Search Results (87)

Lysophosphatidic acid (LPA) is a small bioactive phospholipid which plays an important role during embryonic development and promotes developmental potential of in-vitro-produced (IVP) embryos in several species, including sheep and pigs. In bovines, LPA accelerates IVP blastocyst formation through the Hippo/YAP pathway. However, other LPA effects and its potential receptors during bovine embryo development are less clear. In this study, we used enzyme-linked immunosorbent assay (ELISA) to assess the presence of LPA in bovine oviductal fluid and determine cell apoptosis in embryos after LPA stimulation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). We further evaluated potential receptors of LPA through molecular docking, RNA-seq data analysis and quantitative RT-PCR. LPA was found to be present in oviductal fluid. An increase in total cell number and a decrease in apoptosis levels were detected in day 7 blastocysts after LPA treatment. Among eight LPA receptors (LPARs), GPR87 and LPAR2 showed the highest affinity with LPA and their transcripts were expressed in embryos after the 16-cell stage in RNA-seq and qRT-PCR analysis. However, only the expression of LPAR2 was significantly increased in day 6 blastocysts after LPA stimulation, indicating its potential role in LPA-mediated signaling pathways. Our data highlight the positive effects of LPA on embryos and enrich information of related signaling mediators of LPA during embryonic development. Full article

Background/Objectives: Activation of cannabinoid CB1 or CB2 receptors induces the death of glial progenitors from the chick retina in culture. Here, by using an enriched retinal glial cell culture, we characterized some mechanisms underlying glial death promoted by cannabinoids. Methods and Results: Retinal cultures obtained from 8-day-old (E8) chick embryos and maintained for 12–15 days (C12–15) were used. MTT assays revealed that the CB1/CB2 agonist WIN 55,212-2 (WIN) decreased cell viability in the cultures in a time-dependent manner, with a concomitant increase in extracellular LDH activity, suggesting membrane integrity loss. Cell death was also dose-dependently induced by cannabidiol (CBD), Δ⁹-tetrahydrocannabinol (THC), and CP55940, another CB1/CB2 agonist. In contrast to WIN-induced cell death that was not blocked by either antagonist, the deleterious effect of CBD was blocked by the CB2 receptor antagonist SR144528, but not by PF514273, a CB1 receptor antagonist. WIN-treated cultures showed glial cells with large vacuoles in cytoplasm that were absent in cultures incubated with WIN plus 4-phenyl-butyrate (PBA), a chemical chaperone. Since cannabinoids induced the phosphorylation of eukaryotic initiation factor 2-alfa (eIF2α), these results suggest a process of endoplasmic reticulum (ER) swelling and stress. Incubation of the cultures with WIN for 4 h induced a ~five-fold increase in the number of cells labeled with the ROS indicator CM-H2DCFDA. WIN induced the phosphorylation of JNK but not of p38 in the cultures, and also induced an increase in the number of glial cells expressing cleaved-caspase 3 (c-CASP3). The decrease in cell viability and the expression of c-CASP3 was blocked by salubrinal, an inhibitor of eIF2α dephosphorylation. Conclusions: These data suggest that cannabinoids induce the apoptosis of glial cells in culture by promoting ROS production, ER stress, JNK phosphorylation, and caspase-3 processing. The graphical abstract was created at Biorender.com. Full article

Major histocompatibility complex class I (MHC-I) gene expression in the placenta is modulated to tailor the maternal immune response to fetal antigens during pregnancy. This study evaluated MHC-I expression through immunohistochemistry (IHC) using an anti-mouse preimplantation embryo development (PED) clone Qa-2 and anti-bovine leukocyte antigen I (BoLA) monoclonal antibody clone IL-A88 (n = 23), as well as RT-qPCR (n = 17) for classical and non-classical (BoLA-NC) genes in control and cloned bovine placentomes during early and near-term gestation. Control samples showed minimal Qa-2 protein expression in early gestation, with intense labeling in trophoblasts and the maternal uterine epithelium near term. In contrast, cloned samples exhibited intense Qa-2 labeling in both maternal and trophoblastic epithelia at both stages, while trophoblast giant cells (TGCs), located apposed to the maternal epithelium, showed no labeling. Control samples exhibited intense IL-A88 labeling in the maternal epithelium at both stages. In cloned samples, weak to no labeling was observed in early gestation, with intense labeling in maternal and fetal epithelium near term. RT-qPCR revealed significant upregulation of BoLA-NC3 in early gestation, with sustained elevated expression in cloned samples in the near term. These findings suggest that altered BoLA protein expression and gene regulation in cloned pregnancies may contribute to pregnancy complications and increased losses. Full article

As a highly sensitive vibrational technique, Raman spectroscopy (RS) can provide valuable chemical and molecular data useful to characterise animal cell types, tissues and organs. As a label-free, rapid detection method, RS has been considered a valuable asset in forensics, biology and medicine. The technique has been applied to zebrafish for various purposes, including physiological, biochemical or bioaccumulation analyses. The available data point out its potential for the early diagnosis of detrimental effects elicited by toxicant exposure. Nevertheless, no baseline spectra are available for zebrafish embryos and larvae that could allow for suitable planning of toxicological assessments, comparison with toxicant-elicited spectra or mechanistic understanding of biochemical and physiological responses to the exposures. With this in mind, this work carried out a baseline characterisation of Raman spectra of zebrafish embryos and larvae throughout early development. Raman spectra were recorded from the iris, forebrain, melanocytes, heart, muscle and swim bladder between 24 and 168 h post-fertilisation. A chemometrics approach, based on partial least-squares discriminant analysis (PLS-DA), was used to obtain a Raman characterisation of each tissue or organ. In total, 117 Raman bands were identified, of which 24 were well represented and, thus, retained in the data analysed. Only three bands were found to be common to all organs and tissues. The PLS-DA provided a tentative Raman spectral fingerprint typical of each tissue or organ, reflecting the ongoing developmental dynamics. The bands showed frequencies previously assigned to collagen, cholesterol, various essential amino acids, carbohydrates and nucleic acids. Full article

Exogenous DNA damage represents one of the most harmful outcomes produced by environmental, physical, or chemical agents. Here, a comparative analysis of DNA fragmentation was carried out on Paracentrotus lividus sea urchin embryos exposed to four common pollutants of the marine environment: vanadium, cadmium, gadolinium and selenium. Using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, fragmented DNA was quantified and localized in apoptotic cells mapping whole-mount embryos. This is the first study reporting how different chemicals are able to activate distinctive apoptotic features in sea urchin embryos, categorized as follows: (i) cell-selective apoptosis, showing DNA fragmentation restricted to a subset of extremely damaged cells, acting as an embryo survival mechanism; or (ii) total apoptosis, with fragmented DNA widespread throughout the cells of the entire embryo, leading to its death. Also, this is the first report of the effects of Se exposure on P. lividus sea urchin embryos. These data confirm the TUNEL assay as the most suitable test to study DNA fragmentation in the sea urchin embryo model system. Taken together, this research highlights embryos’ ability to find alternative pathways and set physiological limits for development under stress conditions. Full article

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis. The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated. Full article

Otoliths of the fish’s inner ear serve as a natural chronological recorder because of their continuous formation marked by daily, monthly, and annual increments. Despite their importance, the comprehensive protein content of otoliths remains not fully identified. Using the label-free shotgun proteomics method with one-dimensional liquid chromatography coupled to electrospray ionization-orbitrap tandem mass spectrometry, we quantified a broad range of proteins, with individual otoliths containing between 1341 and 1839 proteins. The identified proteins could potentially serve as a blueprint for fish growth from embryo to adult. We quantified eleven heat-shock proteins (HSPs) in both sexes and several proteins impacted by endocrine disruptors, indicating the otolith’s capacity to reflect environmental stress, potentially linked to climate change effects and altering of hormonal and neuroendocrine functions. Our bioinformatic ontology analysis confirmed the presence of proteins critical for various biological processes, including structural and enzymatic proteins. Protein–protein interaction (PPI) mapping also identified key interactions between the identified proteins. These findings significantly advance our understanding of otolith proteomics, offering a solid foundation for future work. Most of the identified proteins deposited daily and influenced by the environment were not implicated in the biomineralization of otolith, raising the potential for the otolith proteome to recreate details of fish life history at previously unrealized levels. Full article

The shape of a cell as defined by its membrane can be closely associated with its physiological state. For example, the irregular shapes of cancerous cells and elongated shapes of neuron cells often reflect specific functions, such as cell motility and cell communication. However, it remains unclear whether and which cell shape descriptors can characterize different cellular physiological states. In this study, 12 geometric shape descriptors for a three-dimensional (3D) object were collected from the previous literature and tested with a public dataset of ~400,000 independent 3D cell regions segmented based on fluorescent labeling of the cell membranes in Caenorhabditis elegans embryos. It is revealed that those shape descriptors can faithfully characterize cellular physiological states, including (1) cell division (cytokinesis), along with an abrupt increase in the elongation ratio; (2) a negative correlation of cell migration speed with cell sphericity; (3) cell lineage specification with symmetrically patterned cell shape changes; and (4) cell fate specification with differential gene expression and differential cell shapes. The descriptors established may be used to identify and predict the diverse physiological states in numerous cells, which could be used for not only studying developmental morphogenesis but also diagnosing human disease (e.g., the rapid detection of abnormal cells). Full article

Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development. Full article

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days’ post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC. Full article

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein–protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy. Full article

The chromatin-remodeling protein ATRX, which is currently recognized as one of the key genome caretakers, plays an important role in oogenesis and early embryogenesis in mammals. ATRX distribution in the nuclei of mouse embryos developing in vivo and in vitro, including when the embryos are arrested at the two-cell stage—the so-called two-cell block in vitro—was studied using immunofluorescent labeling and FISH. In normally developing two- and four-cell embryos, ATRX was found to be closely colocalized with pericentromeric DNA sequences detected with a probe to the mouse major satellite DNA. The association of ATRX with pericentromeric heterochromatin is mediated by nuclear actin and reduced after the treatment of embryos with latrunculin B. When culturing embryos in vitro, the distribution pattern of ATRX changes, leading to a decrease in the association of this protein with major satellite DNA especially under the two-cell block in vitro. Taken together, our data suggest that the intranuclear distribution of ATRX reflects the viability of mouse embryos and their probability of successful preimplantation development. Full article

Congenital Zika syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they were born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction in PH3+ cells, intermediate progenitor cells, and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV-infected cultures show a reduction in callosal axon length. GFAP labeling showed that an in utero infection compromises glial cells responsible for midline axon guidance. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis, but also axon guidance and growth across the midline. Full article

The widespread applications of ZnO NPs in the different areas of science, technology, medicine, agriculture, and commercial products have led to increased chances of their release into the environment. This created a growing public concern about the toxicological and environmental effects of the nanoparticles. The impact of these NPs on the genetic materials of living organisms is documented in some cultured cells and plants, but there are only a few studies regarding this aspect in animals. In view of this, the present work regarding the assessment of the genotoxicity of zinc oxide nanoparticles using the mosquito Culex quinquefaciatus has been taken up. Statistically significant chromosomal aberrations over the control are recorded after the exposure of the fourth instar larvae to a dose of less than LD₂₀ for 24 h. In order to select this dose, LD₂₀ of ZnO NPs for the mosquito is determined by Probit analysis. Lacto-aceto-orcein stained chromosomal preparations are made from gonads of adult treated and control mosquitoes. Both structural aberrations, such as chromosomal breaks, fragments, translocations, and terminal fusions, resulting in the formation of rings and clumped chromosomes, and numerical ones, including hypo- and hyper-aneuploidy at metaphases, bridges, and laggards at the anaphase stage are observed. The percentage frequency of abnormalities in the shape of sperm heads is also found to be statistically significant over the controls. Besides this, zinc oxide nanoparticles are also found to affect the reproductive potential and embryo development as egg rafts obtained from the genetic crosses of ZnO nanoparticle-treated virgin females and normal males are small in size with a far smaller number of eggs per raft. The percentage frequencies of dominant lethal mutations indicated by the frequency of unhatched eggs are also statistically significant (p < 0.05) over the control. The induction of abnormalities in all of the three short-term assays studied during the present piece of work indicates the genotoxic potential of ZnO NPs, which cannot be labeled absolutely safe, and this study pinpoints the need to develop strategies for the protection of the environment and living organisms thriving in it. Full article

The surge of supervised learning methods for segmentation lately has underscored the critical role of label quality in predicting performance. This issue is prevalent in the domain of medical imaging, where high annotation costs and inter-observer variability pose significant challenges. Acquiring labels commonly involves multiple experts providing their interpretations of the “true” segmentation labels, each influenced by their individual biases. The blind acceptance of these noisy labels as the ground truth restricts the potential effectiveness of segmentation algorithms. Here, we apply coupled convolutional neural network approaches to a small-sized real-world dataset of bovine cumulus oocyte complexes. This is the first time these methods have been applied to a real-world annotation medical dataset, since they were previously tested only on artificially generated labels of medical and non-medical datasets. This dataset is crucial for healthy embryo development. Its application revealed an important challenge: the inability to effectively learn distinct confusion matrices for each expert due to large areas of agreement. In response, we propose a novel method that focuses on areas of high uncertainty. This approach allows us to understand the individual characteristics better, extract their behavior, and use this insight to create a more sophisticated ground truth using maximum likelihood. These findings contribute to the ongoing discussion of leveraging machine learning algorithms for medical image segmentation, particularly in scenarios involving multiple human annotators. Full article