Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.

Peroxisome Proliferator Activated Receptors (PPARs) are transcription factors that regulate processes such as lipid and glucose metabolism. Synthetic PPAR ligands, designed as therapeutics for metabolic disease, provide a tool to assess the relationship between PPAR activity and pancreas development in vivo, an area that remains poorly characterized. Here, we aim to assess the effects of PPAR agonists and antagonists on gene expression, embryonic morphology and pancreas development in transgenic zebrafish embryos. To evaluate developmental perturbations, we assessed gross body and pancreas morphology at 4 days post fertilization (dpf) in response to developmental exposures with PPARα, PPARγ, and PPARß/δ agonists and antagonists at 0, 0.01, 0.1, 1, and 10 µM concentrations. All ligand exposures, with the exception of the PPARα agonist, resulted in significantly altered fish length and yolk sac area. PPARγ agonist and antagonist had higher incidence of darkened yolk sac and craniofacial deformities, whereas PPARα antagonist had higher incidence of pericardial edema and death. Significantly reduced endocrine pancreas area was observed in both PPARγ ligands and PPARα agonist exposed embryos, some of which also exhibited aberrant endocrine pancreas morphology. Both PPARß/δ ligands caused reduced exocrine pancreas length and novel aberrant phenotype, and disrupted gene expression of pancreatic targets pdx1, gcga, and try. Lipid staining was performed at 8 dpf and revealed altered lipid accumulation consistent with isoform function. These data indicate chronic exposure to synthetic ligands may induce morphological and pancreatic defects in zebrafish embryos.

Assessment of the in vitro developmental toxicity of diethylstilbestrol and estradiol in the zebrafish embryotoxicity test.

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17ß-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.

Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages.

Toxicological effects of butylparaben (BuP) and ethylparaben (EtP) on zebrafish (Danio rerio) early-life stages are not well established. The present study evaluated, using zebrafish embryos and larvae, the toxicity of BuP and EtP through benchmark dose (BMD) approach. BuP was more toxic than EtP to zebrafish larvae. In fact, Lethal Concentration 50 (LC50) values at 96 h post-fertilization (hpf) for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. Indeed, BMD confidence interval (lower bound (BMDL) - upper bound (BMDU) was 0.91-1.92 mg/L for BuP and 10.8-17.4 mg/L for EtP. Zebrafish embryos exposed to 1 mg/L, 2.5 mg/L of BuP and 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L of EtP showed several developmental abnormalities and teratological effects compared to negative control. Exposed zebrafish developed reduced heartbeat, reduction in blood circulation, blood stasis, pericardial edema, deformed notochord and misshaped yolk sac. Embryos exposed to the highest concentrations of the chemicals (2.5 mg/L of BuP, 10 mg/L, 20 mg/L and 30 mg/L of EtP) showed the developmental abnormalities at 48 hpf while those treated with 1 mg/L of BuP and 10 mg/L of EtP reported behavioral changes at 72 hpf, including trembling of head, pectoral fins and spinal cord. This research identified the lethal and sublethal effects of BuP and EtP in zebrafish early-life stages and could be helpful to elucidate the developmental pathways of toxicity of parabens.

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD).

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6-7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

DHF-BAHPC molecule exerts ameliorative antioxidant status and reduced cadmium-induced toxicity in zebrafish (Danio rerio) embryos.

In this study, we report the antioxidant and antitoxic potential of chemically synthesized 4-oxo-2-phenyl-4H-chromene-7,8-diyl bis((1-amino-2-hydroxypropyl)carbamate) (DHF-BAHPC) compound using in vitro and in vivo assays. The DHF-BAHPC was synthesized by linking 7,8-Dihydroxyflavone (DHF) with two molecules of Fmoc-threonine and characterized by Ultraviolet-visible spectroscopy (UV-vis), Fourier-transform infrared spectroscopy (FT-IR), 1H NMR, 13C NMR and Mass spectrometry (MS). In vitro, antioxidant assay results revealed that DHF-BAHPC has a dose-dependent radical scavenging potential towards DPPH, ABTS, FRAP and H2O2 radicals with an IC50 range of 15.45, 66.27, 25.71, 4.375 µg/mL, respectively. Furthermore DHF-BAHPC treatment significantly altered cadmium (Cd) intoxicated zebrafish embryos by rescuing the developmental changes associated with severe histological and reduced the level of defensive antioxidant activities (SOD, CAT, GPx and GST). The overall results of the present study represented that DHF-BAHPC may be used as a potential drug in redox-based therapeutics.

Relation between types of yolk sac abnormalities and early embryonic morphology in first-trimester missed miscarriage.

AIM: The aim of this study was to identify yolk sac (YS) abnormalities and analyze their relation with different embryonic morphologies detected by ultrasonography in first-trimester missed miscarriage. MATERIAL AND METHODS: This descriptive study was carried out on 204 women with established first-trimester missed miscarriage. Ultrasonography depicted the YS's diameter, shape, and echogenicity, as well as the embryonic morphologic findings, including normal shape, isolated defects, and growth disorganization types 1 to 4. The findings were qualified and analyzed. RESULTS: Abnormalities in YS diameter were much more common than those in YS shape or echogenicity. YS with normal diameter constituted 30.9% of the cases, as opposed to a vast majority with normal shape and normal echogenicity (93.5% and 94.7%, respectively). YS of normal diameter, shape and echogenicity were most commonly (47.5%) associated with normal embryonic morphology. Absent YS was most commonly (75%) associated with growth disorganization type 1. Too-small YS were most prevalent among normal embryos (44.8%) and type 1 growth disorganization embryos (27.6%). Cystic YS were mostly associated with isolated embryonic anomalies (36.8%). CONCLUSIONS: This study found a significant relation between YS abnormalities and embryonic morphology in missed miscarriage cases. This was most evident with abnormalities in YS diameter rather than the YS shape or appearance. The commonest combinations met in our cases were growth disorganized 1 embryos with an absent YS, normal embryonic morphology with normal or small YS, and isolated embryonic defects with cystic YS.

Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio).

The nitrophenols (NPs) are water-soluble compounds. These compounds pose a significant health threat since they are priority environmental pollutants. In this study, 2-Nitrophenol (2NP) and 2,4-dinitrophenol (DNP) were examined for embryo and early life stage toxicity in zebrafish (Danio rerio). Acute toxicity and teratogenicity of 2NP and DNP were tested for 4 days using zebrafish embryos. The typical lesions observed were no somite formation, incomplete eye and head development, tail curvature, weak pigmentation (≤48 hours postfertilization (hpf)), kyphosis, scoliosis, yolk sac deformity, and nonpigmentation (72 hpf). Also, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and compound-dependent. Of the compounds tested, 2,4-DNP was found to be highly toxic to the fish embryos following exposure. The median lethal concentrations and median effective concentrations for 2NP are 18.7 mg/L and 7.9 mg/L, respectively; the corresponding values for DNP are 9.65 mg/L and 3.05 mg/L for 48 h. The chorda deformity was the most sensitive endpoint measured. It is suggested that the embryotoxicity may be mediated by an oxidative phosphorylation uncoupling mechanism. This article is the first to describe the teratogenicity and embryotoxicity of two NPs to the early life stages of zebrafish.

Partial depletion of yolk during zebrafish embryogenesis changes the dynamics of methionine cycle and metabolic genes.

BACKGROUND: Limited nutrient availability during development is associated with metabolic diseases in adulthood. The molecular cause for these defects is unclear. Here, we investigate if transcriptional changes caused by developmental malnutrition reveal an early response that can be linked to metabolism and metabolic diseases. RESULTS: We limited nutrient availability by removing yolk from zebrafish (Danio rerio) embryos. We then measured genome expression after 8, 24, 32 h post-fertilization (hpf) by RNA sequencing and 48 hpf by microarray profiling. We assessed the functional impact of deregulated genes by enrichment analysis of gene ontologies, pathways and CpG sites around the transcription start sites. Nutrient depletion during embryogenesis does not affect viability, but induces a bias towards female development. It induces subtle expression changes of metabolic genes: lipid transport, oxidative signaling, and glycolysis are affected during earlier stages, and hormonal signaling at 48 hpf. Co-citation analysis indicates association of deregulated genes to the metabolic syndrome, a known outcome of early-life nutrient depletion. Notably, deregulated methionine cycle genes indicate altered methyl donor availability. We find that the regulation of deregulated genes may be less dependent on methyl donor availability. CONCLUSIONS: The systemic response to reduced nutrient availability in zebrafish embryos affects metabolic pathways and can be linked to metabolic diseases. Further exploration of the reported zebrafish model system may elucidate the consequences of reduced nutrient availability during embryogenesis.

Toxicity effects of profenofos on embryonic and larval development of Zebrafish (Danio rerio).

The aim of the present study was to evaluate the developmental toxicity of profenofos to early developing Zebrafish (Danio rerio) embryos (4h post fertilization) in a static system at 1.0 to 2.25mg/L. Median lethal concentrations (LC50) of profenofos at 24-h, 48-h, 72-h and 96-h were determined as 2.04, 1.58, 1.57 and 1.56 mg/L, respectively. The hatching of embryos were recorded at every 12h interval and the median hatching time (HT50) was also calculated for each concentration. In a separate set of experiments, 96-h LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations were used to assess the developmental toxicity in relation to behavior, morphology, and interactions with the targeted enzyme acetylcholinesterase. Live video-microscopy revealed that the profenofos exposed embryos exhibited an abnormal development, skeletal defects and altered heart morphology in a concentration-dependent manner, which leads to alterations in the swimming behavior of hatchlings at 144-h, which indicate that developing zebrafish are sensitive to profenofos.

Termination of pregnancy at very early gestation without visible yolk sac on ultrasound.

INTRODUCTION: Requests for termination of pregnancy (TOP) at very early gestation (≤6 weeks) can prove challenging for abortion services as the ultrasound feature usually accepted as definitive evidence of an intrauterine pregnancy (IUP), the presence of a yolk sac within a gestational sac, may not yet be evident. In 2011 the Edinburgh TOP service introduced a protocol permitting women to proceed to treatment without further investigations provided that ultrasound showed the features of an eccentrically placed gestational sac (≥3 mm) with a decidual reaction, and there were no signs, symptoms or risk factors for ectopic pregnancy. METHODS: A retrospective audit was conducted of outcomes of women presenting for TOP at ≤6 weeks' gestation over a 2-year period using the hospital computerised database. RESULTS: A total of 1155 women presented for TOP with an ultrasound gestational age of ≤6 weeks. Of these, 1030 (89%) had ultrasound evidence of a yolk sac. Eighty-seven women (7.5%) had an eccentrically placed gestational sac with a decidual reaction. All 87 women fulfilled our criteria to proceed to medical TOP, and 66 did so. In the remaining 21 cases, further investigations were performed before they proceeded to medical TOP. Two (0.17%) medical TOPs failed, both in women whose initial ultrasound had shown a yolk sac. CONCLUSION: Women with ultrasound features consistent with a very early IUP (≥3 mm eccentrically placed gestational sac with a decidual reaction) and without signs, symptoms or risk factors for ectopic pregnancy can proceed directly to medical TOP without the need for delay for further ultrasonography.

Abnormal sonographic appearances of the yolk sac: which can be associated with adverse perinatal outcome?

AIMS: The present study aimed to determine whether yolk sacs with abnormal sonographic appearance are associated with adverse perinatal outcomes in both early and late gestation. MATERIAL AND METHODS: A total of 305 viable singleton pregnancies with gestational age of 6 to 9 weeks were prospectively evaluated with respect to perinatal outcomes and sonographic characteristics of the yolk sacs. RESULTS: An abnormal yolk sac was found in 66 pregnancies. In pregnancies with enlarged yolk sacs a miscarriage occurred in 37.5% of cases (3/8). The pregnancies with a yolk sac diameter >/= 5 mm had a significantly higher risk of miscarriage (p = 0.005). The risk of miscarriage was statistically similar between the pregnancies with regular and those with irregular yolk sacs (p = 0.73). Miscarriage occurred in 3.8% of pregnancies with irregular yolk sacs (2/52) and none of pregnancies with echogenic yolk sacs (0/6). Adverse perinatal outcomes were not associated with either irregular or echogenic yolk sacs. CONCLUSIONS: An enlarged yolk sac visualized before the 7th week of gestation is strongly associated with a significantly increased risk for spontaneous miscarriage. The presence of an echogenic or irregular yolk sac appears to be unrelated to adverse perinatal outcome.

Early embryonic morphology sonographically assessed and its correlation with yolk sac in missed abortion.

PURPOSE: To provide additional information about embryo morphology sonographic assessment and its correlation with yolk sac. METHODS: A systematic study in 200 consecutive cases of missed abortion <10 weeks diagnosed by transvaginal ultrasound. RESULTS: In 104 gestations of embryos with morphological abnormalities, 88 (84.6 %) were at least 1 week smaller than expected for gestational age and 16 (15.4 %) were the expected size. From 32 normal morphologic embryos, 7 (21.9 %) were at least 1 week smaller than expected for gestational age, and 25 (78.1 %) were the expected size (p < 0.005). Normal morphologic embryos are linked more frequently with normal yolk sac (62.5 %). Findings in anembryonic gestations (GD1) included an absent yolk sac (46.9 %) and a cystic yolk sac (25 %). Likewise, findings in GD2-3 embryos included more frequently a cystic yolk sac (42.9 %) and an absent yolk sac (32.5 %). GD4 embryos are associated with an echogenic yolk sac (40 %), a relatively small-hypoplastic- (40 %) and a relatively large-cystic- (20 %). In DI embryos, yolk sac appears cystic (62.5 %) or echogenic (37.5 %). CONCLUSIONS: Our study proves the correlation between morphology of conceptuses and yolk sac appearance in cases of missed abortion.

Development of bovine embryos derived from reproductive techniques.

Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

Association between increased yolk sac diameter and abnormal karyotypes.

AIMS: To investigate the association between increased yolk sac diameter and abnormal karyotype. METHODS: Retrospective analysis of 42 patients with no history of diabetes between 6 and 12 weeks of gestation with increased yolk sac diameter measuring ≥6 mm was evaluated by transvaginal ultrasound. Sonographic findings were correlated with karyotype. The Fisher's exact test and exact conditional logistic regression analysis were used for statistical analysis. RESULTS: Chromosome abnormalities were found in 76.2% of chorionic villi samples. A statistically significant relationship between karyotype and missed abortion was detected (P=0.001). None of the patients with a yolk size diameter ≥8 mm and viable pregnancy had a normal karyotype. Trisomy 15 or 16 was strongly associated with missed abortion (unadjusted odds ratio=14.97, P=0.01). Nine patients with viable pregnancy had a yolk sac ≥6 mm (six patients with normal karyotype, one patient with monosomy X, one patient with trisomy 16, and one patient with trisomy 21). CONCLUSION: Our data indicate that enlarged yolk sac may also be visualized in viable pregnancies. Patients with an enlarged yolk sac and normal karyotype require detailed ultrasound evaluation in the second and third trimester.

Histology of a paraumbilical band in a neonate with gastroschisis.

We report a case of gastroschisis in which a paraumbilical band was found at the right margin of the abdominal wall defect and extended into the antimesenteric side of the small intestine. The band consisted of 2 thin cords. Microscopically, 1 band showed a fibrous tissue, and the other 1 revealed a unique vascular structure resembling the vitelline artery and vein, suggesting that the paraumbilical band represents a remnant of the yolk stalk that failed to be incorporated into the umbilical stalk. The origin of the paraumbilical band and an associated pathogenetic hypothesis of gastroschisis are discussed.

Is the yolk sac a new marker for chromosomal abnormalities in early pregnancy?

INTRODUCTION: Abnormal yolk sac size is associated with fetal miscarriage. This is a case report of two pregnancies with abnormal yolk sacs. MATERIALS AND METHODS: In one case, a twofold sac was found; in the other case, the yolk sac was not of spherical form. CONCLUSION: Currently available publications demonstrate a correlation between abnormal yolk sac size and miscarriage. However, in both cases a trisomy was confirmed. It should therefore be discussed whether form and size of the yolk sac could be a marker for chromosomal abnormalities of the fetus.

Deficiency of autotaxin/lysophospholipase D results in head cavity formation in mouse embryos through the LPA receptor-Rho-ROCK pathway.

Autotaxin, encoded by the Enpp2 gene, generates lysophosphatidic acid (LPA) extracellularly, eliciting various cellular responses through specific LPA receptors. Previous studies have revealed that Enpp2(-/-) mice die at E9.5 owing to angiogenic defects in the yolk sac. Moreover, Enpp2(-/-) embryos show growth retardation, allantois malformation, no axial turning, and head cavity formation. We have also demonstrated that lysosome biogenesis is impaired in yolk sac visceral endoderm cells of Enpp2(-/-) embryos as a result of the downregulation of the Rho-ROCK (Rho-associated coiled-coil containing protein kinase)-LIM kinase pathway. In this study, we examine what signaling defect(s) is responsible for head cavity formation and yolk sac angiogenic defects. By using a whole embryo culture system, we show that 10 µM Ki16425, an antagonist for the LPA receptors, induces head cavity formation and yolk sac angiogenic defects in wild-type embryos. Moreover, 1 µM Ki16425 induces both phenotypes in Enpp2 heterozygous embryos at significantly higher incidence than in wild-type embryos, suggesting an interaction between autotaxin and LPA receptor signaling. Furthermore, we show that inhibition of the Rho-ROCK pathway induces head cavity formation, whereas multiple pathways are involved in yolk sac angiogenic defects. These results reveal the signal transduction defects that underlie the abnormalities in Enpp2(-/-) embryos.

RapGEF2 is essential for embryonic hematopoiesis but dispensable for adult hematopoiesis.

RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ~ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1(+) cells were present in RapGEF2(+/+) and RapGEF2(-/-) yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2(-/-) genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2(-/-) cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2(-/-) cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2(-/-) cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2(cko/cko)ER(cre) mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.

Detection of enlarged yolk sac on early ultrasound is associated with adverse pregnancy outcomes.

Pregnancies with mean yolk sac diameter>or=5 mm on early ultrasound require monitoring and counseling about a threefold increased risk for first-trimester loss independent of maternal risk factors such as age, body mass index, polycystic ovary syndrome, smoking, and diabetes. In addition, our study shows for the first time that enlarged yolk sac diameter may be associated with an increased risk of preterm delivery.