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To investigate whether environmental concentrations of fragmented polyethylene terephthalate (PET) microplastics (MPs) have additional or combined effects on endocrine-disrupting activity, Java medaka (Oryzias javanicus) were exposed to 17ß-estradiol (E2; 5, 10, 50, and 100 ng L-1), bisphenol A (BPA; 5, 10, 50, and 100 µg L-1), and E2 and BPA combined with PET MPs (1 and 100 particles L-1) for 200 days. The growth parameters, such as body length and weight, were significantly decreased by the highest concentrations of E2 and BPA. A significant reduction in egg production was observed in female fish exposed to BPA, with an additive toxic effect of PET MPs. A female-biased sex ratio was observed in fish exposed to both chemicals. Exposure to E2 significantly increased the hepatosomatic index (HSI) in both sexes, while no significant effect was observed in the gonadosomatic index (GSI). Exposure to BPA significantly increased the HSI in female fish and decreased the GSI in both sexes of fish. An additive effect of PET MPs was observed on the GSI value of female exposed to BPA. Significant elevations in vitellogenin (VTG) levels were observed in both sexes due to exposure to E2 and BPA. Additive effects of PET MPs were observed on VTG levels in males exposed to E2 and BPA. Taken together, even long-term treatment with PET MPs induced only a negligible additive effect on the endocrine-disrupting activity in Java medaka at environmentally relevant concentrations.
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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants generally found in complex mixtures. PAHs are known to cause pleiotropic effects on living organisms, including developmental defects, mutagenicity, carcinogenicity and immunotoxicity, and endocrine disruptions. The main goal of this study is to evaluate the toxicity of water-accommodated fractions (WAFs) of oils in two life stages of the Japanese medaka, larvae and juveniles. The deleterious effects of an acute exposure of 48 h to two WAFs from Arabian light crude oil (LO) and refined oil from Erika (HO) were analyzed in both stages. Relevant endpoints, including ethoxy resorufin-O-deethylase (EROD) activity, DNA damage (Comet assay), photomotor response, and sensitivity to nervous necrosis virus (NNV) infection, were investigated. Larvae exposed to both oil WAFs displayed a significant induction of EROD activity, DNA damage, and developmental anomalies, but no behavioral changes. Deleterious effects were significantly increased following exposure to 1 and 10 µg/L of LO WAFs and 10 µg/L of HO WAFs. Larval infection to NNV induced fish mortality and sharply reduced reaction to light stimulation. Co-exposure to WAFs and NNV increased the mortality rate, suggesting an impact of WAFs on fish defense capacities. WAF toxicity on juveniles was only observed following the NNV challenge, with a higher sensitivity to HO WAFs than to LO WAFs. This study highlighted that environmentally realistic exposure to oil WAFs containing different compositions and concentrations of oil generated high adverse effects, especially in the larval stage. This kind of multi-marker approach is particularly relevant to characterize the toxicity fingerprint of environmental mixtures of hydrocarbons and PAHs.
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Carbaryl is widely used as a highly effective insecticide which harms the marine environment. This study aimed to assess the reproductive toxicity of chronic carbaryl exposure on female marine medaka and their female offspring. After a 180-day exposure from embryonic period to adulthood, females exhibited reduced attraction to males, decreased ovulation, increased gonadosomatic index and a higher proportion of mature and atretic follicles. These reproductive toxic effects of carbaryl may stem from changes in hormone levels and transcription levels of key genes along the HPG axis. Furthermore, maternal carbaryl exposure had detrimental effects on the offspring. F1 females showed the reproductive disorders similar to those observed in F0 females. The significant changes in the transcription levels of DNA methyltransferase and demethylase genes in the F0 and F1 generations of ovaries indicate changes in their DNA methylation levels. The changes in DNA methylation levels in F1 female marine medaka may lead to changes in the expression of certain reproductive key genes, such as an increase in the transcription level of cyp19a, which may be the reason for F1 reproductive toxicity. These findings indicate that maternal exposure may induce severe generational toxicity through alterations in DNA methylation levels. This study assesses the negative impacts of whole life-cycle carbaryl exposure on the reproductive and developmental processes of female marine medaka and its female offspring, while offering data to support the evaluation of the ecological risk posed by carbaryl in marine ecosystems.
Assuntos
Carbaril , Inseticidas , Oryzias , Reprodução , Poluentes Químicos da Água , Animais , Oryzias/fisiologia , Feminino , Carbaril/toxicidade , Reprodução/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Inseticidas/toxicidade , Exposição Materna/efeitos adversos , Metilação de DNA/efeitos dos fármacos , MasculinoRESUMO
Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.
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Digestão , Técnicas de Inativação de Genes , Larva , Oryzias , Receptores de Neuropeptídeo Y , Animais , Oryzias/genética , Receptores de Neuropeptídeo Y/genética , Larva/genética , Sistemas CRISPR-Cas , Comportamento Alimentar , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismoRESUMO
Bisphenol-A (BPA), a known endocrine-disrupting chemical (EDC) in plastics and resins, has been found to induce heritable health effects in fish and mammals, affecting directly exposed individuals and indirectly their progenies in subsequent generations. It is not clearly understood if subsequent generations of the BPA-exposed ancestors have increased sensitivity to the second hit by the chemicals of emerging concern. To understand this, the present study examined the effects of developmental exposure to perfluorooctanesulfonic acid (PFOS), which has been a global contaminant recently, in embryos whose ancestors were exposed to BPA. Two lineages of medaka (Oryzias latipes) were established: 1) the BPA lineage in which the F0 generation was exposed to 10 µg/L BPA during early development and 2) the control lineage with no BPA exposure in the F0 generation. These lineages were raised up to the F4 generation without further exposure. The embryos of the F4 generation were exposed to PFOS at 0, 0.002, 0.02, 0.2, 2, and 20 mg/L concentrations. Early developmental defects resulting in mortality, delayed hatching, teratogenic phenotypes, and altered gene expression were examined in both lineages. The expression level of genes encoding DNA methyltransferases and genes responsible for oxidative stress defense were determined. Following environmentally relevant PFOS exposure, organisms with a history of BPA exposure displayed significant changes in all categories of developmental defects mentioned above, including increased expression of genes related to oxidative stress, compared to individuals without BPA exposure. The present study provides initial evidence that a history of ancestral BPA exposure can alter sensitivity to developmental disorders following the second hit by PFOS exposure. The variable of ancestral BPA exposure could be considered in mechanistic, medical, and regulatory toxicology, and can also be applied to holistic environmental equity research.
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Ácidos Alcanossulfônicos , Compostos Benzidrílicos , Embrião não Mamífero , Disruptores Endócrinos , Fluorocarbonos , Oryzias , Fenóis , Poluentes Químicos da Água , Animais , Oryzias/embriologia , Fluorocarbonos/toxicidade , Ácidos Alcanossulfônicos/toxicidade , Fenóis/toxicidade , Compostos Benzidrílicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Poluentes Químicos da Água/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
Polycyclic aromatic hydrocarbons (PAHs) accumulate and integrate into aquatic environments, raising concerns about the well-being and safety of aquatic ecosystems. Benzo[a]pyrene (BaP), a persistent PAH commonly detected in the environment, has been extensively studied. However, the broader multifaceted toxicity potential of BaP on the early life stages of marine fish during chronic exposure to environmentally relevant concentrations needs further exploration. To fill these knowledge gaps, this study assessed the in vivo biotoxicity of BaP (1, 4, and 8 µg/L) in marine medaka (Oryzias melastigma) during early development over a 30-day exposure period. The investigation included morphological, biochemical, and molecular-level analyses to capture the broader potential of BaP toxicity. Morphological analyses showed that exposure to BaP resulted in skeletal curvatures, heart anomalies, growth retardation, elevated mortality, delayed and reduced hatching rates. Biochemical analyses revealed that BaP exposure not only created oxidative stress but also disrupted the activities of antioxidant enzymes. This disturbance in redox balance was further explored by molecular level investigation. The transcriptional profiles revealed impaired oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle pathways, which potentially inhibited the oxidative respiratory chain in fish following exposure to BaP, and reduced the production of adenosine triphosphate (ATP) and succinate dehydrogenase (SDH). Furthermore, this investigation indicated a potential connection to apoptosis, as demonstrated by fluorescence microscopy and histological analyses, and supported by an increase in the expression levels of related genes via real-time quantitative PCR. This study enhances our understanding of the molecular-level impacts of BaP's multifaceted toxicity in the early life stages of marine medaka, and the associated risks.
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Benzo(a)pireno , Oryzias , Oxirredução , Poluentes Químicos da Água , Animais , Benzo(a)pireno/toxicidade , Oryzias/genética , Poluentes Químicos da Água/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacosRESUMO
Gills and gut are the two primary osmoregulatory organs in fish. Recently, studies have expanded beyond the osmoregulatory mechanisms of these organs to explore the microbiota communities inhabiting them. It is now known that microbial communities in both organs shift in response to osmotic stress. However, there are limited studies identifying the major contributors and co-occurrence among these microbiota in both organs under seawater and freshwater transfer conditions. The current data mining report performed a bioinformatics analysis on two previous published datasets from our group, aiming to provide insights into host-bacteria relationships under osmotic stress. We divided the samples into four groups: control seawater gills (LSW); control seawater gut (TSW); freshwater transfer gills (LFW); and freshwater transfer gut (TFW). Our results showed that LSW had higher diversities, richness, and evenness compared to TSW. However, both the LFW and LSW did not show any significant differences after the freshwater transfer experiment. We further applied co-occurrence network analysis and, for the first time, reported on the interactions of taxa shaping the community structure in these two organs. Moreover, we identified enriched ectoine biosynthesis in seawater samples, suggesting its potential role in seawater environments. Increased mRNA expression levels of Na+/K+-atpase, and cftr, were observed in gills after 6 h of ectoine treatment. These findings provide a foundation for future studies on host-bacteria interactions under osmotic stress.
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The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish that were not directly exposed, but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% were downregulated, and 20% were upregulated. Of all DEGs, 52.31% of DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared the majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.
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Naphthenic acids (NAs) are important pollutants in marine crude oils and have obvious toxic effects on marine organisms. However, the effects of NAs on the intestine are largely unknown. Thus, we evaluated the effects of NAs exposure in the intestines of marine medaka. Fish were experimentally exposed to NAs (0.5 mg/L, 5 mg/L, and 10 mg/L) for 96 h and monitored for changes in intestinal histology, markers of oxidative stress, and intestinal microbiome responses. Significant mucosal damage, inflammation, and oxidative stress were observed in the intestines of marine medaka after exposure to NAs. In addition, significant changes in the gut microbiota were observed. Specifically, the relative abundance of Proteobacteria decreased, while that of Verrucomicrobiota increased in the high-concentration exposure group. In addition, nutrient synthesis and metabolism in the gut were affected. The results of this study contribute to a better understanding of the ecological risk of different concentrations of NAs to marine organisms. CAPSULE ABSTRACT: Changes in the gut microbial community of marine medaka (Oryzias melastigma) caused by naphthenic acids in the marine environment were investigated through the assessment of gut inflammatory factors and comprehensive analysis using 16S rDNA high-throughput sequencing. The results indicated the induction of intestinal inflammation and changes in the structural composition of the intestinal flora.
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Ácidos Carboxílicos , Disbiose , Microbioma Gastrointestinal , Intestinos , Oryzias , Poluentes Químicos da Água , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Ácidos Carboxílicos/toxicidade , Disbiose/veterinária , Disbiose/induzido quimicamente , Intestinos/efeitos dos fármacos , Intestinos/patologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
BACKGROUND: Chinese medaka (Oryzias sinensis) is widely distributed in freshwater rivers in China. Similar to the medaka (Oryzias latipes), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear. METHODS: Chinese medaka dmrt1 (Osdmrt1) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Osdmrt1)] overexpressing Osdmrt1 were generated to investigate the role of Osdmrt1 in sex determination. Western blot was used to validate the integration of the Osdmrt1 into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Osdmrt1) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes. RESULTS: Osdmrt1 was cloned and identified, and it shared similar evolutionary relationships with medaka dmrt1. Tg(CMV:Osdmrt1) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males. CONCLUSION: The Chinese medaka dmrt1 gene could convert females to males in medaka. GENERAL SIGNIFICANCE: These results not only elucidate the function of Chinese medaka dmrt1, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.
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Animais Geneticamente Modificados , Oryzias , Fatores de Transcrição , Animais , Oryzias/genética , Feminino , Masculino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Processos de Determinação Sexual/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , População do Leste AsiáticoRESUMO
Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.
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Animais Geneticamente Modificados , Edwardsiella , Infecções por Enterobacteriaceae , Doenças dos Peixes , Macrófagos , Oryzias , Fagocitose , Animais , Oryzias/genética , Macrófagos/microbiologia , Macrófagos/metabolismo , Macrófagos/imunologia , Edwardsiella/genética , Infecções por Enterobacteriaceae/veterinária , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/imunologia , Larva/microbiologia , Larva/genética , Larva/imunologiaRESUMO
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.
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Oryzias , Diferenciação Sexual , Animais , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Masculino , Diferenciação Sexual/genética , Feminino , Processos de Determinação Sexual/genética , Testículo/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proliferação de Células , Diferenciação Celular/genética , Células Germinativas/metabolismo , Células Germinativas/citologia , Meiose/genéticaRESUMO
Organophosphorus pesticides (OPs), such as chlorpyrifos (CPF), are the most commonly used pesticides worldwide. Considering that OPs will eventually enter aquatic ecosystems due to runoff from agricultural lands, accidental leakage, and other unforeseen emergencies, monitoring water pollution of those substances is crucial for environmental protection and public health. In this study, Japanese medaka (Oryzias latipes) were exposed to CPF (0.03, 0.06, and 0.12 mg/L) for 6 h, and the time-series variations in their locomotor behavior and vocal traits were investigated. Compared with that measured before exposure, significantly changed locomotor behavior and vocal traits in Japanese medaka exposed to CPF could be observed at 4 h after exposure and thereafter, and the pattern of behavioral changes depends on the CPF concentrations. Exposure to CPF also changed the frequency-sound pressure level curve of Japanese medaka at 6 h after exposure, especially at 0.12 mg/L. Moreover, CPF exposure could significantly inhibit the acetylcholinesterase (AChE) activity in the brains and eyes of medaka, which exhibited significant correlations with the variation of locomotor behavioral and vocal traits. Considering that inhibiting the AChE activity is the primary mechanism underlying the neurobehavioral toxicity of all OPs, our finding suggested that simultaneously monitoring changes in the locomotor behavioral and vocal traits has a high potential to reflect the pollution of organophosphorus substances.
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Clorpirifos , Locomoção , Oryzias , Poluentes Químicos da Água , Animais , Oryzias/fisiologia , Clorpirifos/toxicidade , Poluentes Químicos da Água/toxicidade , Locomoção/efeitos dos fármacos , Vocalização Animal/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Inseticidas/toxicidade , Praguicidas/toxicidade , Comportamento Animal/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Encéfalo/efeitos dos fármacosRESUMO
Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.
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Criopreservação , Oryzias , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Animais , Oryzias/fisiologia , Masculino , Criopreservação/métodos , Espermatozoides/fisiologia , Preservação do Sêmen/métodos , Feminino , Crioprotetores/farmacologiaRESUMO
The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.
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The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.
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The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish who were not directly exposed but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% DEGs were downregulated and 20% DEGs were upregulated. Off all DEGs, 52.31% DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.
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Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.
Assuntos
Motilina , Oryzias , Receptores dos Hormônios Gastrointestinais , Animais , Oryzias/metabolismo , Oryzias/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Motilina/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Animais Geneticamente Modificados , Neurônios Dopaminérgicos/metabolismo , Encéfalo/metabolismoRESUMO
Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4â³4 (+4 bp) and OlNectin4â³7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Nectinas , Nodaviridae , Oryzias , Infecções por Vírus de RNA , Animais , Oryzias/genética , Oryzias/imunologia , Nodaviridae/fisiologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/imunologia , Nectinas/genética , Nectinas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Filogenia , Sequência de Aminoácidos , Imunidade Inata/genética , Alinhamento de Sequência/veterinária , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterináriaRESUMO
Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.