Hypoxia Causes Transgenerational Impairment of Ovarian Development and Hatching Success in Fish.

Hypoxia is a pressing environmental problem in both marine and freshwater ecosystems globally, and this problem will be further exacerbated by global warming in the coming decades. Recently, we reported that hypoxia can cause transgenerational impairment of sperm quality and quantity in fish (in F0, F1, and F2 generations) through DNA methylome modifications. Here, we provide evidence that female fish ( Oryzias melastigma) exposed to hypoxia exhibit reproductive impairments (follicle atresia and retarded oocyte development), leading to a drastic reduction in hatching success in the F2 generation of the transgenerational group, although they have never been exposed to hypoxia. Further analyses show that the observed transgenerational impairments in ovarian functions are related to changes in the DNA methylation and expression pattern of two gene clusters that are closely associated with stress-induced cell cycle arrest and cell apoptosis. The observed epigenetic and transgenerational alterations suggest that hypoxia may pose a significant threat to the sustainability of natural fish populations.

Tissue-specific transcriptome assemblies of the marine medaka Oryzias melastigma and comparative analysis with the freshwater medaka Oryzias latipes.

BACKGROUND: The marine medaka Oryzias melastigma has been demonstrated as a novel model for marine ecotoxicological studies. However, the lack of genome and transcriptome reference has largely restricted the use of O. melastigma in the assessment of in vivo molecular responses to environmental stresses and the analysis of biological toxicity in the marine environment. Although O. melastigma is believed to be phylogenetically closely related to Oryzias latipes, the divergence between these two species is still largely unknown. Using Illumina high-throughput RNA sequencing followed by de novo assembly and comprehensive gene annotation, we provided transcriptomic resources for the brain, liver, ovary and testis of O. melastigma. We also investigated the possible extent of divergence between O. melastigma and O. latipes at the transcriptome level. RESULTS: More than 14,000 transcripts across brain, liver, ovary and testis in marine medaka were annotated, of which 5880 transcripts were orthologous between O. melastigma and O. latipes. Tissue-enriched genes were identified in O. melastigma, and Gene Ontology analysis demonstrated the functional specificity of the annotated genes in respective tissue. Lastly, the identification of marine medaka-enriched transcripts suggested the necessity of generating transcriptome dataset of O. melastigma. CONCLUSIONS: Orthologous transcripts between O. melastigma and O. latipes, tissue-enriched genes and O. melastigma-enriched transcripts were identified. Genome-wide expression studies of marine medaka require an assembled transcriptome, and this sequencing effort has generated a valuable resource of coding DNA for a non-model species. This transcriptome resource will aid future studies assessing in vivo molecular responses to environmental stresses and those analyzing biological toxicity in the marine environment.

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.

Hypoxia turns genotypic female medaka fish into phenotypic males.

Hypoxia caused by eutrophication is amongst the most pressing global problems in aquatic systems. Notably, more than 400 "dead zones" have been identified worldwide, resulting in large scale collapse of fisheries and major changes in the structure and trophodynamics. Recent studies further discovered that hypoxia can also disrupt sex hormone metabolism and alter the sexual differentiation of fish, resulting in male biased F1 generations and therefore posing a threat to the sustainability of natural populations. However, it is not known whether, and if so how, hypoxia can also change the sex ratio in vertebrates that have sex-determining XX/XY chromosomes. Using the Japanese medaka (Oryzias latipes) as a model, we demonstrate, for the first time, that hypoxia can turn genotypic female fish with XX chromosomes into phenotypic males. Over half of the XX females exposed to hypoxia exhibit male secondary sexual characteristics and develop testis instead of ovary. We further revealed that hypoxia can: (a) down-regulate the vasa gene, which controls proliferation of primordial germ cells and gonadal sex differentiation into ovary, and (b) up-regulate the DMY gene which resides at the sex-determining locus of the Y chromosome, and direct testis differentiation. This is the first report that hypoxia can directly act on genes that regulate sex determination and differentiation, thereby turning genotypic females into phenotypic males and leading to a male-dominant F1 population.

Effects of benzophenone-3 and its metabolites on the marine diatom Chaetoceros neogracilis: Underlying mechanisms and environmental implications.

The wide application of benzophenones (BPs), such as benzophenone-3 (BP3), as an ingredient in sunscreens, cosmetics, coatings, and plastics, has led to their global contamination in aquatic environments. Using the marine diatom Chaetoceros neogracilis as a model, this study assessed the toxic effects and mechanisms of BP3 and its two major metabolites (BP8 and BP1). The results showed that BP3 exhibited higher toxicity on C. neogracilis than BP8 and BP1, with their 72-h median effective concentrations being 0.4, 0.8 and 4 mg/L, respectively. Photosynthesis efficiencies were significantly reduced after exposure to environmentally relevant concentrations of the three benzophenones, while cell viability, membrane integrity, membrane potential, and metabolic activities could be further impaired at their higher concentrations. Comparative transcriptomic analysis, followed by gene ontology and KEGG pathway enrichment analyses unraveled that all the three tested benzophenones disrupted photosynthesis and nitrogen metabolism of the diatom through alteration of similar pathways. The toxic effect of BP3 was also attributable to its unique inhibitory effects on eukaryotic ribosome biosynthesis and DNA replication. Taken together, our findings underscore that benzophenones may pose a significant threat to photosynthesis, oxygen production, primary productivity, carbon fixation, and the nitrogen cycle of diatom in coastal waters worldwide.

Bioaccumulation of pollutants in the green-lipped mussel Perna viridis: Assessing pollution abatement in Victoria Harbour and its adjacent aquaculture area, Hong Kong, and the minimal human health risks from mussel consumption.

The green-lipped mussel Perna viridis was utilised for pollution biomonitoring in Victoria Harbour and its adjacent aquaculture area in Hong Kong. P. viridis was collected from a reference site and redeployed at five study sites for five weeks during the dry and wet seasons of 2019. Our study found various polycyclic aromatic hydrocarbons (PAHs) and heavy metals in the mussel tissue, while polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were not detected. P. viridis at the reference site generally displayed lower levels of pollutants. Comparing with previous research in the 1980s and 2000s, we observed substantial reduction in the tissue levels of PAHs, PCBs, OCPs and heavy metals in P. viridis. The human health risks associated with consuming these mussels were determined to be insignificant. Our findings imply that the Harbour Area Treatment Scheme has been effective in improving the water quality in Victoria Harbour and its adjacent aquaculture area.

Embryo developmental toxicity in marine medaka (Oryzias melastigma) due to parental and embryonic 17α-ethinylestradiol exposure.

The synthetic estrogen 17α-ethinylestradiol (EE2) is a common component of hormone therapy and oral contraceptives and has been widely used for nearly 60 years. Numerous studies have shown that exposure to EE2 can affect embryonic development in a number of fish species. The effects of parental and embryonic EE2 exposure on embryo developmental toxicity and the underlying molecular mechanisms, however, have rarely been examined. In this study, embryos collected from parental EE2-exposed adult fish were examined to assess EE2-induecd toxicity during embryo development. The rate of embryo development including heart rate, hatching rate, and larval locomotion were measured to assess embryo developmental toxicity. The embryonic transcriptome was used to delineate the related developmental toxicity pathways. Our results suggest that parental and embryonic EE2 exposure resulted in growth retardation including a reduction in embryo heart rate, a delay in the appearance eye pigmentation, decreased hatching rate and impaired larval locomotion. In addition, gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Ingenuity Pathway Analysis (IPA) of transcriptome revealed that these impairments are controlled by estrogen receptor and related to eye structure, neuronal and synaptic structure, and behaviour. The key factors identified, including PRKAA2, APOB, EPHB2, OXTR, NR2E3, and POU4F2, could serve as biomarkers for assessing EE2-induced embryo developmental toxicity. For the first time, our results show that eye pigmentation is a potentially sensitive marker of EE2-induced embryo developmental toxicity.

Hypoxia in aquatic invertebrates: Occurrence and phenotypic and molecular responses.

Global deoxygenation in aquatic systems is an increasing environmental problem, and substantial oxygen loss has been reported. Aquatic animals have been continuously exposed to hypoxic environments, so-called "dead zones," in which severe die-offs among organisms are driven by low-oxygen events. Multiple studies of hypoxia exposure have focused on in vivo endpoints, metabolism, oxidative stress, and immune responses in aquatic invertebrates such as molluscs, crustaceans, echinoderms, and cnidarians. They have shown that acute and chronic exposure to hypoxia induces significant decreases in locomotion, respiration, feeding, growth, and reproduction rates. Also, several studies have examined the molecular responses of aquatic invertebrates, such as anaerobic metabolism, reactive oxygen species induction, increased antioxidant enzymes, immune response mechanisms, regulation of hypoxia-inducible factor 1-alpha (HIF-1α) genes, and differently expressed hemoglobin/hemocyanin. The genetic basis of those molecular responses involves HIF-1α pathway genes, which are highly expressed in hypoxic conditions. However, the identification of HIF-1α-related genes and understanding of their applications in some aquatic invertebrates remain inadequate. Also, some species of crustaceans, rotifers, sponges, and ctenophores that lack HIF-1α are thought to have alternative defense mechanisms to cope with hypoxia, but those factors are still unclear. This review covers the formation of hypoxia in aquatic environments and the various adverse effects of hypoxia on aquatic invertebrates. The limitations of current hypoxia research and genetic information about the HIF-1α pathway are also discussed. Finally, this paper explains the underlying processes of the hypoxia response and presents an integrated program for research about the molecular mechanisms of hypoxic stresses in aquatic invertebrates.

Chromatin modifiers: A new class of pollutants with potential epigenetic effects revealed by in vitro assays and transcriptomic analyses.

A great variety of endocrine-disrupting chemicals (EDCs) have been used extensively and become widespread in the environment nowadays. Limited mammalian studies have shown that certain EDCs may target chromosome and epigenome of the germline, leading to adverse effects in subsequent generations, despite these progenies having never been exposed to the EDC before. However, the underlying mechanisms of chromosomal changes induced by these pollutants remain poorly known. Using the human ovarian granulosa tumor cell line COV434 as a model, we investigated and compared the transcriptomic changes induced by nine EDCs with diverse chemical structures (i.e. BDE-47, BPA, BP-3, DEHP, DHP, EE2, TCS, TDCPP and NP), to inquire if there is any common epigenetic modification associated with reproductive functions induced by these EDCs. Our results showed that COV434 cells were more responsive to BP-3, NP, DEHP and EE2, and more importantly, these four EDCs altered the expression of gene clusters related to DNA damage response, cell cycle, proliferation, and chromatin remodeling, which can potentially lead to epigenetic modifications and transgenerational inheritance. Furthermore, dysregulation of similar gene clusters was common in DEHP and NP treatments. Bioinformatics analysis further revealed that BP-3 disturbed signaling pathways associated with reproductive functions, whereas alterations in telomere-related pathways were highlighted upon EE2 exposure. Overall, this study highlighted chromatin modifications caused by a class of chemicals which that may potentially lead to epigenetic changes and transgenerational reproductive impairments.

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.

Continuous 17α-ethinylestradiol exposure impairs the sperm quality of marine medaka (Oryzias melastigma).

17α-ethinylestradiol (EE2) is an anthropogenic estrogen that is widely used for hormone therapy and oral contraceptives. It was reported that EE2 exposure induced reproductive impairments through processes affecting reproduction behavior and inducing ovotestis. However, the effects of continuous EE2 exposure on the reproductive performance remain largely unknown. In this study, adult marine medaka fish (Oryzias melastigma) were exposed to EE2 (85 ng/L) for one (F0) and two (F1) generations. Our results indicate that continuous EE2 exposure reduced fecundity and sperm motility. The testicular transcriptome, followed by bioinformatic analysis revealed the dysregulation of pathways related to steroidogenesis, sperm motility, and reproductive system development. Collectively, our findings indicate that continuous EE2 exposure directly affected sperm quality via the alteration of steroidogenesis and dysregulation of reproductive system development. The identified key factors including DNM1, PINK1, PDE7B, and SLC12A7 can serve as biomarkers to assess EE2-reduced sperm motility.

Corrigendum: Low-Dose Radiation Can Cause Epigenetic Alterations Associated With Impairments in Both Male and Female Reproductive Cell.

[This corrects the article DOI: 10.3389/fgene.2021.710143.].

Low-Dose Radiation Can Cause Epigenetic Alterations Associated With Impairments in Both Male and Female Reproductive Cells.

Humans are regularly and continuously exposed to ionizing radiation from both natural and artificial sources. Cumulating evidence shows adverse effects of ionizing radiation on both male and female reproductive systems, including reduction of testis weight and sperm count and reduction of female germ cells and premature ovarian failure. While most of the observed effects were caused by DNA damage and disturbance of DNA repairment, ionizing radiation may also alter DNA methylation, histone, and chromatin modification, leading to epigenetic changes and transgenerational effects. However, the molecular mechanisms underlying the epigenetic changes and transgenerational reproductive impairment induced by low-dose radiation remain largely unknown. In this study, two different types of human ovarian cells and two different types of testicular cells were exposed to low dose of ionizing radiation, followed by bioinformatics analysis (including gene ontology functional analysis and Ingenuity Pathway Analysis), to unravel and compare epigenetic effects and pathway changes in male and female reproductive cells induced by ionizing radiation. Our findings showed that the radiation could alter the expression of gene cluster related to DNA damage responses through the control of MYC. Furthermore, ionizing radiation could lead to gender-specific reproductive impairment through deregulation of different gene networks. More importantly, the observed epigenetic modifications induced by ionizing radiation are mediated through the alteration of chromatin remodeling and telomere function. This study, for the first time, demonstrated that ionizing radiation may alter the epigenome of germ cells, leading to transgenerational reproductive impairments, and correspondingly call for research in this new emerging area which remains almost unknown.

Hypoxia causes sex-specific hepatic toxicity at the transcriptome level in marine medaka (Oryzias melastigma).

Hypoxia, a low environmental oxygen level, is a common problem in the ocean globally. Hypoxia has been known to cause disruption to the endocrine system of marine organisms in both laboratory and field studies. Our previous studies have demonstrated the sex-specific response to hypoxia in the neural and reproductive systems of marine fish. In the current report, we aim to study the sex-specific hepatic response of fish at the transcriptome level to hypoxic stress. By using a comparative transcriptome analysis, followed by a systematic bioinformatics analysis including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA), we found that hypoxia altered expression of genes related to cell proliferation and apoptosis of hepatocytes, which are associated with human pathologies, such as liver inflammation hepatic steatosis and steatohepatitis. Furthermore, we observed sex-specific responses in the livers of fish through different cell signaling pathways. In female fish, hypoxia causes dysregulation of expression of genes related to impairment in endoplasmic reticulum structure and liver metabolism. In male fish, genes associated with redox homeostasis and fatty acid metabolism were altered by hypoxic stress. The findings of this study support the notion that hypoxia could cause sex-specific changes (hepatic toxicity and changes) in marine fish.

Ultra violet filters in the urine of preschool children and drinking water.

Benzophenones (BPs) and other ultra violet (UV) filters (UV-filters) are widely used in sunblock and other personal care products, raising concerns about their adverse health risks to human, especially for children. In the present study, BP-type UV-filters and other four widely used UV-filters were evaluated in the child urinary samples (4-6 years, n = 53), tap water and commercial distilled water in Hong Kong. The results suggested that the target chemicals are ubiquitous in the subject. BP1, BP2, BP3 and BP4 in children urine samples contributed closely to the overall children exposure of UV filters, with detection rates above 58% and geometric means ranging from 44.2 to 76.7 ng/mL. As a contrast, BP3 was the major substance found in the tap water and distilled bottle water, with detection rates of 100% and geometric means of 9.64 and 14.5 ng/L, respectively. There were some significant relationships between urinary UV filters and personal characteristics (BMI values, sex, income level, hand washing frequency, and body location usage), but the health risks associated with UV-filters in Hong Kong children might not be concerning. Only two children applied sun creams in this research, indicating that there were other sources to exposure these chemicals.

Construction of High-Resolution RAD-Seq Based Linkage Map, Anchoring Reference Genome, and QTL Mapping of the Sex Chromosome in the Marine Medaka Oryzias melastigma.

Medaka (Oryzias sp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resource, the marine medaka Oryzias melastigma is also applicable for the marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 single nucleotide polymorphism (SNP) markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with SNP markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics.

Transcriptomic analysis reveals transgenerational effect of hypoxia on the neural control of testicular functions.

There are over 400 hypoxic zones in the ocean worldwide. Both laboratory and field studies have shown that hypoxia causes endocrine disruption and reproductive impairments in vertebrates. More importantly, our recent study discovered that parental (F0) hypoxia exposure resulted in the transgenerational impairment of sperm quality in the F2 generation through the epigenetic regulation of germ cells. In the present study, we aim to test the hypothesis that the brain, as the major regulator of the brain-pituitary-gonad (BPG) axis, is also involved in the observed transgenerational effect. Using comparative transcriptomic analysis on brain tissues of marine medaka Oryzias melastigma, 45 common differentially expressed genes caused by parental hypoxia exposure were found in the hypoxic group of the F0 and F2 generations, and the transgenerational groups of the F2 generation. The bioinformatic analysis on this deregulated gene cluster further highlighted the possible involvement of the brain in the transgenerational effect of hypoxia on testicular structure, including abnormal morphologies of the epididymis and the seminal vesicle, and degeneration of the seminiferous tubule. This finding is concordant to the result of hematoxylin and eosin staining, which showed the reduction of testicular lobular diameter in the F0 and F2 generations. Our study demonstrated for the first time the involvement of the brain in the transgenerational effect of hypoxia.

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.

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 17beta-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 alpha (ojERalpha) 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.

Hypoxia alters steroidogenesis in female marine medaka through miRNAs regulation.

Hypoxia is a worldwide environmental problem in marine ecosystems, leading to serious declines in fishery production over large areas. Our previous studies demonstrated that hypoxia is an endocrine disruptor which can cause reproductive impairment through the regulation of miRNAs, suggesting the functional role of miRNAs in reproductive systems in response to hypoxia. In this study, we used small RNA sequencing to determine the change in miRNA profile in ovary of marine medaka Oryzias melastigma under hypoxic stress. A total of 509 miRNAs were found in the ovary of marine medaka, in which, 33 and 10 miRNAs were found to be statistically significant upregulated and downregulated under hypoxia, respectively. Bioinformatics analysis highlighted that a large number of hypoxia-suppressed miRNAs that target a variety of steroidogenic enzymes including steroidogenic acute regulatory protein, aromatase, and 17-alpha-monooxygenase. Also, estrogen receptor 2 and androgen receptor were found to be targeted by hypoxia-responsive miRNAs. For the first time, our results showed that hypoxia may upregulate specific steroidogenic enzymes and hormone receptors through actions of miRNA, and hence provide a novel mechanism for the observed female reproductive impairment caused by hypoxia.