Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes).

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

The presence of a conspecific induces risk-taking behaviour and enlargement of somata size of dopaminergic neurons in the brain of male medaka fish.

Boldness and risk-taking behaviours in animals are important traits to obtain advantages such as habitation, food resources, reproductive success and social dominance. Risk-taking behaviour is influenced by physiological and environmental conditions; however, whether individual fish become bolder by the presence of conspecifics remains unknown. In this study, a light-dark preference test was conducted using medaka fish (Oryzias latipes) with or without a neighbouring conspecific. It was found that individual medaka male fish preferred a light environment and avoided a dark environment, whereas the display of a neighbouring conspecific enhanced the time the male spent in the dark environment (i.e., this condition encouraged risk-taking). The blood glucose level increased in fish confined to the dark condition but did not increase in light-preferring fish and risk-taking fish. Large somata expressing tyrosine hydroxylase, which is the rate-limiting enzyme in dopamine synthesis, were detected in the telencephalic and diencephalic brain regions in risk-taking medaka, whereas large somata were detected in the diencephalic region in medaka confined to the dark condition. These findings indicated that medaka is a good fish model to explore the central roles of dopaminergic neurons in the telencephalon and the diencephalon, which regulate risk-taking behaviour.

Examination of methods for manipulating serum 17ß-Estradiol (E2) levels by analysis of blood E2 concentration in medaka (Oryzias latipes).

It is widely known that reproduction in vertebrates is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. Although the mechanism of the HPG axis has been well documented in mammals, it cannot be always applied to that in non-mammalian species, which is a great disadvantage in understanding reproduction of vertebrates in general. Recently, transgenic and genome editing tools have rapidly been developed in small teleosts, and thus these species are expected to be useful for the understanding of general mechanism of reproduction in vertebrates. One of the major sex steroid hormones in female vertebrates 17ß-Estradiol (E2) plays crucial roles in the formation of sexual dimorphism and the HPG axis regulation. In spite of the importance of E2 in reproductive regulation, only a few studies have analyzed blood E2 levels in small teleosts that are easily amenable to genetic manipulation. In the present study, we analyzed blood E2 concentration in medaka and demonstrated that female medaka show diurnal changes in blood E2 concentration. We then examined the best method for manipulating the circulating E2. First, we found that ovariectomy (OVX) drastically removes endogenous E2 in a day in female medaka. We examined different methods for E2 administration and revealed that feeding administration of E2-containing food is the most convenient and physiological method for mimicking the diurnal E2 changes of female medaka. On the other hand, the medaka exposed to E2 containing water showed high blood E2 concentrations, which exceeds those of environmental water, suggesting that E2 may cause bioconcentration.

Cytotoxic and genotoxic effects of arsenic on erythrocytes of Oryzias latipes: Bioremediation using Spirulina platensis.

BACKGROUND: Exposure to the environmental pollutants poses a serious threat to aquatic organism. The arsenic exposure in fish increases the risk of developing serious alterations from embryo to adult. OBJECTIVES: The present investigation was done to study the toxic effects of heavy metal arsenic [As(III)] on medaka (Oryzias latipes). Morphological alterations, apoptosis, nuclear abnormalities, and genotoxic biomarkers in erythrocytes were used to determine the stress caused by arsenic (As) exposure. METHODS: Medaka was exposed to As for 15 days at two toxic sublethal concentrations (7 ppm and 10 ppm) in combination with Spirulina platensis (SP) treatment as antioxidant algae at 200 mg/L. RESULTS: Results were consistent with a previous study results on tilapia. Exposure of medaka to As resulted in a dose-dependent increase in most the biomarkers used in the current study. Fish exposed to10 ppm As showed highest level of DNA damage. For the first time to our knowledge, using SP to counter the As toxicity in medaka, DNA damage restored to control levels. CONCLUSION: Accordingly, those results suggests that SP can protect medaka in aquaculture against As-induced damage by its ability as reactive oxygen species (ROS) reducer, antioxidant role, and DNA damage scavenger.

Sensitivity of medaka (Oryzias latipes) to 4-nonylphenol subacute exposure; erythrocyte alterations and apoptosis.

The present study was undertaken to assess the effects of the endocrine-disrupting compound; 4-nonylphenol (4-NP) in medaka (Oryzias latipes). The frequencies of erythrocyte alterations, apoptosis, and micronuclei were used as biological indicators of damage. Medaka were exposed 15 days to 4-NP at three sublethal concentrations (50, 80, and 100 µg/l 4-NP) and results compared with those of a previous study using catfish as an animal model. Exposure of medaka resulted in a dose-dependent increase in the frequency of erythrocyte alterations, apoptosis and micronucleus (MN). Many morphological alterations and nuclear abnormalities were observed, including acanthocytes, lobed nucleus, eccentric nucleus, fragmented nucleus, blebbed nucleus, binuclei, deformed nucleus, notched nucleus, hemolysed cells, crenated cells, teardrop-like cells, and schistocytes. Mortality was recorded after treatment with 80 and 100 µg/l 4-NP, indicating that medaka are more sensitive than catfish to 4-NP exposure. We concluded that, 4-NP causes several malformations in the shape and number of erythrocytes in medaka, indicating its genotoxicity.

Social rank-dependent expression of gonadotropin-releasing hormones and kisspeptin in the medaka brain.

Social interactions regulate the expression of several neuropeptides that have a central role in the reproductive system of mammals. Nonmammalian vertebrates also have these neuropeptides or paralogs, however, studies on the social regulation of reproductive physiology in nonmammalian species are limited. In this study, we examined whether the expression of gonadotropin-releasing hormones (GnRHs) and kisspeptin (Kiss1) is affected by social hierarchy resulting from the outcomes of male-male competition in medaka fish (Oryzias latipes). Four males were introduced to each other in an experimental tank, and classified as the most aggressive dominant or the most submissive subordinate fish, based on the frequency of their aggressive acts during a short-term competition. Dominant and subordinate males maintained their social rank during a long-term competition. Immediately after short-term competition, gonadotropin-releasing hormone-3 (GnRH3) level in the olfactory bulb was significantly higher in subordinate males than in dominant males. After long-term competition, dominant males had high level of gonadotropin-releasing hormone-1 (GnRH1) in the preoptic area, whereas subordinate males had lower Kiss1 level in the nucleus ventral tuberis. On the other hand, the levels of gonadotropin-releasing hormone-2 (GnRH2) in the nucleus lateralis valvulae and Kiss1 in the nucleus posterioris periventricularis, and plasma 11-ketotestosterone (11-KT) concentration did not differ between subordinate and dominant males after the short- and long-term competitions. These results suggest that social hierarchy regulates the expression of GnRH1, GnRH3, and Kiss1 without affecting 11-KT level in male medaka.

Chronic Exposure of Marine Medaka (Oryzias melastigma) to 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) Reveals Its Mechanism of Action in Endocrine Disruption via the Hypothalamus-Pituitary-Gonadal-Liver (HPGL) Axis.

In this study, marine medaka (Oryzias melastigma) were chronically exposed for 28 days to environmentally realistic concentrations of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) (0, 0.76, 2.45, and 9.86 µg/L), the active ingredient in commercial antifouling agent SeaNine 211. Alterations of the hypothalamus-pituitary-gonadal-liver (HPGL) axis were investigated across diverse levels of biological organization to reveal the underlying mechanisms of its endocrine disruptive effects. Gene transcription analysis showed that DCOIT had positive regulatory effects mainly in male HPGL axis with lesser extent in females. The stimulated steroidogenic activities resulted in increased concentrations of steroid hormones, including estradiol (E2), testosterone (T), and 11-KT-testosterone (11-KT), in the plasma of both sexes, leading to an imbalance in hormone homeostasis and increased E2/T ratio. The relatively estrogenic intracellular environment in both sexes induced the hepatic synthesis and increased the liver and plasma content of vitellogenin (VTG) or choriogenin. Furthermore, parental exposure to DCOIT transgenerationally impaired the viability of offspring, as supported by a decrease in hatching and swimming activity. Overall, the present results elucidated the estrogenic mechanisms along HPGL axis for the endocrine disruptive effects of DCOIT. The reproductive impairments of DCOIT at environmentally realistic concentrations highlights the need for more comprehensive investigations of its potential ecological risks.

Endocrine Disruption throughout the Hypothalamus-Pituitary-Gonadal-Liver (HPGL) Axis in Marine Medaka (Oryzias melastigma) Chronically Exposed to the Antifouling and Chemopreventive Agent, 3,3'-Diindolylmethane (DIM).

Despite being proposed as a promising antifouling and chemopreventive agent, the environmental risks of 3,3'-diindolylmethane (DIM) are scarcely investigated. Therefore, this study used adult marine medaka (Oryzias melastigma) as a model organism to examine the toxicological effects and underlying mechanism of DIM throughout the hypothalamus-pituitary-gonadal-liver (HPGL) axis following 28 days of exposure to low DIM concentrations (0 and 8.46 µg/L). The results showed that altered gene transcription in the hypothalamus, pituitary, and gonads contributed to the great imbalance in hormone homeostasis. The lowered estradiol (E2)/testosterone (T) and E2/11-keto-testosterone (11-KT) ratios in female plasma resulted in decreased synthesis and levels of vitellogenin (VTG) and choriogenin in the liver and plasma, and vice versa in males. Subsequently, VTG and choriogenin deficiency blocked the reproductive function of the ovary as indicated by decreased fecundity and offspring viability, whereas in male medaka, DIM mainly targeted the liver and induced severe vacuolization. Proteomic profiling of plasma revealed that the sex-specific susceptibility to DIM could be attributed to the increased detoxification and oxidative defense in males. Overall, this study identified the endocrine disruption and reproductive impairment potency of DIM and first elucidated its mechanisms of action in medaka. The differential responses to DIM (estrogenic activities in the male but antiestrogenic activities in the female) provided sensitive biomarkers characteristic of each sex. Considering the chemical stability and potent endocrine disturbance at low concentration, the application of DIM either as an antifouling or chemopreventive agent should be approached with caution in marine environments.

High levels of plasma cortisol and impaired hypoosmoregulation in a mutant medaka deficient in P450c17I.

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na(+)/K(+)/2Cl(-) cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na(+)/K(+)/2Cl(-) cotransporter, in seawater.

Nuclear and cytoplasmic changes in erythrocytes of p53-deficient medaka fish (Oryzias latipes) after exposure to gamma-radiation.

Previous studies have examined the effects of gamma-radiation on Japanese fish, in particular medaka (Oryzias latipes). In the present work, alterations in erythrocytes were recorded as haematological bio-indicators of exposure to gamma-radiation. After exposure of medaka fish to two different doses of radiation (2 Gy and 10 Gy), many malformations in red blood cells were observed in the irradiated fish compared with control fish. These malformations included acanthocytes, crenated cells, amoeboid cells, and sickle cells. More malformations were seen at the higher radiation dose. No micronuclei were seen in any group, but nuclear abnormalities were observed. We conclude that gamma-radiation causes morphological malformations of erythrocytes and is harmful to medaka fish.

Medaka fish, Oryzias latipes, as a model for human obesity-related glomerulopathy.

Obesity, an ongoing significant public health problem, is a part of complex disease characterized as metabolic syndrome. Medaka and zebrafish are useful aquatic experimental animals widely used in the field of toxicology and environmental health sciences and as a human disease models. In medaka, simple feeding of a high fat diet (HFD) can induce body weight gain, excessive accumulation of visceral adipose tissue, hyperglycemia, hyperlipidemia, and steatohepatitis, which mimics human metabolic syndrome. In the present study, to explore the possibility that the adult medaka fed with HFD (HFD-medaka) can be used as an animal model for human metabolic syndrome-associated glomerular disease, including obesity-related glomerulopathy (ORG), we analyzed structural alterations and protein expression in the mesonephric kidney of HFD-medaka. We found that the histopathology was consistent with glomerulomegaly accompanied by the dilation of glomerular capillaries and proliferative expansion of the mesangium, a condition partially comparable to human ORG. Moreover, expressions of several kinds of kidney disease-related proteins (such as MYH9, SM22α) were significantly elevated. Thus, the HFD-medaka has a high potential as an animal model useful for exploring the mechanism underling human ORG.

Transcriptomic profiles of Japanese medaka (Oryzias latipes) in response to alkalinity stress.

Oryzias latipes (Adrianichthyidae), known as Japanese medaka or Japanese killifish, is a small 2-4 cm long fish common in rice paddies in coastal Southeast Asia and is also a popular aquarium fish. It has been widely used as a research model because of its small size and because it is very easy to rear. Alkalinity stress is considered to be one of the major stressors on fish in saline-alkaline water. As very little is known about molecular genetic responses of aquatic organisms to alkalinity stress, we examined genome-wide gene expression profiles of Japanese medaka in response to carbonate alkalinity stress. Adult fish were exposed to freshwater and high carbonate alkaline water in the laboratory. We designed a microarray containing 26,429 genes for measuring gene expression change in the gills of the fish exposed to high carbonate alkalinity stress. Among these genes, 512 were up-regulated and 501 were down-regulated in the gills. These differentially expressed genes can be divided into gene groups using gene ontology, including biological processes, cellular components and molecular function. These gene groups are related to acid-base and ion regulation, cellular stress response, metabolism, immune response, and reproduction processes. Biological pathways including amino sugar and nucleotide sugar metabolism, porphyrin and chlorophyll metabolism, metabolism of xenobiotics by cytochrome P450, drug metabolism, aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversions, glutathione metabolism, and fructose and mannose metabolism were significantly up-regulated. Alkalinity stress stimulates the energy and ion regulation pathways, and it also slows down the pathways related to the immune system and reproduction.

Salinity-dependent expression of a Na+, K+, 2Cl- cotransporter in gills of the brackish medaka Oryzias dancena: a molecular correlate for hyposmoregulatory endurance.

This study used the brackish medaka (Oryzias dancena) to characterize Na+, K+, 2Cl- cotransporter (NKCC) expression from the genetic to cellular level in gills. Using RT-PCR to survey tissue distribution of nkcc1a, 1b, and 2, we report that gills of brackish medaka prominently express Odnkcc1a. The full-length cDNA of Odnkcc1a was cloned from gill tissue. In situ hybridization indicates that Odnkcc1a was localized to mitochondrion-rich (MR) cells. Higher mRNA levels of Odnkcc1a were found in gills from seawater (SW) and brackish water (BW) medaka when compared to freshwater (FW) fish. Furthermore, higher amounts of NKCC1a-like protein were detected by the monoclonal antibody in gills of SW and BW medaka compared to FW medaka. Double immunofluorescence staining revealed that NKCC1a-like protein colocalizes with Na+, K+-ATPase on the basolateral membrane of MR cells in BW and SW fish. In addition, transfer of brackish medaka from SW to FW revealed that expression of NKCC1a-like protein in gills was retained until 7days, which is a likely mechanism for maintaining hyposmoregulatory endurance. The study illustrates salinity-dependent expression of NKCC1a in branchial MR cells from brackish medaka and suggests a critical role for NKCC1a in hyposmoregulatory endurance of this fish.

Interactions between estrogenic chemicals in binary mixtures investigated using vitellogenin induction and factorial analysis.

Considerable progress has been made in assessing the combined effects of chemicals, but the effect of mixtures remains one of the most daunting challenges in environmental toxicology. In this study, the effects of binary mixtures of estrogenic chemicals were investigated using plasma vitellogenin (VTG) induction in male adult Japanese medaka (Oryzias latipes) as the endpoint. We focused on whether or not the factorial design using rigorous statistical methods was appropriate for the identification of possible interactions. The estrogenic activities of 17beta-estradiol (E2), 4-tert-nonylphenol (NP) and bisphenol A (BPA) were investigated in medaka following 14 d of exposure. Although all chemicals induced concentration-dependent increases in plasma VTG, a complete concentration-response curve was obtained only for E2, while partial curves were obtained for NP and BPA. Therefore, a 3 x 3 factorial design was employed to identify both the individual and interactive effects. A significant difference in mean plasma VTG induction was found when any chemical was considered alone. However, no significant interactions between E2 and NP, E2 and BPA, and NP and BPA were demonstrated. These results suggest that these three model estrogenic chemicals share a common mechanism for inducing VTG synthesis, and that no interactions occur when they act in combination. We also demonstrated the ability of this experimental design to detect interactions between binary mixtures, which will allow the assessment of biological effects of more complex mixtures in future research.

Observation of non-nucleated erythrocytes in the peripheral blood of medaka, Oryzias latipes.

The medaka, Oryzias latipes is a useful animal model for the study of vertebrate developmental genetics. Using May-Grünwald-Giemsa stain, we found non-nucleated erythrocytes in the peripheral blood of medaka. Eleven of 50 fish occasionally showed non-nucleated erythrocytes in their peripheral blood. We expect that this observation will be useful in future studies involving screening for hematologic mutants.

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater.

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na(+)/K(+)-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA alpha-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and alpha-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell numberxcell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.

Monitoring of vasculogenesis-inhibiting activities in sewage effluents by using medaka embryos.

Environmental samples are known to be contaminated with complex chemicals such as estrogens, polycyclic aromatic hydrocarbons, and retinoids. These contaminants have potentially an adverse impact on survival of aquatic animals, because we found previously that medaka (Oryzias latipes) embryos are defective in the development of blood vessels and bones in the presence of these chemicals. Thus, it is important to test whether sewage effluents contain inhibitory activities against the embryonic development. To examine for such activity, medaka embryos were exposed for 48 h to extracts or freeze-evaporated concentrates of effluent samples collected from different municipal sewage treatment plants. We used the transgenic embryos that are hypersensitive to estrogens due to a high-level expression of estrogen receptor for detecting the total (sum of estrogenic and non-estrogenic) vessel-inhibiting activity. The embryos were specifically defective in blood-vessel formation in most effluent samples, showing the activities ranging from 3 to 30 ng of 17beta-estradiol equiv per liter. Detection limit of 17beta-estradiol was 10 ng per liter. For detection of the non-estrogenic vessel-inhibiting activity, we treated the transgenic embryos in the presence of an antiestrogen, tamoxifen, or used the wild-type embryos. The non-estrogenic activities were found in some (7 out of 18) effluents, ranging from half to all of the total activities. Our findings for the first time demonstrate the utility of the vascular assay for monitoring sewage effluents.

The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme.

Full-length cDNA clones encoding two types of hemopexin-like protein, mWap65-1 and mWap65-2, were isolated from the HNI inbred line of medaka Oryzias latipes. The deduced amino acid sequence of mWap65-2 resembled mammalian hemopexins more closely than that of mWap65-1. Histidine residues required for the high affinity of hemopexins for hemes were conserved in mWap65-2, but not in mWap65-1. Surprisingly, mWap65-1, but not mWap65-2, showed heme-binding ability as revealed by hemin-agarose affinity chromatography, even though mWap65-1 lacked the essential histidine residues. Furthermore, RT-PCR analysis of different tissues demonstrated that the transcripts of mWap65-2 were restricted to liver, whereas those of mWap65-1 were found in various tissues including liver, eye, heart and brain. Quantitative RT-PCR revealed that transcripts of mWap65-2 were expressed earlier than those of mWap65-1 during ontogeny. However, the accumulated mRNA levels of both mWap65-1 and mWap65-2 did not differ significantly in fish acclimated to either 10 degrees C or 30 degrees C for 5 weeks. These characteristics suggest that the two proteins have different physiological functions and that mWap65-2 is not a hemopexin.

Effect of UV screens and preservatives on vitellogenin and choriogenin production in male medaka (Oryzias latipes).

Ultra violet (UV) screens and preservatives are widely and increasingly used in cosmetics and pharmaceuticals. In the present study, we examined the estrogenicity of 4-methyl-benzylidene camphor (4-MBC), octyl-methoxycinnamate (OMC), and propyl paraben (n-propyl-p-hydroxy-benzoate; PP), among UV screens and preservatives, using male medaka (Oryzias latipes), in regard to production of vitellogenin (VTG) and choriogenin (CHG) which are known to be estrogen-responsive gene products. First, using a VTG enzyme-linked immunosorbent assay (ELISA) system, we determined the increase in VTG plasma concentration in medaka due to exposure to 4-MBC, OMC, and PP, and compared this concentration to the non-treated control. Next, we found increases in mRNA expression levels of VTG subtypes VTG-1 and VTG-2, and CHG subtypes CHG-L and CHG-H, in liver due to exposure to 4-MBC, OMC, and PP compared to the non-treated control. In addition, we also found increased mRNA expression levels of estrogen receptor (ER) alpha, among sex hormone receptors in the liver, due to exposure to 4-MBC, OMC, and PP compared to the non-treated control. In this study, we showed that 4-MBC, OMC, and PP have estrogenic activity in fish.

Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes).

This study was designed to determine the estrogenic effect of the phytoestrogen genistein on several measures of endocrine function in adult Japanese medaka (Oryzias latipes) relative to 17-beta-estradiol. Adult animals of both sexes were exposed to 75, 750 and 30,000 ng/fish (average fish weight equals 0.26 g) of genistein by i.p. injection, with a positive control group treated with 300 ng/fish of 17-beta-estradiol, while a negative control group received a vehicle-only (corn oil) injection. Content of vitellogenin, the yolk glycoprotein made in the liver in response to estradiol stimulation, was measured using Western blots. Circulating estradiol and testosterone levels were measured using a steroid-enzyme immunosorbant assay. The ability of ovaries and testes to synthesize and release estradiol and testosterone was determined by ex vivo incubation of gonads with 25-hydroxycholesterol. Vitellogenin, while induced by 17-beta-estradiol, was not increased in the liver of individuals treated with genistein. In females, genistein treatment at 750 and 30,000 ng increased the estradiol production of ovaries more than the 17-beta-estradiol treatment. In males, genistein treatment resulted in decreased testosterone production from ex vivo testis and a comparable reduction in circulating testosterone level. The changes in vitellogenin, circulating steroids and ex vivo steroidogenesis in medaka in response to genistein are similar to that of 17-beta-estradiol. However, some endpoints are more sensitive to estradiol treatment (vitellogenin), while others are more sensitive to genistein (male testosterone and ovarian estrogenesis).