Intergenerational toxicity of 17α-ethinylestradiol (EE2): Effects of parental exposure on early larval development and transcriptomic profiles in the Sydney rock oyster, Saccostrea glomerata.

This study exposed adult Sydney rock oysters, of either sex or both, to the synthetic estrogen 17α-ethinylestradiol (EE2) at 50 ng/L for 21 days, followed by an examination of developmental endpoints and transcriptomic responses in unexposed larvae. Reduced survival was observed at 1 day post-fertilisation (dpf) in larvae from bi-parental exposure (FTMT). Motile larvae at 2 dpf were fewer from maternal (FTMC), paternal (FCMT), and FTMT exposures. Additionally, shell length at 7 dpf decreased in larvae from FTMC and FTMT parents. RNA sequencing (RNA-seq) revealed 1064 differentially expressed genes (DEGs) in 1-dpf larvae from FTMT parents, while fewer DEGs were detected in larvae from FTMC and FCMT parents, with 258 and 7, respectively. GO and KEGG analyses showed significant enrichment of DEGs in diverse terms and pathways, with limited overlap among treatment groups. IPA results indicated potential inhibition of pathways regulating energy production, larval development, transcription, and detoxification of reactive oxygen species in FTMT larvae. qRT-PCR validation confirmed significant downregulation of selected DEGs involved in these pathways and relevant biological processes, as identified in the RNA-seq dataset. Overall, our results suggest that the intergenerational toxicity of EE2 is primarily maternally transmitted, with bi-parental exposure amplifying these effects.

Microplastics act as a carrier for wastewater-borne pathogenic bacteria in sewage.

Microplastic pollution, a pressing global environmental problem, has a severe impact on both aquatic ecosystems and public health worldwide. Due to the small size of microplastics, they are able to pass through the filtration systems of municipal wastewater treatment works (WWTWs). In recent years, studies have focused on the environmental abundance and ecotoxicological effects of microplastics, but there are limited studies investigating the colonization of microplastics by bacteria, especially those pathogenic ones. In this study, we examined the colonization and composition of the bacterial communities on polyethylene microbeads after incubation in raw sewage collected from three municipal WWTWs in Hong Kong (Sha Tin Sewage Treatment Works, Stonecutters Island Sewage Treatment Works, and Shek Wu Hui Sewage Treatment Works). Scanning electron microscopy (SEM) results indicate that bacterial cells were colonized on the surfaces of the microbeads and formed biofilms after sewage incubation. Metagenomic sequencing data demonstrated an increase in bacterial diversity after 21 days of sewage incubation when compared to shorter incubation periods of 6, 11 and 16 days. Importantly, human and fish pathogens such as Arcobacter cryaerophilus, Aeromonas salmonicida, Vibrio areninigrae and Vibrio navarrensis were found in the resident bacterial communities. Taken together, our results demonstrate that microplastics could act as a carrier for wastewater-borne pathogenic bacteria in municipal sewage.

The accumulation and distribution of arsenic species and selected metals in the saltmarsh halophyte, spiny rush (Juncus acutus).

This study examined the accumulation of As species, Se, Cu, Zn, Cd and Pb in the halophyte Juncus acutus, collected from three anthropogenically impacted estuaries in NSW, Australia. As concentration ranged from 4 to 22 µg/g at Georges River, 2-16 µg/g at Lake Macquarie and 6 µg/g at Hunter Estuary. Inorganic As was accumulated mainly in roots with low translocation to culm with a greater abundance of AsV. However, AsIII (TF = 0.32) showed greater mobility from the roots to shoots than AsV (TF = 0.04), indicating a higher quantity of AsIII specific transporter assemblages in the plasmalemma of the endodermis or cytoplasmic reduction of AsV to AsIII in culms. Metal(loid)s, including As (90%), were predominantly in root tissues and very limited translocation to culm, indicating the species is a useful phytostabiliser. As and all other metal(loid)s in roots were correlated with sediment loads (p < 0.05, R2 = 0.10-0.52), indicating the species would be an accumulative bioindicator.

Benthic infaunal assemblages adjacent to an ocean outfall in Australian marine waters: Impact assessment and identification of indicator taxa.

An impact assessment of oceanic effluent releases from Belmont wastewater treatment works (WWTW) in Newcastle, Australia, was undertaken. Benthic infaunal assemblages in sandy sediments of ~25 m water depth were examined, at sites adjacent to the release point, and at increasing distances up to 2 km in both a NE and SW direction over five consecutive years (2016-2020). Localised impacts were evident for infaunal assemblages, with sites within 20 m of the outfall ("Impact" site types) exhibiting lower taxa richness and Shannon diversity, higher abundances of polychaetes and/or nematodes, higher polychaete ratios, and shifts in assemblage composition in comparison to sites at greater distances during some years. Taxa with increased localised abundances at the outfall were identified as indicators for monitoring impacts, including deposit-feeding polychaetes (Families Polygordiidae, Paraonidae and Dorvilleidae) and Phylum Nematoda. Future infaunal monitoring could include molecular tools and paired sediment analyses.

Secondary treatment phase of tertiary wastewater treatment works significantly reduces estrogenic load.

Estrogenic compounds enter waterways via effluents from wastewater treatment works (WWTW), thereby indicating a potential risk to organisms inhabiting adjacent receiving waters. However, little is known about the loads or concentrations of estrogenic compounds that enter Australian WWTWs, the efficiency of removing estrogenic compounds throughout the various stages of tertiary WWTW processes (which are common in Australia), nor the concentrations released into estuarine or marine receiving waters, and the associated risk for aquatic taxa residing in these environments. Therefore, seven estrogenic compounds, comprising the natural estrogens estrone (E1), 17ß-estradiol (E2) and estriol (E3), the synthetic estrogen (EE2), and the industrial chemicals bisphenol A (BPA), 4-t-octyl phenol (4-t-OP) and 4-nonyl phenol (4-NP), in wastewater samples were quantified via liquid chromatographic-mass spectrometry (LC-MS) after solid-phase extraction at different stages of wastewater treatment and associated receiving waters. The concentrations of the target compounds in wastewater ranged from < LOQ (limit of quantification) to 158 ng/L for Tanilba Bay WWTW and < LOQ to 162 ng/L for Belmont WWTW. Most target compounds significantly declined after the secondary treatment phase. Appreciable removal efficiency throughout the treatment process was observed with removal from 39.21 to 99.98% of influent values at both WWTWs. The reduction of the natural estrogens (E1, E2 and E3) and 4-t-OP were significantly greater than EE2, BPA, and 4-NP in both WWTWs. Risk quotients (RQs) were calculated to assess potential ecological risks from individual estrogenic compounds. In predicted diluted effluents, no targeted compounds showed any ecological risk (RQ ≤1.65 × 10-2) at both WWTWs. Similarly, all RQs for shore samples at both WWTWs were below 1. Finally, the hazard index (HI), which represents combined estrogenic contaminants' ecological risk, indicated no mentionable risk for predicted diluted effluents (HI = 0.0097 to 0.0218) as well as shoreline samples (HI = 0.393 to 0.522) in the receiving estuarine or marine waters.

Accumulation and distribution of metal(loid)s in the halophytic saltmarsh shrub, Austral seablite, Suaeda australis in New South Wales, Australia.

We examined the patterns of uptake and partitioning of metal(loid)s in Suaeda australis from three highly urbanised estuaries (Sydney Olympic Park, Hunter Wetlands and Lake Macquarie) in NSW, Australia. Of these, Sydney Olympic Park was found to be the most contaminated estuary in terms of combined sediment metal(loid) load, followed by Hunter Wetlands and lowest in Lake Macquarie (via PERMANOVA). Uptake in roots was greater for the essential metals Cu and Zn along with the non-essential metal Cd and the metalloid Se (root BCFs >1) and lower for Pb and As (root BCFs <1). Substantial barriers for translocation from roots to stems were identified for all metal(loid)s (stem TFs; 0.07-0.68). Conversely, unrestricted flow from stems to leaves was observed for all metal(loid)s at unity or higher (leaf TFs ≥ 1). Strong linear relationships between sediment and root for Zn and Pb were observed, indicating roots as a useful bioindicator.

Global patterns of accumulation and partitioning of metals in halophytic saltmarsh taxa: A phylogenetic comparative approach.

The current study represents the first attempt to analyse quantitatively, within a phylogenetic framework, uptake and partitioning patterns of copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb) in extant saltmarsh taxa globally, and to assess associations of these patterns with various plant traits indicative of their halophytic adaptations. Despite saltmarsh being diverse taxonomically, most saltmarsh taxa accumulate metals to roots at, or above, unity (> 1). Further, there is significant translocation from roots to shoot for Cu, Zn and Cd (≤ 1), however, Pb is less mobile (TF = 0.65). Patterns of accumulation were similar among families, except greater Cd accumulation to roots in members of Juncaceae. Patterns of uptake to roots and translocation to leaves were broadly similar among plant type, plant form, habitat and photosynthetic mode. Zinc is lower in the leaves of salt-secreting species for some closely related taxa, suggesting some species co-excrete sodium (Na+) and Zn2+ through glands in leaf tissue. Salinity tolerance has no relationship to metal uptake and translocation. Translocation of Zn is greater at lower Zn sediment exposures, reflecting its active uptake and essentiality, but such bias does not affect outcomes of analyses when included as a covariate.

Exposure to estrogenic mixtures results in tissue-specific alterations to the metabolome of oysters.

The current study investigated the effect of environmentally relevant mixtures of estrogens at levels representative of receiving waters on the metabolome of the Sydney rock oyster, Saccostrea glomerata. Oysters were exposed to a "low" and a "high" mixture of (xeno) estrogens (representative of Australian and global receiving waters respectively) for 7 days and digestive gland, gill, and gonad tissue were sampled for quantification of polar metabolites by 1H NMR spectroscopy. Exposure to both mixtures lowered body mass and altered the metabolite profile in the digestive glands. Comparatively, gills, and ovaries demonstrated lesser sensitivity to the mixtures, with significant metabolomic alterations observed only for the high mixture. The male gonad did not respond to either estrogenic exposure. In the responsive tissues, major metabolites including amino acids, carbohydrates, intermediates of the tricarboxylic acid cycle and ATP were all down-regulated and exhibited tissue-specific patterns of down-regulation with the greatest proportion of metabolites down-regulated due to estrogenic exposure in the digestive gland. Exposure to (xeno) estrogen mixtures representative of concentrations reported in receiving waters in Australia and globally can impact the metabolome and associated energy metabolism, especially in the digestive gland, translating to lower pools of available ATP energy for potential cellular homeostasis, somatic maintenance and growth, reproduction and fitness.

Accumulation and partitioning of metals and metalloids in the halophytic saltmarsh grass, saltwater couch, Sporobolus virginicus.

Remnant endangered saltmarsh communities in Australia often occur in urbanised estuaries where industrial processes have contaminated sediments with metal(loid)s. Despite this issue, virtually nothing is known on local plant species exposure to metal contaminants, nor their ability to uptake and translocate metal(loid)s from contaminated estuarine sediment. In the current study, we assessed the accumulation and partitioning of the metal(loid)s Zn, Cu, Pb, Cd and Se in the dominant saltmarsh primary producer, Sporobolus virginicus, across three urbanised estuaries in NSW Australia. Lake Macquarie was the most contaminated estuary, while Sydney Olympic Park, Port Jackson exhibited intermediate metal(loid) loadings and Hunter Wetlands exhibited the lowest loadings among estuaries. Essential metals (Zn and Cu) were more mobile, with sediment:root bioconcentration factors (BCFs) greater than unity and translocation among plant organs greater than, or equal to, unity. Other metal(loid)s were less mobile, with BCFs equal to unity and translocation factors among organs much reduced. Despite these barriers to translocation, all metal(loid)s were accumulated to roots with dose, and further accumulative relationships between metal(loid)s in roots and culms, and culms and leaves, were evidenced (with the exception of Cu). Along with sediment metal(loid)s, increases in sediment pH predicted Cu uptake in roots and increases in soil organic matter predicted Se uptake in roots. Although significant positive linear relationships were observed between sediment metal(loid)s and plant organ metal(loid)s(withholding Cu), the variance explained was low to intermediate for most metal(loid)s suggesting employing S. virginicus as an accumulative bioindicator would be impractical.

Characterisation of the metallothionein gene in the Sydney rock oyster and its expression upon metal exposure in oysters with different prior metal exposure histories.

The metal-binding protein metallothionein (MT) is widely used as a biomarker of metal contamination. In this study, we cloned a MT gene (sgMT) from the Sydney rock oyster Saccostrea glomerata. The gene encodes a MT-I protein with a classical αß domain structure and is expressed as two transcripts resulting from alternative polyadenylation. The gene promoter contains two putative metal-responsive elements (MREs) which are known to be required for metal-inducible transcription. A specific and efficient qPCR assay was developed to quantify sgMT mRNA expression. Further, we assessed whether prior metal exposure history influences sgMT mRNA expression upon subsequent metal exposure. Oysters with varying prior metal exposure histories (contaminated and reference) were exposed to Cu, Cd and Zn. Expression of sgMT generally increased with metal dose, and oysters with an elevated past metal exposure history exhibited higher sgMT expression under Cd and Zn stress, representing a potential acclimatory response to prior metal exposure.

The utility of vitellogenin as a biomarker of estrogenic endocrine disrupting chemicals in molluscs.

Estrogenic endocrine disrupting chemicals (EDCs) are natural hormones, synthetic compounds or industrial chemicals that mimic estrogens due to their structural similarity with estrogen's functional moieties. They typically enter aquatic environments through wastewater treatment plant effluents or runoff from intensive livestock operations. Globally, most natural and synthetic estrogens in receiving aquatic environments are in the low ng/L range, while industrial chemicals (such as bisphenol A, nonylphenol and octylphenol) are present in the µg to low mg/L range. These environmental concentrations often exceed laboratory-based predicted no effect concentrations (PNECs) and have been evidenced to cause negative reproductive impacts on resident aquatic biota. In vertebrates, such as fish, a well-established indicator of estrogen-mediated endocrine disruption is overexpression of the egg yolk protein precursor vitellogenin (Vtg) in males. Although the vertebrate Vtg has high sensitivity and specificity to estrogens, and the molecular basis of its estrogen inducibility has been well studied, there is growing ethical concern over the use of vertebrate animals for contaminant monitoring. The potential utility of the invertebrate Vtg as a biomonitor for environmental estrogens has therefore gained increasing attention. Here we review evidence providing support that the molluscan Vtg holds promise as an invertebrate biomarker for exposure to estrogens. Unlike vertebrates, estrogen signalling in invertebrates remains largely unclarified and the classical genomic pathway only partially explains estrogen-mediated activation of Vtg. In light of this, in the latter part of this review, we summarise recent progress towards understanding the molecular mechanisms underlying the activation of the molluscan Vtg gene by estrogens and present a hypothetical model of the interplay between genomic and non-genomic pathways in the transcriptional regulation of the gene.

Overexpression and Knockdown of Hypoxia-Inducible Factor 1 Disrupt the Expression of Steroidogenic Enzyme Genes and Early Embryonic Development in Zebrafish.

Hypoxia is an important environmental stressor leading to endocrine disruption and reproductive impairment in fish. Although the hypoxia-inducible factor 1 (HIF-1) is known to regulate the transcription of various genes mediating oxygen homeostasis, its role in modulating steroidogenesis-related gene expression remains poorly understood. In this study, the regulatory effect of HIF-1 on the expression of 9 steroidogenic enzyme genes was investigated in zebrafish embryos using a "gain-of-function and loss-of-function" approach. Eight of the genes, CYP11a, CYP11b2, 3ß-HSD, HMGCR, CYP17a1, 17ß-HSD2, CYP19a, and CYP19b, were found to be differentially upregulated at 24 and 48 hpf following zHIF-1α-ΔODD overexpression (a mutant zebrafish HIF-1α protein with proline-414 and proline-557 deleted). Knockdown of zHIF-1α also affected the expression pattern of the steroidogenic enzyme genes. Overexpression of zHIF-1α and hypoxia exposure resulted in downregulated StAR expression but upregulated CYP11a and 3ß-HSD expression in zebrafish embryos. Conversely, the expression patterns of these 3 genes were reversed in embryos in which zHIF-1α was knocked down under normoxia, suggesting that these 3 genes are regulated by HIF-1. Overall, the findings from this study indicate that HIF-1-mediated mechanisms are likely involved in the regulation of specific steroidogenic genes.

Acclimatory processes are likely responsible for metal tolerance in oyster embryos.

We aimed to determine if offspring of oysters from contaminated locations were more tolerant to metals, and whether this tolerance could be attributed to acclimation. Oysters from 10 estuaries were sampled, representing a gradient in metal contamination. Tolerance to metals of the F1 offspring from adults residing in these estuaries was assessed. Then, adults from these estuaries were translocated to a single estuary and their offspring tolerance reassessed. No linear relationship was found between the Cu concentrations of adults and their offspring's tolerance to Cu. A positive linear relationship was found between the Zn concentration of adults and the Zn EC50's of their offspring. Zn tolerance was lost after translocation. Zn EC50 values of offspring from transplanted adults bore no relation to the Zn EC50's of their location of origin. Thus the initial tolerance observed could be attributed to acclimation transferred to the F1 generation.

The constitutively active estrogen receptor (ER) binds and activates the promoter of the vitellogenin (Vtg) gene in the Sydney rock oyster, Saccostrea glomerata.

Vitellogenin (Vtg) is a well-established biomarker of estrogenic exposure in aquatic animals. In vertebrates, Vtg gene transcription is controlled by the estrogen receptors (ERs). Although an ER ortholog is present in molluscs, its role as a transcriptional regulator remains elusive. Here, we tested the hypothesis that in the Sydney rock oyster, Saccostrea glomerata, the ER ortholog activates Vtg gene transcription through specific interaction with its promoter. Luciferase reporter assays indicated that sgER activated both a minimal promoter containing the consensus estrogen-responsive elements (EREs) and the sgVtg promoter in an estrogen-independent manner. The sgVtg promoter-luciferase activation was significantly reduced when any of three putative ERE half sites (½EREs) in the promoter were mutated. Electrophoretic mobility shift assay (EMSA) confirmed that sgER binds specifically to a 68-bp promoter sequence where these ½EREs reside. Overall, the results suggest that sgER is a constitutively active transcription factor that binds and activates the sgVtg promoter.

Potential mechanisms underlying estrogen-induced expression of the molluscan estrogen receptor (ER) gene.

In vertebrates, estrogens and estrogen mimicking chemicals modulate gene expression mainly through a genomic pathway mediated by the estrogen receptors (ERs). Although the existence of an ER orthologue in the mollusc genome has been known for some time, its role in estrogen signalling has yet to be deciphered. This is largely due to its constitutive (ligand-independent) activation and a limited mechanistic understanding of its regulation. To fill this knowledge gap, we cloned and characterised an ER cDNA (sgER) and the 5'-flanking region of the gene from the Sydney rock oyster Saccostrea glomerata. The sgER cDNA is predicted to encode a 477-amino acid protein that contains a DNA-binding domain (DBD) and a ligand-binding domain (LBD) typically conserved among both vertebrate and invertebrate ERs. A comparison of the sgER LBD sequence with those of other ligand-dependent ERs revealed that the sgER LBD is variable at several conserved residues known to be critical for ligand binding and receptor activation. Ligand binding assays using fluorescent-labelled E2 and purified sgER protein confirmed that sgER is devoid of estrogen binding. In silico analysis of the sgER 5'-flanking sequence indicated the presence of three putative estrogen responsive element (ERE) half-sites and several putative sites for ER-interacting transcription factors, suggesting that the sgER promoter may be autoregulated by its own gene product. sgER mRNA is ubiquitously expressed in adult oyster tissues, with the highest expression found in the ovary. Ovarian expression of sgER mRNA was significantly upregulated following in vitro and in vivo exposure to 17ß-estradiol (E2). Notably, the activation of sgER expression by E2 in vitro was abolished by the specific ER antagonist ICI 182, 780. To determine whether sgER expression is epigenetically regulated, the in vivo DNA methylation status of the putative proximal promoter in ovarian tissues was assessed using bisulfite genomic sequencing. The results showed that the promoter is predominantly hypomethylated (with 0-3.3% methylcytosines) regardless of sgER mRNA levels. Overall, our investigations suggest that the estrogen responsiveness of sgER is regulated by a novel ligand-dependent receptor, presumably via a non-genomic pathway(s) of estrogen signalling.

Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.

Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5'- and 3'-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17ß-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5' CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0-3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs.

Effects of 4-methylbenzylidene camphor (4-MBC) on neuronal and muscular development in zebrafish (Danio rerio) embryos.

The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor (4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3 days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15 µM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15 µM) and in vitro in mammalian Neuro-2A cells (0.1 µM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects.

Evidence for microRNA-mediated regulation of steroidogenesis by hypoxia.

Environmental hypoxia can occur in both natural and occupational environments. Over the recent years, the ability of hypoxia to cause endocrine disruption via perturbations in steroid synthesis (steroidogenesis) has become increasingly clear. To further understand the molecular mechanism underlying hypoxia-induced endocrine disruption, the steroid-producing human cell line H295R was used to identify microRNAs (miRNAs) affecting steroidogenic gene expression under hypoxia. Hypoxic treatment of H295R cells resulted in the downregulation of seven steroidogenic genes and one of these, CYP19A1 (aromatase), was shown to be regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). Using bioinformatic and luciferase reporter analyses, miR-98 was identified to be a CYP19A1-targeting miRNA from a subset of HIF-1-inducible miRNAs. Gain- and loss-of-function analysis suggested that under hypoxia, the increased expression of miR-98 led to the downregulation of CYP19A1 mRNA and protein expression and that it may have contributed to a reduction in estradiol (E2) production. Intriguingly, luciferase reporter assays using deletion constructs of a proximal 5'-flanking region of miR-98 did not reveal a hypoxia-responsive element (HRE)-containing promoter. Overall, this study provided evidence for the role of miRNAs in regulating steroidogenesis and novel insights into the molecular mechanisms of hypoxia-induced endocrine disruption.

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 3ß-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 3ß-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.

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.