Hypoxia Impairs Primordial Germ Cell Migration in Zebrafish (Danio rerio) Embryos

Background: As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear. Methodology/Principal Findings: In the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration. Conclusions/Significance: This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1

Hypoxia affects sex differentiation and development leading to a male-dominated population in zebrafish (Danio rerio)

Hypoxia is affecting thousands of square kilometers of water and has caused declines in fish populations and major changes in aquatic communities worldwide. For the first time, we report that hypoxia can affect sex differentiation and sex development of zebrafish (Danio renio), leading to a male-biased population in the F1 generation (74.4% ± 1.7% males in the hypoxic groups versus 61.9% ± 1.6% males in the normoxic groups, n = 5; p < 0.05, χ2 test). The increase in males was associated with downregulations of various genes controlling the synthesis of sex hormones (i.e., 3β-HSD, CYP11 A, CYP19A, and CYP19B) as well as an increase in the testosterone/estradiol ratio. The male-dominated populations caused by hypoxia will have reduced reproductive success, thereby threatening the sustainability of natural fish populations. © 2006 American Chemical Society.link_to_subscribed_fulltex

Induction of hepatic choriogenin mRNA expression in male marine medaka: A highly sensitive biomarker for environmental estrogens

Teleost choriogenins, precursors of the inner layer subunits of egg envelope, have been recently introduced as sensitive biomarkers for exposure to estrogenic compounds. In this study, two full-length cDNAs-ojChgH and ojChgL which encode the choriogenin H and L forms, respectively, were cloned from the marine medaka, Oryzias javanicus. The deduced protein sequences of ojChgH and ojChgL are highly similar to the corresponding homologues in the freshwater medaka (O. latipes) with identities of 77.2 and 87.6%, respectively. Phylogenetic analysis indicated that ojChgH and ojChgL are members of two different classes of liver-specific ZP-domain containing proteins (ZPB and ZPC, respectively). Computer analysis of ca. 2 kb of the 5′-flanking sequences of ojChgH and ojChgL revealed that both genes contain a number of putative estrogen response elements (EREs) and/or half-site EREs. In vivo mRNA expression patterns of the genes were examined by quantitative real-time RT-PCR. ojChgH is expressed exclusively in the liver while ojChgL is co-expressed in the liver (major) and ovary (minor). Exposure of fish to waterborne 17β-estradiol (E2) at environmentally relevant concentrations (1, 5, 10 and 100 ng/L) resulted in dose-dependent induction of both genes in the liver, with higher sensitivity and magnitude of induction in males than in females. In the male liver, induction of ojChgH is more sensitive to E2 than that of ojChgL and two other estrogen-responsive genes, estrogen receptor α (ojERα) and vitellogenin (ojVTG). The lowest-observed-effect concentration (LOEC) of E2 on induction of hepatic ojChgH mRNA is 1 ng/L. In the ovary, expression of ojChgL is non-responsive to E2 treatment. In conclusion, the present study suggested that induction of hepatic ojChgH mRNA in male fish may be a highly sensitive biomarker for exposure to environmental estrogens. © 2005 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Two genes encoding protein phosphatase 2A catalytic subunits are differentially expressed in rice

Type 2A serine/threonine protein phosphatase (PP2A) plays a variety of regulatory roles in metabolism and signal transduction. Two closely related PP2A catalytic subunit (PP2Ac) genes, OsPP2A-1 and OsPP2A-3, have been isolated from the monocot Oryza sativa. Both genes contain six exons and five introns which intervene at identical locations, suggesting they have descended from a recent duplication event. Their encoded proteins share 97% sequence identity and are highly similar (94-96%) to a PP2Ac subfamily (AtPP2A-1, -2 and -5) identified in Arabidopsis thaliana. Both OsPP2A-1 and OsPP2A-3 are ubiquitously expressed, with the expression levels high in stems and flowers and low in leaves. OsPP2A-1, but not OsPP2A-3, is also highly expressed in roots. Transcript levels of OsPP2A-1 in roots and OsPP2A-3 in stems are elevated at the maturation and young stages, respectively. Drought and high salinity upregulate both genes in leaves, whereas heat stress represses OsPP2A-1 in stems and induces OsPP2A-3 in all organs. These findings indicate that the two PP2Ac genes are subjected to developmental and stress-related regulation. In situ hybridization results show that both transcripts exhibit nearly identical cellular distribution, except in leaves, and are abundant in meristematic tissues including the young leaf blade of stems and the root tip.link_to_subscribed_fulltex

Enhancement of hypoxia-induced gene expression in fish liver by the aryl hydrocarbon receptor (AhR) ligand, benzo[a]pyrene (BaP)

Fish in polluted coastal habitats commonly suffer simultaneous exposure to both hypoxia and xenobiotics. Although the adaptive molecular responses to each stress have been described, little is known about the interaction between the signaling pathways mediating these responses. Previous studies in mammalian hepatoma cell lines have shown that hypoxia-inducible factor (HIF)- and/or aryl hydrocarbon receptor (AhR)-activated gene expression is suppressed following co-exposure to hypoxia and the hallmark AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, whether similar crosstalk exists in the non-tumor liver tissues of fish and whether other non-TCDD ligands also play the same inhibitory role in this crosstalk remain unknown. Here, the in vivo hepatic mRNA expression profiles of multiple hypoxia- and AhR-responsive genes (later gene expression = mRNA expression of the gene) were examined in the orange-spotted grouper (Epinephelus coioides) upon single and combined exposures to hypoxia and benzo[a]pyrene (BaP). Combined exposure enhanced hypoxia-induced gene expression but did not significantly alter BaP-induced gene expression. Protein carbonyl content was markedly elevated in fish subjected to combined exposure, indicating accumulation of reactive oxygen species (ROS). Application of diethyldithiocarbamate (DDC) to hypoxia-treated grouper liver explants similarly exaggerated hypoxia-induced gene expression as in the combined stress tissues in vivo. These observations suggest that ROS derived from the combined hypoxia and BaP stress have a role in enhancing hypoxia-induced gene expression. © 2008 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Effects of PCBs and MeSO2-PCBs on adrenocortical steroidogenesis in H295R human adrenocortical carcinoma cells

Some endocrine disrupting chemicals (EDCs) in the environment have been shown to exert their biological effects through interference with steroidogenesis. In this study, the potential effects of four selected polychlorinated biphenyl (PCB) congeners (PCB101, PCB110, PCB126 and PCB149) as well as several of their environmentally-relevant methylsulfonyl-(MeSO2-) PCB metabolites (3′-MeSO2-CB101, 4′-MeSO2-CB101, 4′-MeSO2-CB110, 3′-MeSO2-CB149 and 4′-MeSO2-CB149) on adrenocortical steroidogenesis were evaluated by in vitro bioassay based on the human adrenocortical carcinoma H295R cell line. The PCBs included in the study represented different structures and potential mechanisms of action. Cells were exposed for 48 h to 10 μM of each PCB congener in the presence or absence of 20% (w/w) of their corresponding MeSO2-PCB metabolite(s). After the chemical treatments, changes in mRNA expression of 11 steroidogenic genes (CYP11A, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, 3β-HSD1, 3β-HSD2, 17β-HSD1, StAR and HMGR) were quantified using molecular beacon-based real-time RT-PCR. Genes coding for enzymes involved in the later or final steps of steroid production (CYP11B1, CYP11B2, CYP19, 3β-HSD1, 3β-HSD2 and 17β-HSD1) were up-regulated to various extents by most PCBs. The greatest transcriptional activations (2.8-29.9-fold) were elicited by PCB110 on CYP11B1, CYP11B2, 3β-HSD2 and CYP19, and PCB149 on CYP11B1, 3β-HSD1 and 17β-HSD1. Increased expression of these steroidogenic genes might ultimately lead to a change in hormonal balance through excessive production of steroid hormones including aldosterone, cortisol and estradiol. In addition, co-treatment with 3′- and 4′-MeSO2-PCB149 resulted in a significant decrease in PCB149-induced 3β-HSD1 and 17β-HSD1 expression. This result indicates that some PCB congeners and their MeSO2-metabolites may affect steroidogenesis via different mechanisms. Overall, these findings suggest that PCBs and PCB metabolites can affect regulation of adrenocortical steroidogenesis. © 2005 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex

Effects of 20 PBDE metabolites on steroidogenesis in the H295R cell line

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that have been found in the environment as well as human tissues. Environmental concentrations of these compounds have been increasing in many parts of the world in recent years. Due to their structural similarity, PBDEs are believed to have similar toxicity to PCBs, but their toxicological properties are still being determined. In this study, the steroidogenic effects of hydroxylated, methoxylated and/or chlorinated derivatives of PBDEs were assessed at both the gene and enzyme/hormone levels in the H295R human adrenocortical carcinoma cell line. The expression levels of 10 steroidogenic genes were measured using quantitative real-time PCR (Q-RT-PCR). Aromatase activity in the cells and sex steroid (testosterone (T) and 17β-estradiol (E2)) concentrations in the culture medium were also measured. CYP11B2, which regulates the synthesis of aldosterone, was the most sensitive gene and was induced by most of the compounds tested in this study. CYP19 gene expression, aromatase activity, and E2 production were also affected by several metabolites, but no consistent relationship was observed between these endpoints. Several PBDE metabolites showed some potential ability to interfere with steroidogenesis, including 5-Cl-6-OH-BDE-47, a biologically relevant BDE-47 metabolite, which significantly decreased aromatase activity and E2 production at a concentration of 10 μM. The results of this study indicate that PBDE metabolites affect steroidogenesis in vitro and that they may have the potential to affect steroidogenesis and reproduction in whole organisms. © 2007 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex

Effects of 20 PBDE metabolites on steroidogenesis in the H295R cell line

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that have been found in the environment as well as human tissues. Environmental concentrations of these compounds have been increasing in many parts of the world in recent years. Due to their structural similarity, PBDEs are believed to have similar toxicity to PCBs, but their toxicological properties are still being determined. In this study, the steroidogenic effects of hydroxylated, methoxylated and/or chlorinated derivatives of PBDEs were assessed at both the gene and enzyme/hormone levels in the H295R human adrenocortical carcinoma cell line. The expression levels of 10 steroidogenic genes were measured using quantitative real-time PCR (Q-RT-PCR). Aromatase activity in the cells and sex steroid (testosterone (T) and 17β-estradiol (E2)) concentrations in the culture medium were also measured. CYP11B2, which regulates the synthesis of aldosterone, was the most sensitive gene and was induced by most of the compounds tested in this study. CYP19 gene expression, aromatase activity, and E2 production were also affected by several metabolites, but no consistent relationship was observed between these endpoints. Several PBDE metabolites showed some potential ability to interfere with steroidogenesis, including 5-Cl-6-OH-BDE-47, a biologically relevant BDE-47 metabolite, which significantly decreased aromatase activity and E2 production at a concentration of 10 μM. The results of this study indicate that PBDE metabolites affect steroidogenesis in vitro and that they may have the potential to affect steroidogenesis and reproduction in whole organisms. © 2007 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex

Acupuncture with or without combined auricular acupuncture for insomnia : a randomised, waitlist-controlled trial

Title on author’s file: Acupuncture and Combined Acupuncture and Auricular Acupuncture for Insomnia: A Randomized, Waitlist-Controlled Trial202312 bckwAccepted ManuscriptOthersHealth and Health Services Research Fund, Food and Health Bureau, Hong Kong SARPublishedGreen (AAM

Leptin-mediated modulation of steroidogenic gene expression in hypoxic zebrafish embryos: Implications for the disruption of sex steroids

Hypoxia can impair reproduction of fishes through the disruption of sex steroids. Here, using zebrafish (Danio rerio) embryos, we investigated (i) whether hypoxia can directly affect steroidogenesis independent of pituitary regulation via modulation of steroidogenic gene expression, and (ii) the role of leptin in hypoxia-induced disruption of steroidogenesis. Exposure of fertilized zebrafish embryos to hypoxia (1.0 mg O 2 L -1) from 0-72 h postfertilization (hpf), a developmental window when steroidogenesis is unregulated by pituitary influence, resulted in the up-regulation of cyp11a, cyp17, and 3β-hsd and the down-regulation of cyp19a. Similar gene expression patterns were observed for embryos exposed to 10 mM cobalt chloride (CoCl 2, a chemical inducer of hypoxia-inducible factor 1, HIF-1), suggesting a regulatory role of HIF-1 in steroidogenesis. Testosterone (T) and estradiol (E2) concentrations in hypoxic embryos were greater and lesser, respectively, relative to the normoxic control, thus leading to an increased T/E2 ratio. Expression of the leptin-a gene (zlep-a) was up-regulated upon both hypoxia and CoCl 2 treatments. Functional assays suggested that under hypoxia, elevated zlep-a expression might activate cyp11a and 3β-hsd and inhibit cyp19a. Overall, this study indicates that hypoxia, possibly via HIF-1-induced leptin expression, modulates sex steroid synthesis by acting directly on steroidogenic gene expression. © 2012 American Chemical Society.link_to_subscribed_fulltex