Thallium is a rare-earth element, but widely distributed in water environments, posing a potential risk to our health. This study was designed to investigate the chronic effects of thallium based on physiological responses, gene expression, and changes in the activity of relevant enzymes in adult zebra fish exposed to thallium at low doses. The endpoints assessed include mRNA expression of metallothionein (MT)2 and heat shock protein HSP70; enzymatic activities of superoxide dismutase (SOD) and Na+/K+-ATPase; and the histopathology of gill, gonad, and liver tissues. The results showed significant increases in HSP70 mRNA expression following exposure to 100 ng/L thallium and in MT2 expression following exposure to 500 ng/L thallium. Significantly higher activities were observed for SOD in liver and Na+/K+-ATPase activity in gill in zebra fish exposed to thallium (20 and 100 ng/L, respectively) in comparison to control fish. Gill, liver, and gonad tissues displayed different degrees of damage. The overall results imply that thallium may cause toxicity to zebra fish at environmentally relevant aqueous concentrations.

[Effect of Bushen Tongdu Capsule on RANK/RANKL/OPG pathway of collagen induced arthritis rats].

OBJECTIVE: To study the effect of Bushen Tongdu Capsule (BTC) on RANK/RANKL/ OPG pathway of collagen induced arthritis (CIA) rats, thereby laying theoretic evidence for treating rheumatic arthritis (RA) by Chinese medicine. METHODS: RA model was induced by CIA. Totally 42 rats were randomly divided into six groups, i.e., the normal control group, the model group, the low dose BTC (BSL) group, the medium dose BTC (BSM) group, the high dose BTC (BSH) group, and the Tripterygium Glycosides (TG) group, 7 in each group. BTC at the daily dose of 120, 240, and 480 mg/kg was given by gastrogavage to rats in the BSL, BSM, and BSH group respectively from the 13th day of modeling. TG at the daily dose of 24 mg/kg was given by gastrogavage to rats in the TG group. All medication was given once daily, 2 mL each time. Two mL normal saline was administered to rats in the normal control group and the model group. All medication lasted for 18 days. Samples were taken at day 31. The TRAP section of the ankle joint was fixed in 10% formalin for TRAP stain. Serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) were detected using ELISA. RESULTS: Compared with the normal control group, positive reactions of pathological ankle joint section, inflammation, and osteoclasia degree were significantly improved in the model group, serum levels of RANKL and M-CSF were up-regulated, levels of OPG and OPG/RANKL were significantly lowered (all P < 0.01). Compared with the model group, positive reactions of pathological ankle joint section, inflammation, and osteoclasia degree also significantly decreased in the BSH group and the TG group (all P < 0.01). RANKL and M-CSF were significantly down-regulated in each medicated group, while levels of OPG and OPG/RANKL were significantly up-regulated (all P < 0.01). Compared with the TG group, M-CSF was lower, but levels of OPG and OPG/RANKL were significantly up-regulated in the normal control group (all P < 0.01). RANKL and M-CSF were significantly up-regulated, while levels of OPG and OPG/RANKL were significantly down-regulated in the model group and each BS group (all P < 0.01). CONCLUSION: BTC could relieve bone damage of CIA rats possibly through regulating and controlling osteoclasts.

A review on the methods of determining ecological thresholds.

The drastic changes or threshold phenomena of ecosystems in response to environmental changes are main research focuses in ecology. However, the difficulty in quantitative detection of ecological thresholds hinders the further research progress and application of this topic. Based on typical cases, we analzyed the driving-response mechanism of S-shaped curve type, subsidy-stress type, step type of the potential ecological thresholds. Seven methods to determine ecological thre-shold were summarized, including locally weighted scatterplot smoothing, piecewise regression, Gaussian model, change-point analyzer, regime shift detection, threshold indicator taxa analysis and system dynamics simulation model. The advantages, disadvantages and applicability of those methods were also addressed. This study could provide references for quantitative detection of ecological threshold.

Levonorgestrel and dydrogesterone affect sex determination via different pathways in zebrafish.

Levonorgestrel (LNG) and dydrogesterone (DDG) are two commonly used synthetic progestins that have been detected in aquatic environments. They could affect fish sex differentiation, but the underlying mechanisms remain unknown. Here we investigated the effects of LNG (5 ng L-1 and 50 ng L-1), DDG (100 ng L-1) and their mixtures on gonadal differentiation and sex determination in zebrafish at transcriptomic and histological levels from 2 hours post-fertilization (eleutheroembryos) to 144 days post-fertilization (sexual maturity). Germ cell development and oogenesis pathways were significantly enriched in LNG and the mixture of LNG and DDG treatments, while insulin and apoptosis pathways in the DDG treatment. LNG and the mixture of LNG and DDG strongly decreased transcripts of germ cell development and oogenesis related genes, while DDG increased the transcripts of insulin and apoptosis related genes at 28 days post fertilization (dpf) and 35 dpf. Furthermore, DDG caused ∼ 90% males, and LNG and the mixture of LNG and DDG resulted in 100% males on all sampling dates. Specifically, most males in LNG and the mixture of LNG and DDG treatments were "Type I" males without juvenile oocytes at 28 dpf and 35 dpf, while those in DDG treatment were "Type II" and "Type III" males with a few juvenile oocytes. These results indicated that LNG and DDG promoted testicular differentiation via different pathways to cause male bias. LNG and DDG mixtures have similar effect on testicular differentiation to LNG alone. The findings from this study could have significant ecological implications to fish populations.

[Research basis of ecological thresholds and regime shifts in China].

Non-linear changes widely occur in ecosystems, which manifest as sudden changes in system state with increasing pressure. To explain these changes, ecologists proposed the concepts of ecological threshold and regime shift, and continuously improved the theory and method system. Mechanistic and empirical studies further deepen our understanding for evolution mechanism of the complex system, which begun to be implemented into the environmental management. In recent decades, a large number of quantitative studies on stress-response in various types of ecosystems in China. Results from those studies are closely related to the theory paradigms of ecological threshold and regime shift in essence. Based on the "China Ecological Thresholds and Alternative Stable States Database", we screened and summarized the relevant ecological thresholds and explained the mechanisms underlying regime shift for 10 ecosystem types, including river, lake, wetland, forest, grassland, estuary and ocean, farmland, desert, city, and tundra. We linked case studies with theory paradigms of ecological threshold and regime shift with the aim to integrate multi-field scientific achievements and resources as the basis for ecosystem complexity research, and promote their application in ecological monitoring, ecological security early warning, and ecological standards innovation.

Medroxyprogesterone acetate affects sex differentiation and spermatogenesis in zebrafish.

Medroxyprogesterone acetate (MPA) is a widely used synthetic progestin and it has been frequently detected in aquatic environments. However, its effects on aquatic organisms remain largely unknown. Here we investigated the chronic effects of MPA on sex differentiation and gonad development in zebrafish. Zebrafish larvae at 20 days post fertilization (dpf) were exposed to 4.32, 42.0, and 424 ng L-1 of MPA until they reached 140 dpf. The results showed that chronic exposure to 42.0 ng L-1 of MPA caused 60% proportion of males as well as significant up-regulation of dmrt1 (˜1.79 fold) and hsd17b3 (˜1.92 fold). Histological analysis showed MPA significantly increased the frequency of immature spermatocytes accompanied with the increased transcription of dmrt1 (˜2.06 fold) and ar (˜1.73 fold) in the testes. Meanwhile, MPA exposure significantly increased the transcription of lhb at all exposure concentrations in the males. In contrast, it significantly suppressed the transcription of lhb (˜-8.06-fold) and fshb (˜-6.35-fold) at 42.0 ng L-1 in the females. Collectively our results demonstrated that MPA had androgenic activity, and could affect sex differentiation and spermatogenesis in zebrafish at environmentally relevant concentrations. The findings from this study suggest that MPA in the aquatic environment may pose potential androgenic risks to fish populations.

Immune response induced by major environmental pollutants through altering neutrophils in zebrafish larvae.

Environmental pollutants may cause adverse effects on the immune system of aquatic organisms. However, the cellular effects of pollutants on fish immune system are largely unknown. Here, we exploited the transgenic zebrafish Tg(lysC:DsRed2) larva as a preliminary screening system to evaluate the potential inflammatory effects of environmental pollutants. Tg(lysC:DsRED2) larvae aged 7-day-postfertilization (7 dpf) were treated with selected environmental chemicals for 24 h (24 h) and the number of neutrophils were quantified using both image analysis and fluorescence activated cell sorting (FACS). We found that the numbers of neutrophils in the Tg(lysC:DsRED2) larvae were significantly increased by most of the organic chemicals tested, including E2 (17ß-estradiol), BPA (Bisphenol-A), NDEA (N-nitrosodiethylamine), 4-NP (4-Nitrophenol) and Lindane (γ-hexachlorocyclohexane). Neutrophil numbers were also increased by all the metals tested (Na2HAsO4· 7H2O, Pb(NO3)2, HgCl2, CdCl2, CuSO4·5H2O, ZnSO4, and K2Cr2O7). The only exception was TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which significantly reduced the number of neutrophils after exposure. Additionally, the transcription of genes (lyz, mpo, tnfα and il8) related to fish immune system were significantly modulated upon exposure to some of the selected chemicals such as E2, TCDD, Cu and Cd. This study revealed that representatives of major categories of environmental pollutants could cause an acute inflammatory response in zebrafish larvae as shown by alterations in the neutrophils, which may imply a common immunotoxicity mechanism for most environmental pollutants. This study has also demonstrated that Tg(lyz:DsRed2) transgenic zebrafish is an excellent tool for screening environmental chemicals with potential inflammatory effects through FACS-facilitated neutrophil counting.

The progestin norethindrone affects sex differentiation and alters transcriptional profiles of genes along the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes in juvenile zebrafish Dario renio.

Natural and synthetic progestins may pose a threat to wild fish populations living in receiving waters. In this study, the effects of norethindrone (NET) on the sex differentiation of zebrafish (Dario renio) and the mechanisms underlying these effects were investigated. Juvenile zebrafish (20 days post fertilization, pdf) were exposed to environmentally relevant concentrations (5, 50, 500, and 1000 ng L-1) for 45 d. Sex ratio of the NET-exposed populations, the histology of the gonads and the transcriptional profile of the regulatory genes involved in sex differentiation and steroidogenesis were examined. The results showed that a significantly higher ratio of male/female was induced in the zebrafish populations exposed to NET at concentrations higher than 32.3 ng L-1. Exposure to NET caused acceleration of sexual mature in males and a delay in ovary maturation in female zebrafish. Among the genes regulating sexual differentiation, transcripts of Dmrt1 showed a dose-dependent increase while transcripts of Figa and Fox12 showed a dose-dependent decrease in response to exposure to NET. For genes regulating the steroidogenesis, the expressions of Cyp11a1, Cyp17, Cyp19a1a, and Cyp11b were significantly down-regulated by exposure to NET, while Hsd17b3 expression was significantly up-regulated by exposure to NET at 421.3 and 892.9 ng L-1. For the receptor genes in the gonads, the transcriptional expression of Pgr, Ar, and Mr was significantly up-regulated at 421.3 and 892.9 ng L-1 of NET. For genes involved in the hypothalamic-pituitary axis, the transcriptional expression of Gnrh3 and Pomc was significantly up-regulated by exposure to NET with the exception for Gnrh3 at 4.2 ng L-1. The results demonstrated that exposure to NET at the juvenile stage could affect gonad differentiation and sex ratio, which might be accounted for by the alterations of the transcriptional expressions of genes along the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes.

Alterations of secondary sex characteristics, reproductive histology and behaviors by norgestrel in the western mosquitofish (Gambusia affinis).

Synthetic hormones in wastewater effluents released into the aquatic environments may interfere with the normal endocrine systems of fish in receiving streams. Norgestrel (NGT) is a synthetic progestin widely used in oral contraceptives and frequently detected in wastewater effluents. In this study, adult female mosquitofish (Gambusia affinis) were exposed to three environmentally relevant concentrations of norgestrel (NGT) (i.e., 3.6, 35.8, and 368.0 ng L-1) for 42 d, fin morphology, histology of the ovary, and reproductive behaviors were evaluated. The results showed that NGT at all three concentrations caused an increased frequency of atretic follicular cells in ovaries and impaired mating behaviors exhibited by males toward the NGT-exposed females. In mosquitofish exposed to NGT at 35.8 and 368 ng L-1, the anal fin of females had an increased length ratio of ray4/ray 6, an increased width of ray 3, and increased number of segments in ray 3. The histopathological analysis showed that exposure to NGT increased the incidence of spermatogenesis in ovaries. Mating behavior was impaired 58.4%, 65.7%, and 76.4% (P < 0.01 in all cases) when mosquitofish were exposed to NGT at 3.6, 35.6 and 368.0 ng L-1, respectively. The rapid masculinization, the increased frequency of atretic follicles, the incidence of spermatogenesis in the ovary of female fish, and the altered reproductive behaviors suggest that wild populations of mosquitofish could be similarly affected inhabiting in NGT contaminated environments.

Altered development and reproduction in western mosquitofish (Gambusia affinis) found in the Hanxi River, southern China.

The current study investigated the development and reproductive health of western mosquitofish (Gambusia affinis) in the Hanxi River, which has been heavily contaminated by municipal wastewaters from towns in Dongguan of southern China. Western mosquitofish collected from four study sites, Songmu (SM), Yangwu (YW), Hengli (HL), and Zhangcun (ZC) of the Hanxi River, as well as a reference site (LX) of the Liuxi River, were dissected and analyzed for development parameters (total length, wet body mass, liver mass, gonad index, and population composition), reproductive parameters (oocyte count and weight of females, and sperm count and viability of males), and morphology (anal fin in males and females, and hemal spines in males). With the exception of the origin site (SM), mosquitofish from the Hanxi River exhibited significantly decreasing development and reproduction levels. Significant correlations were found for the collected mosquitofish between the morphological and reproductive indexes (ratio of perpendicular distance to tip to vertical distance from the tip on the 16th hemal spine) versus the sperm count of males; ratio 16P:16D versus sperm viability of males; segments of anal fin ray 3 versus oocyte count of females; and segments of anal fin ray 3 versus average oocyte mass of females. The results demonstrated that both male and female mosquitofish in the Hanxi River were affected by the discharged wastewaters, as reflected in their morphological changes in comparison with those of mosquitofish from the reference site.