Gsdf is not indispensable for male differentiation in the medaka species Oryzias hubbsi.

Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.

High-fat diet in early life triggers both reversible and persistent epigenetic changes in the medaka fish (Oryzias latipes).

BACKGROUND: The nutritional status during early life can have enduring effects on an animal's metabolism, although the mechanisms underlying these long-term effects are still unclear. Epigenetic modifications are considered a prime candidate mechanism for encoding early-life nutritional memories during this critical developmental period. However, the extent to which these epigenetic changes occur and persist over time remains uncertain, in part due to challenges associated with directly stimulating the fetus with specific nutrients in viviparous mammalian systems. RESULTS: In this study, we used medaka as an oviparous vertebrate model to establish an early-life high-fat diet (HFD) model. Larvae were fed with HFD from the hatching stages (one week after fertilization) for six weeks, followed by normal chow (NC) for eight weeks until the adult stage. We examined the changes in the transcriptomic and epigenetic state of the liver over this period. We found that HFD induces simple liver steatosis, accompanied by drastic changes in the hepatic transcriptome, chromatin accessibility, and histone modifications, especially in metabolic genes. These changes were largely reversed after the long-term NC, demonstrating the high plasticity of the epigenetic state in hepatocytes. However, we found a certain number of genomic loci showing non-reversible epigenetic changes, especially around genes related to cell signaling, liver fibrosis, and hepatocellular carcinoma, implying persistent changes in the cellular state of the liver triggered by early-life HFD feeding. CONCLUSION: In summary, our data show that early-life HFD feeding triggers both reversible and persistent epigenetic changes in medaka hepatocytes. Our data provide novel insights into the epigenetic mechanism of nutritional programming and a comprehensive atlas of the long-term epigenetic state in an early-life HFD model of non-mammalian vertebrates.

Adenylate kinase 1 overexpression increases locomotor activity in medaka fish.

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

Contribution of sox9b to pigment cell formation in medaka fish.

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a-/- ; sox10b-/- double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b-/- ; sox10a-/- and sox10a-/- ; sox10b-/- double mutants, but their numbers were significantly reduced in the sox9b-/- ; sox10a-/- mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.

Developmental toxicity in marine medaka (Oryzias melastigma) embryos and larvae exposed to nickel.

As an important trace metal, nickel (Ni) has been reported extensively in the studies on freshwater animals. However, the toxic effects of Ni on marine organisms are not clearly understood. Therefore, in order to investigate the toxic effects of Ni on the early development of marine fish, the marine medaka (Oryzias melastigma) embryos and larvae were immersed in 0.13-65.80 mg/L Ni solution. The results showed that Ni exposure changed the egg size and heart rate of the embryos, lowered the hatchability, increased the deformity rate, and shortened the total body length of newly hatched larvae. Besides, it was found that before organogenesis and post-hatching periods were the sensitive periods of embryos to Ni. The 25 d LC50 value of embryos was 49.28 mg/L, and the 5 d LC50 of larvae was 55.92 mg/L, indicating that the embryos were more sensitive to Ni than the larvae. Furthermore, the expressions of the metallothionein (MT) gene, the skeletal development-related gene (Cyp26b1) and the cardiac development-related genes (ATPase, smyd1, cox2 and bmp4) were determined, and the results showed that the expressions of ATPase and smyd1 were up-regulated, while MT, Cyp26b1 and cox2 were significantly down-regulated at 9 days post-fertilization (dpf). Overall, Ni exposure caused a significant toxic effect on the early development of the O. melastigma embryos and larvae. Our findings could provide an important supplement to the toxicity data of tropical Ni and provide a reference for the exploration of the molecular mechanisms of Ni toxicity.

Transcriptome analysis provides insights into long noncoding RNAs in medaka gonads.

Long non-coding RNAs (lncRNAs) are gradually regarded as regulators in sex determination and gonad development of various animals. Medaka (Oryzias latipes) is an excellent reproductive research model with sex-determining genes. However, the regulation of gonadal lncRNAs on medaka reproductive development remains unknown. Here, 5317 lncRNAs were obtained from medaka ovary and testis by Illumina HiSeq4000, among which 177 lncRNAs were up-regulated and 120 lncRNAs were down-regulated in the testis compared to the ovary. In addition, 6904 cis-regulated target genes were predicted from 3099 lncRNAs. GO and KEGG enrichment analysis showed that these target genes were mainly involved in phosphorylation, metabolic, metabolism of xenobiotics by cytochrome P450, insulin secretion, and GnRH signaling pathways. Furthermore, six highly expressed lncRNAs were randomly selected to verify the sequencing data by quantitative real time PCR (qRT-PCR). Next, in situ hybridization revealed that one of the sex-biased lncRNA MSTRG.14827.1 was highly expressed in immature germ cells, indicating MSTRG.14827.1 may play a key role in gametogenesis. Taken together, this study provides emerging lncRNA libraries and opens new avenues for future investigation of lncRNAs in medaka.

The endocannabinoid system and retinoic acid signaling combine to influence bone growth.

Osteoporosis is an increasing burden on public health as the world-wide population ages and effective therapeutics are severely needed. Two pathways with high potential for osteoporosis treatment are the retinoic acid (RA) and endocannabinoid system (ECS) signaling pathways. We sought to elucidate the roles that these pathways play in bone development and maturation. Here, we use chemical treatments to modulate the RA and ECS pathways at distinct early, intermediate, and late times bone development in zebrafish. We further assessed osteoclast activity later in zebrafish and medaka. Finally, by combining sub-optimal doses of AR and ECS modulators, we show that enhancing RA signaling or reducing the ECS promote bone formation and decrease osteoclast abundance and activity. These data demonstrate that RA signaling and the ECS can be combined as sub-optimal doses to influence bone growth and may be key targets for potential therapeutics.

Igf signaling couples retina growth with body growth by modulating progenitor cell division.

How the body and organs balance their relative growth is of key importance for coordinating size and function. This is of particular relevance in organisms, which continue to grow over their entire life span. We addressed this issue in the neuroretina of medaka fish (Oryzias latipes), a well-studied system with which to address vertebrate organ growth. We reveal that a central growth regulator, Igf1 receptor (Igf1r), is necessary and sufficient for proliferation control in the postembryonic retinal stem cell niche: the ciliary marginal zone (CMZ). Targeted activation of Igf1r signaling in the CMZ uncouples neuroretina growth from body size control, and we demonstrate that Igf1r operates on progenitor cells, stimulating their proliferation. Activation of Igf1r signaling increases retinal size while preserving its structural integrity, revealing a modular organization in which progenitor differentiation and neurogenesis are self-organized and highly regulated. Our findings position Igf signaling as a key module for controlling retinal size and composition, with important evolutionary implications.

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka.

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that ndrg1b is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.

Inhibition of swim bladder inflation in Japanese medaka (Oryzias latipes) embryos following exposure to select pharmaceuticals alone and in combination.

This study leveraged the Japanese medaka fish embryo model for the assessment of effects of select contaminants on early development in fish. Fish embryos were exposed to various pharmaceutical contaminants including synthetic hormones and non-steroidal anti-inflammatory drugs and their effects on development were observed. Initial screening determined that swim bladder inflation failure was the most common endpoint detected. Swim bladder inflation failure was first explored in a study demonstrating that medaka require access to the air-water interphase to inflate their swim bladders in a time-dependent manner, and swim bladder inflation failure was correlated with mortality. Fish embryos were exposed 24-hours post fertilization until hatch to concentration ranges of various pharmaceutical contaminants including: 17ß-estradiol, 17α-ethinylestradiol, and levonorgestrel (1 to 1000 µg/L), or diclofenac (0.32 to 100 mg/L). The main effect observed across all four compounds was a significant increase in failure of swim bladder inflation with increasing exposure concentration (24 to 72-hours post-hatch). Following single compound experiments combinatorial exposures using no-observed-effect concentrations were conducted. The main effect observed was a significant decrease in inflation success 24-hours post-hatch following a binary mixture of levonorgestrel and 17α-ethinylestradiol, as well as a significant decrease in swim bladder inflation success at all times following exposure to a quaternary mixture of all four compounds. This study demonstrated that embryonic exposure to pharmaceutical compounds, both alone and in combination, resulted in failure of swim bladder inflation in larval Japanese medaka.

Stage-dependent and regioselective toxicity of 2- and 6-hydroxychrysene during Japanese medaka embryogenesis.

Exposure to oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene being more potent than 6-hydroxychrysene. To better characterize this phenomenon, embryos of Japanese medaka (Oryzias latipes) were exposed to 2- or 6-hydroxychrysene (0.5, 2, or 5 µM) from 4 h-post-fertilization (hpf) to 7 d-post-fertilization. Following exposure, hemoglobin concentrations were quantified by staining fixed embryos with o-dianisidine (a hemoglobin-specific dye) and stained embryos were imaged using brightfield microscopy. Exposure to 2-hydroxychrysene resulted in a concentration-dependent decrease in hemoglobin relative to vehicle-exposed embryos, while only the highest concentration of 6-hydroxychrysene resulted in a significant decrease in hemoglobin. All tested concentrations of 2-hydroxychrysene also caused significant mortality (12.2 % ± 2.94, 38.9 % ± 14.4, 85.6 % ± 11.3), whereas mortality was not observed following exposure to 6-hydroxychrysene. Therefore, treatment of embryos with 2-hydroxychrysene at various developmental stages and durations was subsequently conducted to identify key developmental landmarks that may be targeted by 2-hydroxychrysene. A sensitive window of developmental toxicity to 2-hydroxychrysene was found between 52-100 hpf, with a 24 h exposure to 10 µM 2-hydroxychrysene resulting in significant anemia and mortality. Since exposure to 2-hydroxychrysene from 52 to 100 hpf, a window that includes liver morphogenesis in medaka, resulted in the highest magnitude of toxicity, liver development and function may have a role in 2-hydroxychrysene developmental toxicity.

Toxicity effects of hydrophilic algal lysates from Coolia tropicalis on marine medaka larvae (Oryzias melastigma).

Coolia tropicalis is a species of benthic and epiphytic toxic algae, which can produce phycotoxins that intoxicate marine fauna. In this study, the potential toxic effects of C. tropicalis on fish were investigated using larval marine medaka (Oryzias melastigma) as a model to evaluate fish behavior, physiological performance, and stress-induced molecular responses to exposure to two sublethal concentrations (LC10 and LC20) of hydrophilic algal lysates. Exposure to C. tropicalis lysates inhibited swimming activity, activated spontaneous undirected locomotion, altered nerve length ration, and induced early development abnormalities, such as shorter eye diameter, body as well as axon length. Consistent with these abnormalities, changes in the expression of genes associated with apoptosis (CASPASE-3 and BCL-2), the inflammatory response (IL-1ß and COX-2), oxidative stress (SOD), and energy metabolism (ACHE and VHA), were also observed. This study advances our understanding of the mechanisms of C. tropicalis toxicity in marine fish in the early life stages and contributes to future ecological risk assessments of toxic benthic dinoflagellates.

Exposure to 4-nonylphenol induces a shift in the gene expression of gsdf and testis-ova formation and sex reversal in Japanese medaka (Oryzias latipes).

The branched isomer mixture 4-nonylphenol (4-NP) has been used worldwide as a surfactant, and can have endocrine-disrupting effects on aquatic organisms. For instance, 4-NP induces the formation of testis-ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine-disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4-NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 µg/L 4-NP, testis-ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4-NP exposure at 1-100 µg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 µg/L-exposed group was significantly lower than that in the controls. The 4-NP concentration at which gsdf expression was suppressed was equal to that at which testis-ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.

Bucky ball induces primordial germ cell increase in medaka.

Balbaini body (Bb) plays a vital role in germ plasm (GP) assembly and dorsoventral pattern, which is of critical important in germline specification and development. Bucky ball (buc) is reported to be essential for boosting primordial germ cell (PGC) through Bb in previous research. In the present study, a buc homolog (Olbuc) was identified in medaka (Oryzias latipes), and the roles of Olbuc on PGC development were further elucidated. The full length of Olbuc was 2148 bp, which contains a 1724 bp CDS (Coding sequence), a 167 bp 5' UTR (Untranslated region), and a 257 bp 3' UTR. By RT-PCR, the Olbuc RNA expression was maternally provided during embryogenesis and was restricted in the ovary of adult tissues. By in situ hybridization, Olbuc RNA was abundant in oocyte of meiotic stage, but gradually decreased as the oogenesis proceeded. Surprisingly, Olbuc was not co-localized with dazl, the marker gene of Bb. Interestingly, GFP can be specifically and stably expressed through the induction of Olbuc 3'UTR in PGCs. Furthermore, overexpression of Olbuc mRNA could increase PGC number and generate ectopic PGC in medaka and zebrafish embryos. In summary, our results showed that Olbuc performs a conserved function in PGC development in medaka.

Silver nanocolloid affects hindbrain vascular formation during medaka embryogenesis.

Angiogenesis is essential for the normal development of an embryo. Silver nanocolloid (SNC) is known to induce vascular malformation in the medaka embryo. We focused on the development of the central arteries (CtAs) in the hindbrain of Japanese medaka. The CtAs and the basilar artery from which they branch are essential for transporting the blood and nutrients necessary to support the hindbrain parenchyma and the development of the pons and cerebellum from the hindbrain. We exposed medaka embryos at developmental stage 21 (6 somite stage), to 0, 0.5, 5, or 10 mg/L SNC and evaluated hatching rate, number of thrombi per embryo, head size (length and width), body length, and angiogenesis. Although all SNC-exposed embryos hatched, their head size and body length were small in comparison to controls; in addition, the number of thrombi in the head increased and head size and body length decreased as the SNC concentration increased. To evaluate vasculogenic abnormalities, we performed whole-mount in situ hybridization using a vascular marker (eg, fl7) and visualized the CtAs in medaka embryos. In control embryos, CtAs started to sprout at stage 32 (somite completion stage) and their extension was complete by stage 35 (pectoral fin blood circulation stage). In contrast, CtAs failed to sprout in SNC-exposed embryos, and thrombi were present. Furthermore, qRT-PCR analysis showed that SNC significantly suppressed the egfl7 expression level at stage 35. Together, our findings suggest that SNC induced decreased developments of head and body in medaka embryos due to insufficient angiogenesis and hindbrain vascular formation.

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)-a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor ß superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Teratogenic effects of environmentally relevant concentrations of phenanthrene on the early development of marine medaka (Oryzia melastigma).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in marine environments and have arouse great concern since they pose adverse effects to marine ecosystem. To determine the potential impacts of environmentally relevant PAHs on early life stages of marine fish, this study exposed embryos of marine medaka (Oryzias melastigma) to 0, 2, 10, 50, and 250 µg/L of phenanthrene (Phe), one of the most abundant PAHs. The results demonstrated that Phe exposure decreased hatching rates, delayed hatching time of embryos, and increased deformity rate of newly-hatched larvae. Exposure to 10 and 50 µg/L Phe decreased the survival rate of marine medaka larvae at 28 days post-fertilization (dpf), and no embryo successfully hatched in 250 µg/L Phe exposure group. Morphology results showed that 10, 50, and 250 µg/L Phe exposure significantly retarded the development of embryos, and 2, 10, and 50 µg/L caused yolk sac edema and pericardial edema in newly-hatched larvae, indicating that low concentrations of Phe could induce developmental cardiac toxicity. Furthermore, the changes in the expression of heart development-related genes were determined, and the results showed that Phe-induced cardiac malformation might be related with fgf8, bmp4, smyd1, ATPase and gata4 genes. Overall, environmentally relevant PAHs could disrupt heart morphogenesis and hatching process of marine medaka, which might have profound consequences for sustainability of fish population.

Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model.

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.

Peroxisome proliferator-activated receptor alpha is involved in the temperature-induced sex differentiation of a vertebrate.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Establishment of a Japanese medaka (Oryzias latipes) transgenic line expressing Takifugu rubripes pufferfish saxitoxin and tetrodotoxin binding protein 1, and evaluation of tributyltin toxicity via in ovo nanoinjection.

Pufferfish saxitoxin and tetrodotoxin binding proteins (PSTBPs) play an important role in the toxification of certain species of pufferfish. Recombinant Takifugu rubripes PSTBP1 (rTrub.PSTBP1) is reported to bind to tributyltin, and so it has been suggested that rTrub.PSTBP1 may reduce the toxicity of tributyltin. However, the role of PSTBP1 in vivo remains to be elucidated. Here, we established a transgenic medaka line showing whole-body Renilla reniformis green fluorescent protein and Trub.PSTBP1 expression, as confirmed by real-time polymerase chain reaction and mRNA-Seq analysis. mRNA-Seq analysis also showed that cytochrome P450 superfamily genes and the gene encoding ATP-binding cassette sub-family G member 2 were highly expressed in the transgenic medaka. Using embryos of the transgenic medaka line, we conducted an in ovo nanoinjection test to examine the effect of Trub.PSTBP1 in vivo, and obtained data suggesting that Trub.PSTBP1 expression may have reduced the toxicity of tributyltin in our transgenic medaka line. Our findings will be useful for future functional analyses of Trub.PSTBP1.