Medaka Fish Model Research Articles

Reproductive toxicity of DDT in the Japanese medaka fish model: Revisiting the impacts of DDT+ on female reproductive health

The organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) is an endocrine-disrupting compound (EDC) that has been banned by most countries for decades. However, it continues to be detected in nearly all humans and wildlife due to its biological and environmental persistence. The ovarian dysgenesis syndrome hypothesis speculates that exposure to EDCs during sensitive developmental windows such as early gonadal differentiation lead to reproductive disorders later in life. Yet, mechanisms by which DDT affects developing gonads remain unclear due to the inherent challenge of getting developmental exposure data from adults presenting with reproductive disease. The Japanese medaka (Oryzias latipes) is a valuable fish model for sex-specific toxicological studies due to its chromosomal sex determination, external embryonic development, short generation time, and extensively mapped genome. It is well documented that medaka exposed to DDT and its metabolites and byproducts (herein referred to as DDT+) at different developmental time points experience permanent alterations in gonadal morphology, reproductive success, and molecular and hormonal signaling. However, the overwhelming majority of studies focus primarily on functional and morphological outcomes in males and females and have rarely investigated long-term transcriptional or molecular effects. This review summarizes previous experimental findings and the state of our knowledge concerning toxic effects DDT + on reproductive development, fertility, and health in the valuable medaka model. It also identifies gaps in knowledge, emphasizing a need for more focus on molecular mechanisms of ovarian endocrine disruption using enhanced molecular tools that have become increasingly available over the past few decades. Furthermore, DDT forms a myriad of over 45 metabolites and transformation products in biota and the environment, very few of which have been evaluated for environmental abundance or health effects. This reinforces the demand for high throughput and economical in vivo models for predictive toxicology screening, and the Japanese medaka is uniquely positioned to meet this need.

The fungal grid: Fungal communication via electrical signals has inspired the hypothesis of a Wood Wide Web of plants and fungi: Fungal communication via electrical signals has inspired the hypothesis of a Wood Wide Web of plants and fungi.

The observation that soil-dwelling fungi seem to exchange information via electrical impulses has raised new interest about their interactions with plants and their ecological significance.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Functionalized calcium phosphate nanoparticles to direct osteoprotegerin to bone lesion sites in a medaka (Oryzias latipes) osteoporosis model.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

Ubiquitylation of BBSome is required for ciliary assembly and signaling

Bardet‐Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR‐cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf‐like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation‐defective BBS1 mutant (BBS1K143R) affects the trafficking of G‐protein‐coupled receptors (GPCRs) and Shh‐dependent gene transcription. Expression of BBS1K143Rin vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model.

Determination of the Earliest time Point for Heat-shock Inducing Osteoporosis-like Phenotype in the rankl:HSE:CFP Medaka Fish Model for Osteoporosis

The transgenic medaka rankl:HSE:CFP expressing Rankl, a stimulator for osteoclastogenesis - the formation and activation of osteoclasts, bone “eating” cells, under control of a heat inducible promotor has been established as a model for osteoporosis to evaluate antiosteoporosis effects of substances. Transgenic larvae were usually heat-shocked for 90 minutes at 39oC when they were at 9 days post fertilization (dpf) and osteoporosis-like phenotype was analysed when they were at 11 dpf. In this study we investigated whether osteoporosis-like phenotype could be induced in the transgenic larvae when heat-shock was applied at earlier time points, when larvae were at 1, 2, 3, 4, 5, or 6 dpf. Results showed that heat-shocks from the time point of 3 dpf onwards resulted in osteoporosis-like phenotype while heat-shock at 1 or 2 dpf did not affect mineralized bone matrix in 11 dpf larvae. These provide important evidence for study on onset of Rankl induced osteoclasts in fish and help improving experimental procedures using this fish model for osteoporosis.

Stiffness measurement is a biomarker of skin ageing in vivo.

Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin ageing is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin ageing. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through ageing in these models, can be a new biomarker of skin ageing, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin ageing in vivo.

Icariin reduces bone loss in a Rankl-induced transgenic medaka (Oryzias latipes) model for osteoporosis.

Given the limitations and side effects of many synthetic drugs, natural products are an important alternative source for drugs and medications for many diseases. Icariin (ICA), one of the main flavonoids from plants of the Epimedium genus, has been shown to ameliorate osteoporosis and improve bone health in preclinical studies. Those studies have used different in vivo models, mostly rodents, but the underlying mechanisms remain unclear. The present study shows, for the first time, that ICA reduces bone damage in a Rankl-induced medaka fish (Oryzias latipes), a non-rodent osteoporosis model. Live imaging was previously performed in this model to characterize antiresorptive and bone-anabolic properties of drugs. Here, a new quantification method (IM ) was established based on the length of mineralized neural arches to quantify levels of bone mineralization damage and protection in early post-embryonic fish. This method was validated by quantification of three levels of bone damage in three independent Rankl fish lines, and by the determination of different degrees of severity of osteoporosis-like phenotypes in one Rankl line exposed to variable Rankl induction schemes. IM was also used to quantify the efficacy of alendronate and etidronate, two common anti-osteoporotic bisphosphonates, and revealed comparable bone protective effects for ICA and alendronate in this fish osteoporosis model. This study's data support the value of the medaka fish model for bone research and establish a method to screen for novel osteoprotective compounds.

Inhibition of sodium-glucose cotransporter 2 ameliorates renal injury in a novel medaka model of nonalcoholic steatohepatitis-related kidney disease.

The effect of sodium‐glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH‐related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASH‐related kidney disease, which was developed by feeding the d‐rR/Tokyo strain a high‐fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH‐related renal injury and the usefulness of this novel animal model for research into NASH‐related complications.

Effect of histidine on sorafenib-induced vascular damage: Analysis using novel medaka fish model

BackgroundSorafenib (SFN) is an anti-angiogenic chemotherapeutic that prolongs survival of patients with hepatocellular carcinoma (HCC); its side effects, including vascular damages such as hand-foot syndrome (HFS), are a major cause of therapy discontinuation. We previously reported that maintenance of peripheral blood flow by intake of dried bonito broth (DBB) significantly prevented HFS and prolonged the administration period. The amino acids contained in DBB probably contribute to its effects, but the mechanism has not been clarified. We hypothesized that histidine, the largest component among the amino acids contained in DBB, has effects on SFN-induced vascular damage, and evaluated this possibility using a novel medaka fish model. MethodsThe fli::GFP transgenic medaka fish model has a fluorescently visible systemic vasculature. We fed the fish with SFN with and without histidine to compare blood flow and vascular structure among the differently fed models. The vascular cross-sectional area of each fish was measured to determine vascular diameter changes. ResultsOur results demonstrated that SFN-fed medaka developed a narrower vascular diameter. In addition, this narrowing was counteracted by addition of histidine to the medaka diet. We observed no positive effect of histidine on regeneration of cut vessels or on cell growth of endothelial cells and HCC cell lines. ConclusionWe proved the efficacy of the medaka model to assess vascular changes after administration of specific chemicals. And our results suggest that SFN causes vascular damage by narrowing peripheral vessel diameter, and that histidine effectively counteracts these changes to maintain blood flow.

Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo.

Non-coding RNAs provide additional regulatory layers to gene expression as well as the potential to being exploited as therapeutic tools. Non-coding RNA-based therapeutic approaches have been attempted in dominant diseases, however their use for treatment of genetic diseases caused by insufficient gene dosage is currently more challenging. SINEUPs are long antisense non-coding RNAs that up-regulate translation in mammalian cells in a gene-specific manner, although, so far evidence of SINEUP efficacy has only been demonstrated in in vitro systems. We now show that synthetic SINEUPs effectively and specifically increase protein levels of a gene of interest in vivo. We demonstrated that SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human disorder leading to amelioration of the disease phenotype. Our results demonstrate that SINEUPs act through mechanisms conserved among vertebrates and that SINEUP technology can be successfully applied in vivo as a new research and therapeutic tool for gene-specific up-regulation of endogenous functional proteins.

Novel method for analysis of allele specific expression in triploid Oryzias latipes reveals consistent pattern of allele exclusion.

Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance.

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.

Biosensor Medaka for Monitoring Intersex Caused by Estrogenic Chemicals

Estrogenic chemicals can induce intersex in fish species leading to disturbance of spermatogenesis and impairment of reproductive success. To overcome the shortcomings of conventional histopathological observation on intersex (low sensitivity, relatively poor accuracy, long experimental periods, as well as laborious and time-consuming), we generated a pMOSP1-EGFP transgenic medaka fish model. In this transgenic fish, the green fluorescence protein (GFP) reporter gene was derived by the regulatory elements of the OSP1 gene, which is a specific and sensitive molecular biomarker for indicating intersex occurrence in male medaka fish exposed to estrogenic chemicals. The transgenic GFP was faithfully expressed in ovaries and in testes with intersex, perfectly mimicking the expression pattern of endogenous OSP1. In intersex testis, the diameters of primary oocytes which could be distinguished by GFP fluorescence observation were as small as 10 μm, lower than that (more than 20 μm) which is observable by histopathology. Using the novel transgenic medaka fish, intersex was observed after 90-day exposure to 0.75 ng/L 17α-ethinyloestradiol (EE2) (0-90 dph), but only at concentrations of at least 1.38 ng/L EE2 by histopathology. An effectiveness of a short-term in vivo assay for screening estrogenic chemicals that can monitor intersex appearance at early sex developmental stage (about 30 dph) in male medaka fish was also demonstrated by assessing the intersex induction of EE2, 17β-estradiol and 4-nonylphenol. This newly developed assay provides an enhanced ability for screening and testing estrogenic chemicals with the potential to induce intersex and studying their biological impacts.

Proteomic modification in gills and brains of medaka fish ( Oryzias melastigma) after exposure to a sodium channel activator neurotoxin, brevetoxin-1

Although brevetoxins (PbTxs) produced by the marine dinoflagellate Karenia brevis are known to be absorbed across gill membranes and exert their acute toxic effects through an ion-channel mediated pathway in neural tissue, the exact biochemical mechanism concerning PbTxs neurotoxicity in neural tissue and gas-exchange organs has not been well elucidated. In this study, we calculated the LC 50 value of PbTx-1 using the medaka fish model, and presented the molecular responses of sub-acute exposure to PbTx-1 with proteomic method. By adopting two-dimensional electrophoresis, the abundances of 14 and 24 proteins were found to be remarkably altered in the gills and brains, respectively, in response to toxin exposure. Thirteen gill and twenty brain proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis. These proteins could be categorized into diverse functional classes such as cell structure, macromolecule metabolism, signal transduction and neurotransmitter release. These findings can help to elucidate the possible pathways by which aquatic toxins affect marine organisms within target organs.

Sperm Nuclear Transfer and Transgenic Production in the Fish Medaka

Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.

Generation of Medaka Fish Haploid Embryonic Stem Cells

Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. Here, we generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Hence, haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Mosaic oocytes created by combining a mitotic nucleus and a meiotic nucleus can generate fertile fish offspring. Haploid ES cells may offer a yeast-like system for analyzing recessive phenotypes in numerous cell lineages of vertebrates in vitro.

Induction of ras oncogene mutations and hepatocarcinogenesis in medaka ( Oryzias latipes) exposed to diethylnitrosamine

Medaka ( Oryzias latipes), a fish model for assessing the carcinogenic impact of environmental exposure to genotoxic chemicals, were subjected to 390 ppm of diethylnitrosamine (DEN) in an aqueous bath for 48 h. Histological examinations, at 5 and 7 months post-exposure, revealed that 11 of 17 fish developed hepatic neoplasms (hepatocellular carcinomas, cholangiomas and mixed hepato-cholangiocellular carcinomas). Genomic DNA extracted from the livers of normal medaka was used to obtain and sequence the structure of ras gene exon 1 and exon 2. Comparing PCR-amplified products from exon 1 and 2 by hybridization with specific mutation containing probes and/or by sequencing of liver DNA from control and exposed fish revealed an association of ras oncogene mutations and DEN-induced tumor formation. While none of the control fish showed any mutations, three of 11 fish with tumors had ras gene mutations. All three fish had mutations at codon 12 replacing glycine with aspartic acid, while one fish had an additional mutation at codon 16 replacing lysine with threonine. In addition to identifying DEN-induced mutations at codon 12 of the medaka ras oncogene, the present study represents a first report of an in vivo association of hepatocarcinogenesis, in any species, with a mutation at codon 16. Mutation at codon 16 and tumorigenesis has been previously predicted and artificially produced. Our study also supports the relevance of the medaka fish model for in vivo detection of the carcinogenic potential of environmental pollutants in sediments and biota of ambient water systems.