Effects of Acute and Developmental Exposure to Bisphenol S on Chinese Medaka (Oryzias sinensis).

Bisphenol S (BPS), one of the substitutes for bisphenol A (BPA), is widely used in various commodities. The BPS concentrations in surface water have gradually increased in recent years, making it a predominant bisphenol analogue in the aquatic environment and raising concerns about its health and ecological effects on aquatic organisms. For this study, we conducted a 96 h acute toxicity test and a 15-day developmental exposure test to assess the adverse effects of BPS exposure in Chinese medaka (Oryzias sinensis), a new local aquatic animal model. The results indicate that the acute exposure of Chinese medaka embryos to BPS led to relatively low toxicity. However, developmental exposure to BPS was found to cause developmental abnormalities, such as decreased hatching rate and body length, at 15 dpf. A transcriptome analysis showed that exposure to different concentrations of bisphenol S often induced different reactions. In summary, environmental concentrations of BPS can have adverse effects on the hatching and physical development of Chinese medaka, and further attention needs to be paid to the potential toxicity of environmental BPS.

Effects of Chronic Roundup Exposure on Medaka Larvae.

The use of glyphosate-based herbicides is increasing yearly to keep up with the growing demands of the agriculture world. Although glyphosate-based herbicides target the enzymatic pathway in plants, the effects on the endocrine systems of vertebrate organisms, mainly fish, are widely unknown. Many studies with glyphosate used high-exposure concentrations (mg/L), and the effect of environmentally relevant or lower concentrations has not been clearly understood. Therefore, the present study examined the effects of very low, environmentally relevant, and high concentrations of glyphosate exposure on embryo development and the thyroid system of Japanese medaka (Oryzias latipes). The Hd-rR medaka embryos were exposed to Roundup containing 0.05, 0.5, 5, 10, and 20 mg/L glyphosate (glyphosate acid equivalent) from the 8 h post-fertilization stage through the 14-day post-fertilization stage. Phenotypes observed include delayed hatching, increased developmental deformities, abnormal growth, and embryo mortality. The lowest concentration of glyphosate (0.05 mg/L) and the highest concentration (20 mg/L) induced similar phenotypes in embryos and fry. A significant decrease in mRNA levels for acetylcholinesterase (ache) and thyroid hormone receptor alpha (thrα) was found in the fry exposed to 0.05 mg/L and 20 mg/L glyphosate. The present results demonstrated that exposure to glyphosate formulation, at a concentration of 0.05 mg/L, can affect the early development of medaka larvae and the thyroid pathway, suggesting a link between thyroid functional changes and developmental alteration; they also showed that glyphosate can be toxic to fish at this concentration.

Transcriptome Analysis of the Developmental Effects of Bisphenol F Exposure in Chinese Medaka (Oryzias sinensis).

Bisphenol F (BPF) has been used in the syntheses of polymers, which are widely used in coatings, varnishes, adhesives, and other plastics. During the past decades, BPF contamination in the aquatic environment has dramatically increased due to its release from manmade products. Concerns have driven much attention to whether it may adversely impact aquatic lives or human beings. The present study performed an acute toxic exposure experiment and a 15 d developmental exposure of BPF at environmental concentrations (20, 200, and 2000 ng/L) using Chinese medaka (Oryzias sinensis). In the acute toxic exposure, the LC50 of BPF to Chinese medaka is 87.90 mg/L at 96 h. Developmental exposure induced a significant increase in the frequency of larvae with abnormalities in the 2000 ng/L BPF group compared to the control group. Transcriptomic analysis of the whole larvae revealed 565 up-regulated and 493 down-regulated genes in the 2000 ng/L BPF exposure group. Analysis of gene ontology and KEGG pathways enrichments indicated endocrine disorders to be associated with BPF-induced developmental toxicity. The present results suggest that BPF is developmentally toxic at 2000 ng/L concentration in Chinese medaka and causes endocrine-related aberrations in the transcriptional network of genes.

Transcriptional Alterations Induced by Delta-9 Tetrahydrocannabinol in the Brain and Gonads of Adult Medaka.

With the legalization of marijuana smoking in several states of the United States and many other countries for medicinal and recreational use, the possibility of its release into the environment cannot be overruled. Currently, the environmental levels of marijuana metabolites are not monitored on a regular basis, and their stability in the environment is not well understood. Laboratory studies have linked delta 9-tetrahydrocannabinol (Δ9-THC) exposure with behavioral abnormalities in some fish species; however, their effects on endocrine organs are less understood. To understand the effects of THC on the brain and gonads, we exposed adult medaka (Oryzias latipes, Hd-rR strain, both male and female) to 50 ug/L THC for 21 days spanning their complete spermatogenic and oogenic cycles. We examined transcriptional responses of the brain and gonads (testis and ovary) to Δ9-THC, particularly molecular pathways associated with behavioral and reproductive functions. The Δ9-THC effects were more profound in males than females. The Δ9-THC-induced differential expression pattern of genes in the brain of the male fish suggested pathways to neurodegenerative diseases and pathways to reproductive impairment in the testis. The present results provide insights into endocrine disruption in aquatic organisms due to environmental cannabinoid compounds.

In vivo DNA methylation editing in zebrafish.

The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.

Ancestral BPA exposure caused defects in the liver of medaka for four generations.

Environmental chemicals can induce liver defects in experimental animals due to their direct and acute exposure. It is not clear whether environmental chemical exposures result in the transgenerational passage of liver defects in subsequent generations living in an uncontaminated environment. Bisphenol A (BPA), a plasticizer chemical, has been ubiquitous in the environment in the recent decade. Every organism is exposed to this chemical at some point during its lifetime. Literature suggests that direct BPA exposure can result in several metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). Despite the phasing out of BPA from several consumer goods, it is unclear whether ancestral BPA exposure causes liver health problems in the unexposed future generations. Here, we demonstrate an advanced stage of NAFLD in the grandchildren (F2 generation) of medaka fish (Oryzias latipes) due to embryonic BPA exposure in the grandparental generation (F0), which persists for five generations (F4) even in the absence of BPA. The severity of transgenerational NAFLD phenotype included steatosis together with perisinusoidal fibrosis and apoptosis of hepatocytes. Adult females developed more severe histopathological conditions in the liver than males. Genes encoding enzymes involved in lipolytic pathways were significantly decreased. The present results suggest that ancestral BPA exposure can result in transgenerational metabolic diseases that can persist for five generations and that the NAFLD trait is sexually dimorphic. Given that ancestral BPA exposure can lead to altered metabolic health outcomes in the subsequent unexposed generations, the development of the methods and strategies to mitigate the transgenerational onset of metabolic diseases seem imperative to protect future generations.

Genome-wide transcriptome analysis of the orphan crop tef (Eragrostis tef (Zucc.) Trotter) under long-term low calcium stress.

Calcium (Ca2+) is one of the essential mineral nutrients for plant growth and development. However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.

Potassium perchlorate effects on primordial germ cells of developing medaka larvae.

Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Exposure of humans to perchlorate can occur in the theater of war and areas adjacent to military training grounds. A high concentration of perchlorate has been found to affect reproduction in vertebrates, including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs), the founders of sperm and eggs, is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 µg/L potassium perchlorate exposure on the embryonic development of medaka and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, and increased developmental deformities in the larvae. The 10 and 20 mg/L concentrations of perchlorate were lethal to embryos, whereas vitamin C co-treatment (1 mg/L) completely blocked perchlorate-induced mortality. RNA-seq analysis of isolated PGCs showed a non-linear pattern in expression profiles of differentially altered genes. Significantly upregulated genes were found in PGCs from the 10 and 1000 µg/L groups, whereas the 100 µg/L groups showed the highest number of significantly downregulated genes. Gene ontology analysis predicted differentially expressed genes to be involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity. Among the cellular components, extracellular, intracellular, sarcoplasmic, and 6-phosphofructokinase and membrane-bounded processes were affected. Ingenuity Pathway Analysis of PGC transcriptomes revealed thyroid hormone signaling to be affected by all concentrations of perchlorate. The present results suggested that perchlorate affected the development of medaka larvae and vitamin C was able to ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate altered the global transcriptional network in PGCs in a non-linear fashion suggesting its potential effects on developing germ cells and fertility.

Analysis of miRNAs responsive to long-term calcium deficiency in tef (Eragrostis tef (Zucc.) Trotter).

MicroRNAs (miRNAs) play an important role in growth, development, stress resilience, and epigenetic modifications of plants. However, the effect of calcium (Ca2+) deficiency on miRNA expression in the orphan crop tef (Eragrostis tef) remains unknown. In this study, we analyzed expression of miRNAs in roots and shoots of tef in response to Ca2+ treatment. miRNA-seq followed by bioinformatic analysis allowed us to identify a large number of small RNAs (sRNAs) ranging from 17 to 35 nt in length. A total of 1380 miRNAs were identified in tef experiencing long-term Ca2+ deficiency while 1495 miRNAs were detected in control plants. Among the miRNAs identified in this study, 161 miRNAs were similar with those previously characterized in other plant species and 348 miRNAs were novel, while the remaining miRNAs were uncharacterized. Putative target genes and their functions were predicted for all the known and novel miRNAs that we identified. Based on gene ontology (GO) analysis, the predicted target genes are known to have various biological and molecular functions including calcium uptake and transport. Pairwise comparison of differentially expressed miRNAs revealed that some miRNAs were specifically enriched in roots or shoots of low Ca2+-treated plants. Further characterization of the miRNAs and their targets identified in this study may help in understanding Ca2+ deficiency responses in tef and related orphan crops.

Exogenous Expression of pou5f3 Facilitates the Development of Somatic Cell Nuclear Transfer Embryos in Zebrafish at the Early Stage.

Enucleated oocytes can reprogram differentiated nuclei to totipotency after somatic cell nuclear transfer (SCNT), which is valuable in understanding nuclear reprogramming and generating genetically modified animals. To date, reprogramming efficiency is low and the development of SCNT embryos is not going as well as anticipated. To further disclose the reprogramming mechanisms during SCNT zebrafish embryo development, we examined the expression patterns of transcription regulation factors and regulated them by mRNA and morpholino microinjection. In this study, we show that stem cell-related transcription factors are downregulated in zebrafish SCNT embryos at the blastula stage. Exogenous expression of pou5f3 at the single-cell stage improves SCNT embryo development from the blastula to the gastrula stage. We also found that exogenous expression of klf4 or sox2 decreases SCNT embryo development from the blastula to the gastrula stage, while expression of nanog is necessary for the development of SCNT embryos. Our results conclude that zebrafish pou5f3 facilitates the development of SCNT embryos from the blastula to gastrula stage.

Histopathology and transcriptome reveals the tissue-specific hepatotoxicity and gills injury in mosquitofish (Gambusia affinis) induced by sublethal concentration of triclosan.

Triclosan (TCS), a ubiquitous antimicrobial agent, has been frequently detected in wild fish, leading to concerns regarding TCS safety in the aquatic environment. The present work aims to investigate the TCS-mediated effects on various tissues (the liver, gills, brain, and testes) of wild-sourced adult mosquitofish based on histological analysis and transcriptome. Severe morphological injuries were only found in the liver and gills. The histopathological alterations in the liver were characterized by cytoplasmic vacuolation and degeneration, eosinophilic cytoplasmic inclusions, and nuclear polymorphism. The gill lesions contained epithelial lifting, intraepithelial edema, fusion and shortening of the secondary lamellae. Consistently, the numbers of differently expressed genes (DEGs) identified by transcriptome were in the order of liver (1627) > gills (182) > brain (9) > testes (4). Trend-aligned histopathological and transcriptomic changes in the 4 tissues, suggesting the tissue-specific response manner of mosquitofish to TCS, and the liver and gills were the target organs. TCS interrupted many biological pathways associated with lipogenesis and lipid metabolism, transmembrane transporters, protein synthesis, and carbohydrate metabolism in the liver, and it induced nonspecific immune response in the gills. TCS-triggered hepatotoxicity and gills damnification may lead to inflammation, apoptosis, diseases, and even death in mosquitofish. TCS showed moderate acute toxicity and bioaccumulative property on mosquitofish, suggesting that prolonged or massive use of TCS may pose an ecological risk.

Bisphenol A and 17α-ethinylestradiol-induced transgenerational gene expression differences in the brain-pituitary-testis axis of medaka, Oryzias latipes†.

Endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 17α-ethinylestradiol (EE2), can have far reaching health effects, including transgenerational abnormalities in offspring that never directly contacted either chemical. We previously reported reduced fertilization rates and embryo survival at F2 and F3 generations caused by 7-day embryonic exposure (F0) to 100 µg/L BPA or 0.05 µg/L EE2 in medaka. Crossbreeding of fish in F2 generation indicated subfertility in males. To further understand the mechanisms underlying BPA or EE2-induced adult onset and transgenerational reproductive defects in males, the present study examined the expression of genes regulating the brain-pituitary-testis (BPT) axis in the same F0 and F2 generation male medaka. Embryonic exposure to BPA or EE2 led to hyperactivation of brain and pituitary genes, which are actively involved in reproduction in adulthood of the F0 generation male fish, and some of these F0 effects continued to the F2 generation (transgenerational effects). Particularly, the F2 generation inherited the hyperactivated state of expression for kisspeptin (kiss1 and kiss2) and their receptors (kiss1r and kiss2r), and gnrh and gnrh receptors. At F2 generation, expression of DNA methyltransferase 1 (dnmt1) decreased in brain of the BPA treatment lineage, while EE2 treatment lineage showed increased dnmt3bb expression. Global hypomethylation pattern was observed in the testis of both F0 and F2 generation fish. Taken together, these results demonstrated that BPA or EE2-induced transgenerational reproductive impairment in the F2 generation was associated with alterations of reproductive gene expression in brain and testis and global DNA methylation in testis.

Distinct expression patterns of seven crucial microRNAs during early embryonic development in medaka (Oryzias latipes).

MicroRNAs (i.e. miRNAs) are small non-coding RNAs that play essential modulation roles in embryonic development in vertebrates. Paternal and maternal miRNAs contribute to the development of post-fertilization embryo and zygotic genome activation. The pattern of expression and their roles in embryonic development of medaka are not clearly understood. The present study, therefore, examined a temporal expression of seven miRNAs, ola-let-7a, ola-miR-202-3p, ola-miR-126-3p, ola-miR-122, ola-miR-92a, ola-miR-125a-3p and ola-miR-430a in sperm, oocytes, and embryos during early developmental stages. Three unique expression patterns of miRNAs were observed. ola-let7a, ola-miR-202-3p and ola-miR-126-3p showed both paternal and maternal expression, and ola-miR-122, ola-miR-92a, ola-miR-125a-3p showed maternal expression only. The expression of six out of seven miRNAs significantly decreased after maternal-zygotic transition (MZT), whereas ola-miR-430a expression initiated only after MZT. The temporal dynamic expression of these miRNAs suggests their potential roles in early embryogenesis and genome-zygotic activation in medaka.

Developmental abnormalities and epigenetic alterations in medaka (Oryzias latipes) embryos induced by triclosan exposure.

Triclosan (TCS), an antibacterial and antifungal agent present in some consumer products, has been detected in the environment at varying concentrations. TCS exposure has been found to cause developmental abnormalities and endocrine disruption in various species of fish. It is not clearly understood whether TCS exposure causes epigenetic alterations in developing embryos and their germ cells. In the present study, we examined the effects of TCS exposure (0, 50, 100 and, 200 µg/L) on embryonic development and primordial germ cells (PGCs), which are precursors of sperm and eggs, in medaka (Oyzias latipes). Developmental TCS exposure from 8 h post-fertilization through 15 days post-fertilization (dpf) resulted in several developmental abnormalities, including enlarged yolk sac, decreased head trunk angle (HTA), and severe edema in the pericardial region. The male ratio increased in the 100 µg/L TCS exposure group, which was negatively correlated with the expression of cyp19ala (a gene encoding aromatase) and arα (androgen receptor alpha). Developmental 50 µg/L TCS exposure resulted in global hypomethylation in the whole body but not in the isolated PGCs. Expression of the gene encoding DNA methyltransferases (dnmt1 and dnmt3aa) was decreased by 50 µg/L TCS exposure both in the whole body and PGCs. TCS altered the expression of genes encoding enzymes involved in DNA methylation and demethylation in PGCs, suggesting epigenetic effects on germ cells. The present results demonstrate that the embryos exposed to the tested concentrations of TCS develop deformities during the early life stages and that the TCS within this range possesses endocrine disrupting properties potential enough to alter sex ratios of developing embryos.

DNA methylation reprogramming in medaka fish, a promising animal model for environmental epigenetics research.

DNA methylation is a major epigenetic modification that undergoes dramatic changes in two epigenetic reprogramming windows during development: first in preimplantation embryos and second in primordial germ cell (PGC) specification. In both windows, DNA methylation patterns are reprogrammed genome-wide, and the majority of inherited methylation marks are erased, generating cells with broad developmental potential. Recent studies reported that the reprogramming of genome methylation in medaka is similar to human and mouse, suggesting that medaka may serve as a suitable biomedical model for comparative studies focused on the epigenetic and transgenerational inheritance of phenotypic traits. In this mini review, we will discuss how somatic and germ cells in post-fertilization stage embryos are epigenetically reprogrammed in mammals and fishes with a particular focus on DNA methylation dynamics.

Transcriptome analysis of testis reveals the effects of developmental exposure to bisphenol a or 17α-ethinylestradiol in medaka (Oryzias latipes).

Endocrine disrupting chemicals (EDCs) can induce abnormalities in organisms via alteration of molecular pathways and subsequent disruption of endocrine functions. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are ubiquitous EDCs in the environment. Many aquatic organisms, including fish, are often exposed to varying concentrations of BPA and EE2 throughout their lifespan. Both BPA and EE2 can activate estrogenic signaling pathways and cause adverse effects on reproduction via alteration of pathways associated with steroidogenesis. However, transcriptional pathways that are affected by chronic exposure to these two ubiquitous environmental estrogens during embryonic, larval, and juvenile stages are not clearly understood. In the present study, we examined transcriptional alterations in the testis of medaka fish (Oryzias latipes) chronically exposed to a low concentration of BPA or EE2. Medaka were exposed to BPA (10 µg/L) or EE2 (0.01 µg/L) from 8 h post-fertilization (as embryos) to adulthood 50 days post fertilization (dpf), and transcriptional alterations in the testis were examined by RNA sequencing (RNA-seq). Transcriptomic profiling revealed 651 differentially expressed genes (DEGs) between BPA-exposed and control testes, while 1475 DEGs were found between EE2-exposed and control testes. Gene ontology (GO) analysis showed a significant enrichment of "intracellular receptor signaling pathway", "response to steroid hormone" and "hormone-mediated signaling pathway" in the BPA-induced DEGs, and of "cilium organization", "microtubule-based process" and "organelle assembly" in the EE2-induced DEGs. Pathway analysis showed significant enrichment of "integrin signaling pathway" in both treatment groups, and of "cadherin signaling pathway", "Alzheimer disease-presenilin pathway" in EE2-induced DEGs. Single nucleotide polymorphism (SNP) and insertion-deletion (Indel) analysis found no significant differences in mutation rates with either BPA or EE2 treatments. Taken together, global gene expression differences in testes of medaka during early stages of gametogenesis were responsive to chronic BPA and EE2 exposure.

The dynamics of DNA methylation during epigenetic reprogramming of primordial germ cells in medaka (Oryzias latipes).

Primordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.

Bisphenol A and 17α-ethinylestradiol-induced transgenerational differences in expression of osmoregulatory genes in the gill of medaka (Oryzias latipes).

Embryonic bisphenol A (BPA) and 17α-ethinylestradiol (EE2) exposure can have far reaching health effects in fish, including adult onset transgenerational reproductive abnormalities, anxiety, and cardiac disorders. It is unknown whether these two environmental estrogens can induce transgenerational abnormalities in the gill. The present study examined transgenerational effects of BPA or EE2 exposure on genes that are critical for osmoregulation in fish. Medaka (Oryzias latipes) embryos were exposed to either BPA (100 µg/L) or EE2 (0.05 µg/L) for the first 7 days of embryonic development and never thereafter for the remainder of that generation (F0) and in subsequent generations of this study (F1, F2, and F3). Expression of osmoregulatory genes (NKAα1a, NKAα1b, NKAα1c, NKAα3a, NKAα3b, NKCC1a, and CFTR) were examined in gills of the first-generation (F0) adults which were directly exposed as embryo and in the fourth-generation adults (F3), which were never exposed to either of these environmental estrogens. Significant alterations in expression of osmoregulatory genes were observed in both F0 and F3 generations. Within the F0 generation, a sex-specific expression pattern was observed with a downregulation of osmoregulatory genes in males and an upregulation of osmoregulatory genes in females. At the F3 generation, this pattern reversed with the majority of the osmoregulatory genes upregulated in males and downregulated in females, suggesting that exposure to BPA and EE2 during embryonic development induced transgenerational impairment in molecular events associated with osmoregulatory functions in subsequent generations. These adverse outcomes may have impacts on physiological functions related to osmoregulation of fish inhabiting contaminated aquatic environments.

DNA methylation dynamics during epigenetic reprogramming of medaka embryo.

Post-fertilization epigenome reprogramming erases epigenetic marks transmitted through gametes and establishes new marks during mid-blastula stages. The mouse embryo undergoes dynamic DNA methylation reprogramming after fertilization, while in zebrafish, the paternal DNA methylation pattern is maintained throughout the early embryogenesis and the maternal genome is reprogrammed in a pattern similar to that of sperm during the mid-blastula transition. Here, we show DNA methylation dynamics in medaka embryos, the biomedical model fish, during epigenetic reprogramming of embryonic genome. The sperm genome was hypermethylated and the oocyte genome hypomethylated prior to fertilization. After fertilization, the methylation marks of sperm genome were erased within the first cell cycle and embryonic genome remained hypomethylated from the zygote until 16-cell stage. The DNA methylation level gradually increased from 16-cell stage through the gastrula. The 5-hydroxymethylation (5hmC) levels showed an opposite pattern to DNA methylation (5-mC). The mRNA levels for DNA methyltransferase (DNMT) 1 remained high in oocytes and maintained the same level through late blastula stage and was reduced thereafter. DNMT3BB.1 mRNA levels increased prior to remethylation. The mRNA levels for ten-eleven translocation methylcytosine dioxygenases (TET2 & TET3) were detected in sperm and embryos at cleavage stages, whereas TET1 and TET3 mRNAs decreased during gastrulation. The pattern of genome methylation in medaka was identical to mammalian genome methylation but not to zebrafish. The present study suggests that a medaka embryo resets its DNA methylation pattern by active demethylation and by a gradual remethylation similar to mammals.

[Nuclear transfer and reprogramming in fish].

As an important sub-field in the study of animal cloning, fish nuclear transfer was first established in the early 1960s by Chinese embryologists. Due to its advantages, zebrafish has become a unique animal model to study the mystery of reprogramming in nuclear transfer. This article summarizes the history and current situation in fish nuclear transfer technology and discusses the factors that may influence the development of the cloned embryos. A comprehensive understand-ing of the mechanism for epigenetic modification following nuclear transfer, such as genomic DNA methylation and histone acetylation and/or methylation, will likely increase the success rate and eventually lead to the future freedom of cloning technique.