| In the risk assessment of chemical,the reproduction toxicity were concern of the public and the regulatory agencies of chemicals.The hypothalamic-pituitary-gonadal axis(reproduction axis)of vertebrates is a feasible target of contaminants.US Environmental Protection Agency recommends the fish short-term reproduction assay to identify and evaluate the potential reproductive endocrine-disrupting chemicals in environemtal media.Many model chemlcals have been systematically assessed in fish short-term reproduction assays,with the goal of explicitly linking mode of action to apical outcomes of reproduction.Compensation responses 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 to consider the dynamic nature of the system when developing approaches to assess potential risks of reproduction endocrine-active chemicals.In order to explore characterization of dose-dependent compensatory responses along the HPG axis and effects of circadian rhythm on responses of the HPG axis and linkage between mode of action of reproduction toxicity and devepmental toxicity of offspring,the present study utilized sexual mature zebrafish(Danio rerio)and the model chemical prochloraz(aromatase inhibitor)to perform these experiments.Firstly,we conducted acute exposure of zebrafish to study dose-dependent compensation responses of HPG axis and circadian rhythm on responses of the HPGL axis.Secondly,we observed developmental endpoint of offspring derived from adult zebrafish exposed to prochloraz through the short-term fish reproduction assay,and further measured the sex hormones and vitellogenin(vtg)concentrations in plasma,vtg content in ova,prochloraz concentration in ova,zebrafish embryo microinjection,ovum transcriptomic and proteomic sequencing to systematically elucidate potential molecular mechanisms leading to the effects of maternal transfer following prochlolraz exposure(mode of action of anti-estrogenic effect).The present study provide evidence for the comprehensive knowledge on compensation responses of HPG axis and provide new insights in predicting toxic effects of reproductive endocrine-disrupting chemicals.The main results were as follows:1.Transcriptional profiles of the hypothalamic-pituitary-gonadal(HPG)axis and concentrations of 17β-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 10 g/L),medium(30 or 100 g/L)or high concentrations(300 or 1000 g/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 lh-β)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 er2β,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.2.In this study,profiles of expression of genes along the HPGL axis and concentrations of E2 in blood plasma of female zebrafish were compared at two sampling times of day(8:00 AM and 8:00 PM).Prochloraz(PCZ)was selected as a model chemical to evaluate differences in responses of the HPGL axis at these two times of day.Profiles of responses of concentrations of E2 in plasma and expressions of genes along the HPGL axis genes were different between the two times of sampling.Concentrations of E2 were less,and abundances of m RNA for several genes along the HPGL axis were significantly greater or lesser when samples were collected at 8:00 PM than they were when samples were collected at 8:00 AM.Exposure to three concentrations of PCZ(3,30 or 300 μg/L)for 48 h resulted in significantly lesser concentrations of plasma E2 and caused compensatory up-regulation of genes included in hypothalamus,pituitary and ovary.Expressions of genes along the HPGL were more responsive to PCZ at 8:00 AM than they were when samples were collected at 8:00 PM.Correlations among parameters in samples collected at the two times indicated the effects might be due to different concentrations of E2 in plasma due to exposure to PCZ.3.In this study,two exposure experiments were performed.First,adult zebrafish were exposed to aromatase inhibitor PCZ(0,3,30,or 300 μg/L)for 21 days,and the developmental toxicity of larval offspring was evaluated.PCZ markedly decreased E2 and vitellogenin(vtg)concentrations in plasma of females and suppressed spawned egg numbers.PCZ exposure caused bioaccumulation in ova and resulted in a significant decrease in the body length of larval offspring in 300 μg/L PCZ,but unexpectedly,it did not alter the vtg content of ova.Second,adult zebrafish were exposed to 0 or 300 μg/L PCZ for 21 days,and responses of the transcriptome and proteome in ova were characterized.Although a 21 day treatment with PCZ resulted in bioaccumulation of PCZ in ova,it was not responsible for the observed inhibition of body length growth of larval offspring as evidenced by PCZ microinjection.Furthermore,exposure of adult zebrafish to PCZ significantly downregulated m RNA expression of retinol binding protein 4(rbp4)and changed protein concentrations related to mitochondrial energy metabolism in ova,and those changes might be responsible for the inhibition of growth of larval offspring. |
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