Adult Female Medaka Research Articles

Maternal Transfer of 2-Ethylhexyl Diphenyl Phosphate Leads to Developmental Toxicity Possibly by Blocking the Retinoic Acid Receptor and Retinoic X Receptor in Japanese Medaka (Oryzias latipes).

2-Ethylhexyl diphenyl phosphate (EHDPP) has been detected in wild fish with high concentrations, which may pose a risk in the embryo development considering its potential maternal transfer. In this study, EHDPP was demonstrated to elicit antagonistic activity to medaka retinoic acid receptor (mRAR) and retinoic X receptor (mRXR) with 50% inhibitory concentration of 18 and 36 μM, respectively. After adult female medaka were exposed to EHDPP at 156, 405, and 1161 ng/L for 35 days, the embryonic EHDPP concentrations (364-4824 ng/g lipid weight (lw)) were higher than those in the maternal tissues (15.0-4166 ng/g lw), showing notable maternal transfer. The embryonic concentration of EHDPP decreased limitedly during 1-2 day post-fertilization (dpf, the main developmental window of eye) but then decreased sharply after 2 dpf. The transcript abundance of cyp26a1 was inhibited and subsequent increasing embryonic all-trans RA level was observed in embryos, showing RAR/RXR antagonistic activity. These results may specifically contribute to the increased eye deformity incidences in all exposure groups (up to 8.0%; 51/637) relative to the control (1.0%, 7/733). The response behavior of the larvae to light stimulation was impaired in a dose-dependent manner, demonstrating a vision disorder. Because such developmental toxicities were observed at the environmental level, EHDPP may pose a threat to the survival of wild larvae and therefore a population risk for wild fish.

Transient exposure to environmentally realistic concentrations of di-(2-ethylhexyl)-phthalate during sensitive windows of development impaired larval survival and reproduction success in Japanese medaka

Di-(2-ethylhexyl) phthalate (DEHP) is a well-known endocrine disruptor and it is ubiquitously distributed in the environment. However, very few studies have investigated the effects of short-term exposure to environmentally realistic concentrations of DEHP during early developmental stages and its chronic effects. This study monitored the long-term effects of transient exposure to DEHP in early life stages (F0 generation) and its subsequent fertilization success in F1 generation using Japanese medaka, Oryzias latipes, as model organism. Embryos (4 h post-fertilization, 4 hpf) of Japanese medaka were exposed to 0.001 ppb, 0.1 ppb, or 10 ppb DEHP for 21 days and returned to control water (without DEHP) for maturation (4 months old). At day 9 of the exposure study, mortality was significantly increased in medaka embryos (before hatching) treated with 0.001 ppb and 10 ppb DEHP. Continual exposure of young hatchlings for an additional 12 days (a total of 21 days of exposure) resulted in a significant increase in mortality in fish exposed to 0.001 ppb, 0.1 and 10 ppb DEHP. Significant reduction in egg production was observed in adult female medaka (4 months old) with prior exposure to 0.1 ppb and 10 ppb DEHP for 21 days during early development. Fertilization and hatching success were also significantly reduced in breeding pairs with prior exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP during early life stage. Histological analysis of adult male gonads revealed a significant decline in mature sperm count accompanied by an increase in interstitial space in fish exposed to 0.1 ppb and 10 ppb DEHP during early development. Likewise, the amount of vitellogenic (mature) oocytes observed in the ovaries of adult female with transient exposure to 0.1 ppb and 10 ppb DEHP was significantly reduced when compared with the solvent control group. Our data suggest that transient exposure to ultra low concentrations of DEHP during sensitive time windows of development results in irreversible reproductive impairment which may impact fish populations negatively.

Gnrh1-Induced Responses Are Indirect in Female Medaka Fsh Cells, Generated Through Cellular Networks.

Reproductive function in vertebrates is stimulated by GnRH that controls the synthesis and release of the two pituitary gonadotropins, FSH and LH. FSH and LH, which regulate different stages of gonadal development, are produced by two different cell types in the fish pituitary. This is in contrast to the situation in mammals and birds, and it enables investigation of their differential regulation. In the present study, we used fluorescence in situ hybridization to show that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This result was confirmed by patch-clamp recordings and by cytosolic Ca2+ measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, responded to Gnrh1 by biphasic alteration in action-potential frequencies and cytosolic Ca2+ levels. In contrast, both Fsh and Lh cells are able to respond to Gnrh1 in brain-pituitary tissue slices both electrically and by elevating the cytosolic Ca2+ levels. Using Ca2+ uncaging in combination with patch-clamp recordings and cytosolic Ca2+ measurements, we show that Fsh and Lh cells form homotypic and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka are indirect and probably mediated via Lh cells.

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes)

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 μg·L−1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 μg L−1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

γ-H2AX foci as indication for the DNA damage in erythrocytes of medaka (Oryzias latipes) intoxicated with 4-nonylphenol

The present study aimed to investigate the genotoxicity in erythrocytes induced after exposure of medaka (Oryzias latipes) to 4-nonylphenol (4-NP). Adult female medaka fish were exposed to 4-NP at three sublethal concentrations for 15days to compare their sensitivity with that of catfish as an aquatic model. Comet assay and γ-H2AX were used as biomarkers to detect DNA damage in erythrocytes. Exposure to 4-NP resulted in an increase in the tail moment in a dose-dependent manner. The highest level of DNA damage was recorded after exposure to 100μg/l 4-NP. The number of foci was increased after exposure to 4-NP, indicating damage to DNA. The present results confirmed the high level of morphological alterations and apoptosis of erythrocytes detected in the first part of this study. 4-NP induced genotoxic effects in medaka, which were found to be more sensitive than catfish after exposure to 4-nonylphenol. Graphical abstract.

Immunostaining of UVA-induced DNA damage in erythrocytes of medaka (Oryzias latipes)

Some authors have recently reported that UVA induces double-strand breaks (DSBs) in DNA. Only a few researchers have reported on the induction of DSBs upon UVA exposure, as measured using the Comet assay and γ-H2AX as markers of DSB formation. In the present study, we have investigated for the first time the dose-dependent induction of DSBs by UVA in medaka (Oryzias latipes) erythrocytes. Adult female medaka fish were exposed to UVA for 15, 30, and 60min/day for three continuous days; an unirradiated control group was kept in the same laboratory conditions. At 0h and 24h after UVA exposure, blood was collected to detect DNA damage and repair. The number of γ-H2AX foci was higher than the control value at 0h after UVA exposure and decreased within a 24h. the comet assay showed that DNA repair began during the recovery period. These findings confirm our pervious findings of genotoxic effects after UVA exposure in medaka erythrocytes and suggest that the replication-independent formation of UVA-induced DSBs is mediated through the generation of reactive oxygen species. In conclusion, these results suggest that DNA damage and repair occur after UVA exposure in medaka fish. UVA is the main component of solar UV radiation and is used for artificial UV exposure. Our results may have implications for skin cancer research.

Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation

Purpose: Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24 hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24 h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation.Materials and methods: Sexually mature wild-type, WT (Hd-rR) and p53(−/−) adult female medaka (O. latipes) were exposed to 4 Gy dose of γ-IR and sampling were collected after 4, 8 and 24 h and 14 days.Results: Apoptosis and morphological alterations were observed from 4 h after irradiation and remarkably increased 8 h after irradiation in the wild-type. Apoptotic cell death has been observed 8 h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation.Conclusions: We conclude that apoptosis and malformations caused by 4 Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4–24 h and the initial response until 8 h was p53-dependent.

Trenbolone acetate metabolites promote ovarian growth and development in adult Japanese medaka (Oryzias latipes)

Trenbolone acetate, a synthetic androgen, has been used as a growth promoter in beef cattle in the US since 1987. While several teleost studies have investigated the masculinization effects of the metabolite 17β-trenbolone, few have focused on the reproductive impacts of all three trenbolone acetate (TBA) metabolites including trendione. Adult female medaka (Oryzias latipes) were exposed to TBA metabolites (10, 100, and 1000ng/L) for 14days (n=3). Histological examination revealed that TBA metabolites (1000ng/L) significantly reduced the percentage of primary ovarian follicles and increased the percentage of vitellogenic follicles compared to control fish. 17α-Trenbolone significantly increased whereas trendione reduced whole body levels of estradiol-17β. Testosterone was significantly reduced by trendione treatment and only the highest dose of 17β-trenbolone and lowest dose of trendione altered 11-ketotestosterone. Additionally, TBA metabolites may be further broken down and/or metabolized or converted by the animal influencing both sex steroid levels and ovarian development.

Effects of metolachlor on transcription of thyroid system-related genes in juvenile and adult Japanese medaka (Oryzias latipes)

Metolachlor (MT) is one of the most important pesticides applied to corn and other crops for controlling broadleaf and grass weeds. However, the effects of MT on the thyroid system in fish remain to be elucidated. In the present experiment, transcription of genes related to the thyroid system, including thyrotropin releasing hormone (Trh), deiodinase 2 (Dio2), thyroid hormone receptor α (Thrα), and thyroid hormone receptor β (Thrβ), were induced by MT in a sex-, developmental stage-, and tissue- specific manner when medaka were exposed to various concentrations of MT for 14days. The transcriptional levels of the genes were only significantly altered in both juvenile and adult female medaka in response to MT exposure. And the lowest concentrations able to significantly induce transcription of the selected genes were 10 and 100μg/L in juvenile and adult female medaka, respectively. In adult female medaka, a significant up-regulation of these genes was detected only in the brain, with little or no effect in the liver. Furthermore, MT-induced (100μg/L) transcription of thyroid system-related genes was enhanced significantly in male juvenile medaka in the presence of estrogen (E2) (50 and 100ng/L). Moreover, the mRNA levels of Thrα and Thrβ in males increase with the combined treatments of 100μg/L MT and 100ng/L E2. Dio2 increased when exposed to 100μg/L MT and 50 or 100ng/L E2. The information obtained in the present study suggests that MT has the potential to influence several steps of the hypothalamus–pituitary–thyroid (HPT) axis homeostasis and to disrupt the thyroid system in medaka.

Interconversion of Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in Japanese Medaka

Polybrominated diphenyl ethers (PBDEs), hydroxylated (OH) and methoxylated (MeO), have been widely detected in aquatic environments. However, relationships among these structurally related compounds in exposed organisms are unclear. To elucidate biotransformation relationships among BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47, dietary accumulation, maternal transfer, and tissue distribution of these compounds and their transformation products were investigated in sexually mature Japanese medaka (Oryzias latipes). In addition, transformation of each compound was determined in vitro using liver microsomes of medaka. OH-PBDEs and MeO-PBDEs were not detected in fish exposed to BDE-47. However, significant concentrations of 6-OH-BDE-47 were detected in medaka or microsomes exposed to 6-MeO-BDE-47. Significant concentrations of 6-MeO-BDE-47 were also measured in fish exposed to 6-OH-BDE-47, but 6-MeO-BDE-47 was not detected in microsomes exposed to 6-OH-BDE-47. Similar patterns of transformation products were observed in medaka eggs from adult fish during exposure. This study presents direct in vivo evidence of biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47. In addition, this is the first study to demonstrate biotransformation of 6-OH-BDE-47 to 6-MeO-BDE-47. Demethylation of 6-MeO-BDE-47 was the primary transformation pathway leading to formation of 6-OH-BDE-47 in medaka, while the previously hypothesized formation of OH-PBDEs from synthetic BDE-47 did not occur. Biotransformation products formed in adult female medaka were transferred to eggs.

Environmental cues influence EDC-mediated endocrine disruption effects in different developmental stages of Japanese medaka ( Oryzias latipes)

Temperature and photoperiod are the two most important environmental cues in the regulation of the annual cycles of circulating sex steroid hormones and reproduction in fish. Thus, these variables may alter the endocrine disruption effects induced by environmental endocrine disrupting chemicals (EDCs). In the present study, we found that the temperature and photoperiod significantly influence the transcription of the estrogen-responsive genes, vitellogenin1 (Vtg1), vitellogenin2 (Vtg2) and estrogen receptor-α (ERα) after a 7-day exposure to environmentally relevant concentrations of 17β-estradiol (E2) and nonylphenol (NP) in different developmental stages of Japanese medaka. In general, gene transcription levels increased as temperature and photoperiod length increased. The Vtg1 mRNA levels in 20 °C–12 L group were 10–30 times higher than those in 10 °C–10 L group when larvae, both sexes of juvenile or male adult medaka were exposed to 50 ng/L E2. In addition, juveniles of both sexes and adult males were more sensitive to temperature and photoperiod changes than larval and adult female medaka. Specifically, two-way analysis indicated that both E2 and NP-mediated induction of Vtg1 mRNA (the most effective) expression interacted with environmental cues (temperature and photoperiod) and exposure concentrations in a developmental stage- and sex-specific manner. The results obtained in the present study demonstrate that the endocrine disrupting effects of environmental chemicals can be modified by seasonality and life stage in which the exposure occurs and that Vtg1 transcription is a good biomarker to evaluate these interactions.

Increased cellular apoptosis after chronic aqueous exposure to nonylphenol and quercetin in adult medaka ( Oryzias latipes)

Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka ( Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.

Spontaneous ovarian tumour in a medaka ( Oryzias latipes)

Swelling of the abdomen was found in an adult female medaka (Oryzias latipes). Multiple tumour masses occupying most of the abdominal cavity were found at necropsy. Histologically, the tumour consisted of homogeneous round cells arranged in islands and occasional trabecular structures with thin capsules. Ova at various stages of maturation were seen in the periphery of the tumour mass. Metastasis was observed in the vicinity of the thyroid gland with accompanying infiltration by lymphocytic cells and also abdominal muscles. The tumour cells were characterized by a large ovoid nucleus with no aggregation of heterochromatin, occasional mitotic figures and prominent nucleoli. Examination by electron microscopy showed cells with scant cytoplasm but many ribosomes and a few other organelles. Annulate lamellae and dense cytoplasmic masses were also frequently observed. Based on the above features, the tumour was diagnosed as an ovarian dysgerminoma, the first reported case in a medaka.

A photosensitive daily rhythm in the female medaka, Oryzias latipes

Adult female medaka, Oryzias latipes, with regressed ovaries were exposed to 7 h of continuous light plus an additional hour of light at different times during the dark period of a 24-h cycle (7 + 1 light: 16 dark) at 24 ± 1 °C. When this additional hour of light falls on the 16th h, counting the onset of the light period as the 0 h, ovarian development was induced. This ovarian development is enhanced when the exposure period is increased and when the fish are preexposed to a warm temperature. The use of a photosensitive daily rhythm to measure photoperiod and to time the spawning season in the medaka is suggested.