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.

Organophosphate flame retardants and bisphenol A in children's urine in Hong Kong: has the burden been underestimated?

The urine levels of organophosphate flame retardants (PFRs) and bisphenol A (BPA) in kindergarten children (n = 31, 4-6 years old, sampling performed in 2016) in Hong Kong were measured. The detection frequency of the target PFRs, tri(2-chloroethyl)phosphate (TCEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(chloroisopropyl)phosphate (TCIPP), triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) ranged from 52% to 84%. The 95th percentile urinary concentrations of TPHP, TDCIPP, TCIPP, EHDPP and TCEP were 1.70, 0.24, 0.03, 0.05, 0.68 and 0.03 ng/mL, respectively. The median urine level of BPA was 1.69 ng/mL, with a detection frequency of 77%. Due to the lack of metabolism information, two scenarios were used to calculate the estimated daily intake (EDI) of these compounds. Back-calculated EDIs of PFRs using the urinary excretion rates from in vivo animal data (scenario 2) were up to 2.97 µg/kg/d (TDCIPP), which was only a little less than that observed in a sample of American infants, and the reference dose (RfD), meaning that the potential health risk of TDCIPP cannot be ignored. Dust ingestion was suggested to be the major pathway of exposure to PFRs, but when the levels in dust and air particles in kindergartens in Hong Kong were used to predict EDIs, these values were nearly half as much as those predicted from urinary TDCIPP in this study. This suggested that children's PFRs burden may be underestimated when considering only PFR levels in dust or air. There is thus a need for further studies with large-scale surveys and investigation of exposure routes.

Heavy metal contamination along the China coastline: A comprehensive study using Artificial Mussels and native mussels.

A comprehensive study was carried out to assess metal contamination in five cities spanning from temperate to tropical environment along the coastal line of China with different hydrographical conditions. At each of the five cities, Artificial Mussels (AM) were deployed together with a native species of mussel at a control site and a polluted site. High levels of Cr, Cu and Hg were found in Qingdao, high level of Cd, Hg and Pb was found in Shanghai, and high level of Zn was found in Dalian. Furthermore, level of Cu contamination in all the five cities was consistently much higher than those reported in similar studies in other countries (e.g., Australia, Portugal, Scotland, Iceland, Korea, South Africa and Bangladesh). Levels of individual metal species in the AM showed a highly significant correlation with that in the native mussels (except for Zn in Mytilus edulis and Cd in Perna viridis), while no significant difference can be found between the regression relationships of metal in the AM and each of the two native mussel species. The results demonstrated that AM can provide a reliable time-integrated estimate of metal concentration in contrasting environments over large biogeographic areas and different hydrographic conditions, and overcome the shortcomings of monitoring metals in water, sediment and the use of biomonitors.

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 disrupts gene modulation along the brain-pituitary-gonad (BPG)-liver axis.

Hypoxia alters sex hormone concentrations leading to reproductive impairment in fish; however the mechanisms underlying these effects remain largely unknown. Using zebrafish (Danio rerio), this study is the first to demonstrate that hypoxia causes endocrine disruption by simultaneously acting on multiple targets along the brain-pituitary-gonadal (BPG)-liver axis in fish. Alterations in the expression of key genes associated with reproductive endocrine pathways in the brain (sGnRH), pituitary (FSHß and LHß), gonads (FSH-R, LH-R, HMGR, StAR, CYP19A, CYP11A, CYP11ß and 20ß-HSD), and liver were correlated with significant reductions of estradiol in females and testosterone in males. Hypoxia also induced sex-specific and tissue-specific changes in the expression of estrogen, androgen, and membrane progestin receptors along the BPG axis, suggesting disruption of the feedback and synchronization of hormone signals. Furthermore, the hypoxia-induced upregulation of hepatic sex hormone-binding globulin suggests an increase in hormone transport and reduced bioavailability in blood, while upregulation of hepatic CYP3A65 and CYP1A in females suggests an increase in estrogen biotransformation and clearance. Given that the regulation of reproductive hormones and the BPG-liver axis are highly conserved, this study provides new insights into the hypoxia-induced endocrine disrupting mechanisms and reproductive impairment in other vertebrates.

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.

Proteomic response of marine invertebrate larvae to ocean acidification and hypoxia during metamorphosis and calcification.

Calcifying marine invertebrates with complex life cycles are particularly at risk to climate changes as they undergo an abrupt ontogenetic shift during larval metamorphosis. Although our understanding of the larval response to climate changes is rapidly advancing, the proteome plasticity involved in a compensatory response to climate change is still unknown. In this study, we investigated the proteomic response of metamorphosing larvae of the tubeworm Hydroides elegans, challenged with two climate change stressors, ocean acidification (OA; pH 7.6) and hypoxia (HYP; 2.8 mg O2 l(-1)), and with both combined. Using a two-dimensional gel electrophoresis (2-DE)-based approach coupled with mass spectrometry, we found that climate change stressors did not affect metamorphosis except under OA, but altered the larval proteome and phosphorylation status. Metabolism and various stress and calcification-related proteins were downregulated in response to OA. In OA and HYP combined, HYP restored the expression of the calcification-related proteins to the control levels. We speculate that mild HYP stress could compensate for the negative effects of OA. This study also discusses the potential functions of selected proteins that might play important roles in larval acclimation and adaption to climate change.

Whole spectrum of cytochrome P450 genes and molecular responses to water-accommodated fractions exposure in the marine medaka.

Water-accommodated fractions (WAFs) of crude oil include chemicals that are potent toxicants in fish. Increasing oil pollution thus demands a better understanding of molecular mechanisms for detoxification, metabolism, toxicity, and adaptation of fish. Previous studies with fish show modulation of expression of key genes in relation to stress response against WAF exposure, but there is still a lack of studies on responses of cytochrome P450 (CYP) genes and changes in biotransformation upon WAF exposure. In this study, we used the full spectrum of CYP genes of the marine medaka, Oryzias melastigma, to understand their potential mode of action on WAFs-triggered molecular mechanisms. We also analyzed further CYP-involved detoxification and endogenous steroidogenic metabolism after exposure to different concentrations of WAFs over different time courses in the marine medaka. Also, detoxification- and antioxidant-related enzymes' activities were analyzed with different concentrations of WAFs. As a result, the WAF exposure induced CYP-involved detoxification mechanism but reduced CYP-involved steroidogenic metabolism in the marine medaka. These data suggest that whole CYP profiling can be a way of understanding and uncovering the mode of action particularly with respect to emerging chemicals such as WAF exposure with the new finding that WAFs have dual functions on CYP-involved metabolisms.

Luteinizing hormone receptor (lhcgr) as a marker gene for characterizing estrogenic endocrine-disrupting chemicals in zebrafish ovarian follicle cells.

The adverse effects of endocrine-disrupting chemicals (EDCs) have been well documented; however, the action mechanisms of many EDCs remain elusive and controversial. Furthermore, the highly diversified chemical structures and low environmental concentrations of EDCs present a major challenge to their chemical detection. Clearly, there is an urgent need for simple and reliable bioassays to detect EDCs in the environment and unravel their action mechanisms. We have recently identified luteinizing hormone receptor (lhcgr) as a robust estradiol (E2)-responsive gene in cultured zebrafish ovarian follicle cells. The expression of lhcgr exhibited a distinct biphasic response to E2 over a 24-h time-course treatment, making this a unique system for characterizing estrogenic EDCs. This study was undertaken to validate this platform by testing a wide range of EDCs, including 17α-ethinylestradiol (EE2), diethylstilbestrol (DES), bisphenol A (BPA), genistein (GEN), 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p'-DDT), vinclozolin (VIN), bis(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Diethylstilbestrol (DES), EE2 and o,p'-DDT mimicked E2 and induced a biphasic expression of lhcgr while BPA and GEN stimulated a monophasic expression in the 24-h time-course. In contrast, BDE-47, DEHP and VIN had no effect, whereas TCDD decreased lhcgr expression. Dose-response experiment showed that E2, EE2 and DES had the highest potency, which was followed by GEN, BPA and o,p'-DDT. The effects of estrogenic EDCs were further confirmed by their potentiation of hCG-induced activin ßA2 subunit (inhbab) expression. In conclusion, the present study showed that the expression of lhcgr in cultured zebrafish follicle cells and its biphasic response to estrogens provide a unique in vitro platform for screening and categorizing estrogenic substances and deciphering their action mechanisms.

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.

Polybrominated diphenyl ethers (PBDEs) alter larval settlement of marine intertidal organisms across three phyla via reducing bacterial abundance on the biofilms.

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants over the last three decades, and are now ubiquitous in the marine environment. While the harmful effects of PBDEs on the abnormal development and reproductive impairment in mammals and fish are well documented, the effects on marine invertebrates remain virtually unknown. Using three model intertidal species accross three phyla, including the polychaete Hydroides elegans (Phylum Annelida), the gastropod Crepidula onyx (Phylum Mollusca), and the barnacle Balanus amphitrite (Phylum Arthopoda), this study demonstrated that (a) chronic exposure to BDE-47 (at spiking concentrations up to 1000 ng L(-1)) throughout the entire larval stage did not affect settlement, development or growth of all three species per se, despite bioaccumulation was clearly evident (measured body burden ranging from approximately 7000 to 13 000 ng BDE-47 g(-1) lipid), and (b) BDE-47, at measured concentrations of 15 and 113 ng g(-1) lipid, reduced the bacterial abundance in biofilms and resulted in a concomitant change in larval settlement pattern of all the model intertidal species across three phyla.

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.

Estrogenic potential of benzotriazole on marine medaka (Oryzias melastigma).

This study, for the first time, assessed the reproductive effects of benzotriazoles, widely used industrial chemicals, on marine fish. Marine medakas (Oryzias melastigma) were exposed to 0.01, 0.1, and 1mg/L benzotriazole for periods of four and 35 days. The results that are obtained showed that the expression levels of CYP1A1 were down-regulated in the liver, gills and intestines of both males and females. Vitellogenin (VTG) was highly induced in the liver, gills and intestine of both male and female marine medaka, and CYP19a was up-regulated in the ovaries especially after being exposed for 35 days. Most importantly, the results of the present study suggest that even at environmentally relevant concentrations detected in the aquatic environment, 0.01 mg/L, benzotriazole also caused notable changes in expression levels of VTG, CYP1A1 and CYP19a. More concerns about the toxicity of benzotriazoles on marine animals should be raised.

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.

Hypoxia-induced epigenetic transgenerational miRNAs dysregulation involved in reproductive impairment of ovary.

Hypoxia is a potent endocrine disruptor that is posing serious problems to the fish reproductive systems. Our previous studies reported that hypoxia could cause a transgenerational impairment of ovarian development and interfere hatching success in F2 offspring of marine medaka fish (Oryzias melastigma) through epigenetic regulation. As part of the epigenetic regulation, we investigated the involvement of microRNAs (miRNAs) in hypoxia-induced transgenerational reproductive impairments. In the present study, we used comparative small RNA sequencing to reveal that hypoxia caused miRNA dysregulation in ovaries of F0 hypoxia group and F2 transgenerational group. We found 4 common dysregulated miRNA in the F0 and F2 generations. Furthermore, integrated miRNA-mRNA analysis, followed by gene ontology enrichment analysis on the hypoxia-dysregulated miRNA-target genes further highlighted the importance of these dysregulated miRNAs in biological processes related to reproduction. More importantly, we identified 3 miRNA-mRNA pairs (novel miRNA-525-DIAPH2, novel miRNA-525-MYOCD, and novel miRNA-525-RAI14) that might play epigenetic roles in hypoxia-induced reproductive impairment. For the first time, our findings suggested the involvement of miRNA in hypoxia-induced reproductive impairments may be inherited via a transgenerational manner.