Stage Embryos Research Articles

Embryology outcomes of a device-based sperm separation technique compared to density gradient centrifugation using thawed spermatozoa-a sibling donor oocyte study.

To evaluate whether the ZyMōt™ Multi 850μl sperm separation device (SSD) effectively recovers motile spermatozoa from cryopreserved ejaculates and compare its effect on key embryology outcomes including fertilization, cleavage stage, and total and top-quality blastocyst formation rates to the traditional Density Gradient Centrifugation (DGC) method. In this prospective, single-center, controlled study, we used fresh sibling donor oocytes and non-donor cryopreserved ejaculates. In total, 150 couples participated in this study. At least eight MII donor oocytes were allocated to each couple split into two arms. One arm underwent ICSI with the control DGC-processed sample, and the other arm processed with SSD. No significant difference on fertilization and cleavage stage embryo rates was observed between the two techniques. We observed a significant increase in the percentage of total (SSD: 74.03 ± 23.47% vs. DGC: 67.86 ± 23.92%; p = 0.016) and top-quality (SSD: 66.38 ± 24.94% vs. DGC: 60.98 ± 24.40%; p = 0.035) blastocysts formed post-SSD processing. Sub-analysis showed that this increase remained significant for the WHO-normal group (n = 118), but not for the WHO-abnormal group (n = 32). The SSD was successfully applied in all 150 cases, providing adequate numbers of spermatozoa to undergo ICSI. Additionally, SSD significantly improved blastocyst development rates; however, this was of limited clinical impact considering the minor improvement on the average number of top-quality blastocysts. It can be hypothesized that this positive contribution may be stronger and clinically significant when a larger number of oocytes is used or in homologous oocyte ICSI cycles, where the repair mechanisms of the oocytes may insufficient for promoting healthy embryo development.

Silver nano-colloid particles embedded on Langmuir-Blodgett film matrix of stearic acid serving as a SERS active sensor for detecting the herbicide ‘Paraquat’

The herbicide Paraquat, widely used for efficient weed control, poses significant health risks to humans viz., severe toxicity to vital organs and induction of neurodegenerative disorder like Parkinson’s disease, underscoring the urgent need for developing sensitive detection methods for the herbicide. This study aims at fabricating a novel SERS-active substrate SA-LB/Ag (silver nano-colloids adsorbed on Langmuir-Blodgett film of stearic acid), as a SERS based sensor having high sensitivity, uniformity, and reproducibility to detect ultra-trace amounts of paraquat. The sensor was built up by immersing bilayer LB films of SA in silver nano-colloid suspension, prepared by mixing silver nitrate and sodium borohydride with vigorous stirring. Quantitative evaluations establish a significant linear regression across the concentration spectrum (100 ppm to 75 ppb) of paraquat, with a remarkable 15 ppb limit of detection (LOD) value. In practical terms, this SERS based sensor exhibits notable proficiency in detecting paraquat residues in (a) agricultural water from rice and vegetable fields and environmental water from adjoining water-logged areas, and (b) soil extracts of the agricultural fields. Moreover, the minimum toxic concentration of paraquat at the developmental stage of zebrafish embryos and larvae is found to be approximately 20 ppb, implying that the SA-LB/Ag sensor is sensitive enough to determine the limiting toxic level of paraquat for animal systems. Thus, the designed sensor holds far-reaching implications in ensuring food safety, monitoring the environment, and framing regulatory measures for paraquat use.

Regulation of α-Ketoglutarate levels by Myc affects metabolism and demethylation in porcine early embryos

The Myc family is essential for cell proliferation, differentiation, and metabolism, particularly in embryonic development and stem cell functions. However, the specific role of Myc in porcine early embryonic development is not fully understood. This study observed high Myc expression during the four-cell stage of porcine embryos. Inhibition of Myc using 10058-F4 impaired embryonic development, disrupted energy metabolism, and increased DNA methylation. Mechanistically, these effects were dependent on α-KG, a TCA cycle intermediate and cofactor for TET demethylation enzymes. Sequencing analysis of four-cell embryos post-Myc inhibition revealed downregulation of key metabolic enzymes related to α-KG, such as CS, IDH2, leading to reduced α-KG levels. Supplementation with α-Ketoglutarate (α-KG) mitigated the negative effects of Myc inhibition, including lower blastocyst rates, decreased ATP levels, and increased 5 mC levels. In conclusion, Myc regulates the expression of key metabolic enzymes during the four-cell stage, influencing early embryonic metabolism and epigenetic reprogramming.

Nanopore-Based Sequencing of the Full-Length Transcriptome of Male and Female Cleavage-Stage Embryos of the Chinese Mitten Crab (Eriocheir sinensis).

The cleavage stage plays a crucial role in embryo development, characterized by a swift surge in cell proliferation alongside the accurate genetic material transmission to offspring. To delve into the characteristics of sex development during the cleavage stage of embryos, we generated the full-length transcriptome of Eriocheir sinensis male and female cleavage-stage embryos using Oxford Nanopore Technologies (ONT). Notably, this investigation represents the first sequencing effort distinguishing between genders in E. sinensis embryos. In the transcriptome structure analysis, male and female cleavage-stage embryos, while not clustered, exhibited a comparable frequency of alternative splicing (AS) occurrences. We also successfully identified 2875 transcription factors (TFs). The quantitative analysis showed the top 150 genes, in which the highly expressed genes in male embryos predominantly related to protein synthesis and metabolism. Further investigation unveiled 500 differentially expressed genes (DEGs), of which 7 male-biased ribosomal protein genes (RPGs) were particularly noteworthy and further confirmed. These analyses suggest that there may be a more active protein synthesis process in male E. sinensis cleavage-stage embryos. Furthermore, among the 2875 identified TFs, we predicted that 18 TFs could regulate the differentially expressed RPGs, with most TFs belonging to the zf-C2H2 and Homeobox families, which are crucial for embryonic development. During the cleavage stage of E. sinensis, the differential RPGs between genders were intricately linked to energy metabolism. We proposed that these RPGs exert regulatory effects on gene expression in E. sinensis, thereby regulating the difference of development between male and females. Our research sheds light on the developmental mechanisms of E. sinensis during the embryo stage and establishes a groundwork for a deeper understanding of sex development in E. sinensis. The results also provide comprehensive full-length transcriptome data for future gene expression and genetic studies in E. sinensis.

Novel application of metabolic imaging of early embryos using a light-sheet on-a-chip device: a proof-of-concept study.

Is it feasible to safely determine metabolic imaging signatures of nicotinamide adenine dinucleotide [NAD(P)H] associated auto-fluorescence in early embryos using a light-sheet on-a-chip approach? We developed an optofluidic device capable of obtaining high-resolution 3D images of the NAD(P)H autofluorescence of live mouse embryos using a light-sheet on-a-chip device as a proof-of-concept. Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. Indeed, in recent years, metabolic imaging has been investigated using highly advanced microscopy technologies such as fluorescence-lifetime imaging and hyperspectral microscopy. The potential safety of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67 h post metabolic imaging at the two-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system non-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). The microstructures were fabricated following a standard UV-photolithography process integrating light-sheet fluorescence microscopy into a microfluidic system, including on-chip micro-lenses to generate a light-sheet at the centre of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce two-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between the study groups. A convolutional neural network (ResNet 34) model using metabolic images was also trained. The optofluidic device was capable of obtaining high-resolution 3D images of live mouse embryos that can be linked to their metabolic activity. The system's design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet and fast imaging of the embryos (<2 s), while keeping a low dose of light exposure (16 J · cm-2 and 8 J · cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable of obtaining 30 times higher signal-noise-ratio images than images obtained with a confocal system (P < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (P > 0.05; Yate's chi-squared test). Additionally, embryos with inhibited metabolic activity showed a decreased blastocyst formation rate of 22.2% compared to controls, as well as a 47% reduction in metabolic activity measured by metabolic imaging (P < 0.0001; t-test). This indicates that the optofluidic device was capable of producing metabolic images of live embryos by measuring NAD(P)H autofluorescence, allowing a novel and affordable approach. The obtained metabolic images of two-cell embryos predicted blastocyst formation with an AUC of 0.974. N/A. The study was conducted using a mouse model focused on early embryo development assessing illumination at the two-cell stage. Further safety studies are required to assess the safety and use of 405 nm light at the blastocyst stage by investigating any potential negative impact on live birth rates, offspring health, aneuploidy rates, mutational load, changes in gene expression, and/or effects on epigenome stability in newborns. This light-sheet on-a-chip approach is novel and after rigorous safety studies and a roadmap for technology development, potential future applications could be developed for ART. The overall cost-efficient fabrication of the device will facilitate scalability and integration into future devices if full-safety application is demonstrated. This work was partially supported by an Ideas Grant (no 2004126) from the National Health and Medical Research Council (NHMRC), by the Education Program in Reproduction and Development (EPRD), Department Obstetrics and Gynaecology, Monash University, and by the Department of Mechanical and Aerospace Engineering, Faculty of Engineering, Monash University. The authors E.V-O, R.N., V.J.C., A.N., and F.H. have applied for a patent on the topic of this technology (PCT/AU2023/051132). The remaining authors have nothing to disclose.

RNA Extraction from Rice Immature Embryo Using Laser Capture Microdissection.

Laser capture microdissection (LCM) enables the selective isolation of organs, tissues, and cells from surrounding tissues. Total RNA extracted from small tissue sections can be used for a variety of subsequent analysis such as RNA-seq analysis. Here, we describe a method for isolating embryos from rice ovary sections using LCM and extracting total RNA. We successfully obtained sufficient amount of total RNA from a single rice embryo at the globular embryo stage for RNA-seq analysis.

Embryotrophic effect of exogenous protein contained adipose-derived stem cell extracellular vesicles

BackgroundExtracellular vesicles (EVs) regulate cell metabolism and various biological processes by delivering specific proteins and nucleic acids to surrounding cells. We aimed to investigate the effects of the cargo contained in EVs derived from adipose-derived stem cells (ASCs) on the porcine embryonic development.MethodsASCs were isolated from porcine adipose tissue and characterized using ASC-specific markers via flow cytometry. EVs were subsequently extracted from the conditioned media of the established ASCs. These EVs were added to the in vitro culture environment of porcine embryos to observe qualitative improvements in embryonic development. Furthermore, the proteins within the EVs were analyzed to investigate the underlying mechanisms.ResultsWe observed a higher blastocyst development rate and increased mitochondrial activity in early stage embryos in the ASC-EVs-supplemented group than in the controls (24.8% ± 0.8% vs. 28.6% ± 1.1%, respectively). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of blastocysts also revealed significantly reduced apoptotic cells in the ASC-EVs-supplemented group. Furthermore, through proteomics, we detected the proteins in ASC-EVs and blastocysts from each treatment group. This analysis revealed a higher fraction of proteins in the ASC-EVs-supplemented group than in the controls (1,547 vs. 1,495, respectively). Gene analysis confirmed that ASC-EVs showed a high expression of tyrosine-protein kinase (SRC), whereas ASC-EVs supplemented blastocysts showed a higher expression of Cyclin-dependent kinase 1 (CDK1). SRC is postulated to activate protein kinase B (AKT), which inhibits the forkhead box O signaling pathway and activates CDK1. Subsequently, CDK1 activation influences the cell cycle, thereby affecting in vitro embryonic development.ConclusionASC-EVs promote mitochondrial activity, which is crucial for the early development of blastocysts and vital in the downregulation of apoptosis. Additionally, ASC-EVs supply SRC to porcine blastocysts, thereby elongating the cell cycle.

Lysosomal catabolic activity promotes the exit of murine totipotent 2-cell state by silencing early-embryonic retrotransposons.

During mouse preimplantation development, a subset of retrotransposons/genes are transiently expressed in the totipotent 2-cell (2C) embryos. These 2C transcripts rapidly shut down their expression beyond the 2C stage of embryos, promoting the embryo to exit from the 2C stage. However, the mechanisms regulating this shutdown remain unclear. Here, we identified that lysosomal catabolism played a role in the exit of the totipotent 2C state. Our results showed that the activation of embryonic lysosomal catabolism promoted the embryo to exit from the 2C stage and suppressed 2C transcript expression. Mechanistically, our results indicated that lysosomal catabolism suppressed 2C transcripts through replenishing cellular amino-acid levels, thereby inactivating transcriptional factors TFE3/TFEB and abolishing their transcriptional activation of 2C retrotransposons, MERVL (murine endogenous retrovirus-L)/MT2_Mm. Collectively, our study identified that lysosomal activity modulated the transcriptomic landscape and development in mouse embryos and identified an unanticipated layer of transcriptional control on early-embryonic retrotransposons from lysosomal signaling.

The presence of inclusions in blastodiscs of coho salmon embryos (Oncorhynchus kisutch) is associated with low rates of fertility and embryo survival

Characteristics of the first blastomeres in fish have been evaluated in previous studies as a tool for predicting egg and larva quality. Nevertheless, there are still few studies of the presence of inclusions in fish blastodiscs or blastomeres. The object of the present study was to describe the relationship between cellular inclusions and some embryo quality parameters (symmetry, fertility rate, survival rate) in farmed coho salmon. Eggs from 261 females were fertilized with 400 × 103 spermatozoa/egg and incubated in the dark in separated flow-through incubators. Embryos at the four-cell stage were fixed in Stockard's solution and the blastodiscs were classified as: i) unfertilized, ii) fertilized with inclusion, and iii) without inclusion. The blastomere symmetry was also recorded. Embryos from 30 females (15 with and 15 without inclusions) were incubated until pigmented eye embryo stage to determine the survival rate. Cell inclusions were detected in 8.81 % of the females evaluated, with 24.1 ± 24.2 of the eggs from each of those females presenting inclusions. Egg samples with this abnormality showed significantly lower fertility, blastomere symmetry, and survival rate when compared with eggs without inclusions.

SHORT-ROOT specifically functions in the chalazal region to modulate assimilate partitioning into seeds.

Nourishing the embryo with endosperm and enclosing both embryo and endosperm in the seed coat are two important evolutionary innovations. Seed coat is conventionally viewed as a protective layer that functions after the seed has matured. Here, we challenge this notion by showing that a subregion of the seed coat, termed the chalazal seed coat (CZSC), is geared to gate seed nutrition loading in developing seeds. The CZSC develops the coordinative system comprising the apoplastic isolation, mediated by the restricted suberization, and the active transport, mediated by the specific expression of a variety of transporters, at as early as the globular embryo stage in both Arabidopsis and soybean seeds. This coordinated system in the CZSC disrupts the vascular continuum to the maternal tissues and forces the nutrient transport into selective and active absorption. We further reveal that the precision of the spatiotemporal suberin deposition and transporter expression is controlled by the regulatory hierarchy of SHR-MYBs cascades. Our results provide a mechanistic insight into the assimilate accumulation in dicot seeds.

Induction and maturation of somatic embryogenesis in mangaba tree a multipurpose Apocynaceae

This study reported the first results of in vitro somatic embryogenesis induction in Hancornia speciosa Gomes (mangaba tree, Apocynaceae). To induce callus, the TC accession leaf and nodal segments were cultivated in culture medium I (Murashige and Skoog; MS salts, putrescine, 45 µM 2,4-dichlorophenoxyacetic acid; 2,4-D and 22.19 µM 6-benzylaminopurine; BA). For the next stage (multiplication), the callus masses were sub-cultured into a culture medium II (MS salts, 22.62 µM 2,4-D and 22.19 µM BA). After 90 days, embryogenic calluses were inoculated to somatic embryo maturation medium culture III (MS salts, polyethylene glycol; PEG (0 and 2%) and BA (44.39 and 66.58 µM). Pro-embryos and somatic embryos in the maturation stage were assessed after 60 days. The rstatix package from R software with non-parametric tests (p &lt; 0.05) were used to analysis data of experiments I and II. The Tukey’s test (p &lt; 0.05) was performed on the data of experiment III. To induce and multiply embryogenic callus, 45 µM 2,4-D and 22.19 µM BA were efficient. More pro- and somatic embryo development were seen in the nodal explant cultured in the presence of 44.39 and 66.58 µM BA and 2% PEG. Putrescine does not promote the embryogenic masses of callus derived from leaf explants in the TC accession. Keywords: Hancornia speciosa Gomes, micropropagation, somatic embryo.

Reduced Sox7 contributes to abnormal valve development resulting in congenital aortic stenosis

Abstract Background Abnormal valve development is the most common congenital heart malformation. During valve remodeling, abnormal changes in valve cell proliferation, apoptosis, and extracellular matrix synthesis affect valve development, resulting in malformations. The molecular mechanisms underlying pathophysiology of aortic stenos is remain unclear. Purpose We would like to explore how Sox7 is involved in aortic valve development. Methods and Results We proved Sox7 was mainly expressed in endocardial and mesenchymal cells of aortic valves by immunofluorescence staining determined by at different stages of mice (E10.5～8 weeks) and human embryos and RNA-seq of E9.5 mouse embryonic hearts. We constructed a conditional Sox7 floxed allele in endothelial cells using an endocardial specific Cre. Echocardiography data showed that Nfat-KO mice had increased blood velocity, pressure gradient across aortic valves, left ventricular mass and dilated aortic roots. Then, HE and Sirius red staining to examine histological changes in aortic valves of Nfat-KO mice were performed, and we observed a remarkable increase in the size and increased staining of fibrotic cells in aortic valves of KO mice. Next, we collected embryonic hearts of Sox7 CNC-specific knockout mice (WNT1-KO) and controls at E16.5. According to HE staining results and echocardiography analysis, there was no difference in aortic valves between WNT1-KO mice and controls. Meanwhile, we collected Nfat-KO mice and control embryos at E11.5 days. HE staining results showed that EndMT process was not impaired. Interestingly, leaflets and cusps of Nfat-KO mice showed significantly augmented proliferation levels, decreased apoptosis levels, and calcium deposition in fibrin layer. A significant decrease in MMP9 levels were observed in both mRNA and protein levels. Co-transfection of Sox7-expressing vector and MMP9 luciferase in MEEC suggested that Sox7 significantly activated MMP9. We used adenovirus to overexpress Sox7 in valvular interstitial cells. Compared with that in control group, the Sox7-overexpression group increased protein level of MMP9 and cleaved caspase-3, and increased Tunel-positive rate and lower Ki-67 positive rate. Finally, we detected expression of Sox7 protein in human fetal aortic valves derived from patients with aortic stenosis using immunofluorescence. The results demonstrated that the number of Sox7 positive mesenchymal cells in human fetal aortic valves of patients was significantly lower than that in control group. Conclusion We demonstrated that targeted inactivation of Sox7 in the endocardium of mice affects valve development and leads to aortic stenosis. Mechanistically, no influence of Sox7 on the EndMT process of the OFT cushion. Moreover, CNC-derived Sox7 was not involved in aortic valve development. Interestingly, we found that Sox7 regulated ECM remodeling through matrix MMP9 and Sox7 had an impact on apoptosis, proliferation, and calcification.

Entire loosening stage monitoring of bolted joints via nonlinear electro-mechanical impedance spectroscopy

This article proposes a nonlinear electro-mechanical impedance spectroscopy (NEMIS) methodology for the entire loosening stage monitoring of bolted joints. Unlike the conventional electro-mechanical impedance spectroscopy (EMIS) implemented in the frequency domain, NEMIS utilizes a temporal interrogating signal to procure the developed nonlinear impedance spectrum. It can harness the structural resonance information akin to the conventional EMIS, while concurrently capturing the contact acoustic nonlinearity (CAN) features with only one solitary Piezoelectric Wafer Active Sensor (PWAS). To elucidate the underlying principles of NEMIS, a comparative analysis juxtaposing the conventional EMIS, nonlinear ultrasonic techniques, and the proposed NEMIS was conducted. Subsequently, a reduced-order analytical model was established to simulate the rough contact interfaces of the bolted joints under various looseness states. Parametric studies were carried out to demonstrate the resonance deviation and nonlinear features resulting from the bolt looseness. Ultimately, the experimental implementation of NEMIS for the entire loosening stage monitoring of the bolted joint was performed compared with the conventional EMIS method. It was proved that NEMIS could detect both the incipient bolt loosening at an embryo stage and severe loosening of the bolted joint at the terminal period, integrating the continuous monitoring capability of conventional EMIS and the sensitivity of nonlinear ultrasonic methodology. The quantification on the severity of bolt loosening was accomplished via both linear and nonlinear damage indices, exhibiting a consistent trend with the gradations of bolt looseness. The article culminates in a summary, concluding remarks, and suggestions for future work.

Hyperosmolality tolerance of freshwater largemouth bass Micropterus salmoides to brackish in early ontogenetic stages of eggs, embryos, and yolk-sac larvae

Hyperosmolality tolerance of freshwater largemouth bass Micropterus salmoides to brackish in early ontogenetic stages of eggs, embryos, and yolk-sac larvae

Regulation Mechanisms of the Glutamate Transporter in the Response of Pacific Oyster upon High-Temperature Stress.

Glutamate transporters (GLTs) are integral to the glutamatergic system, modulating glutamate homeostasis to enhance resilience and resistance against environmental stress. There are six GLTs identified in the Pacific oyster (Crassostrea gigas), which were categorized into two subfamilies: excitatory amino acid transporters (CgEAATs) and vesicular glutamate transporters (CgVGLUTs). The CgEAATs harbor a GltP domain, while CgVGLUTs feature an MFS domain, both with conserved sequence and structural characteristics. The expression of CgGLTs is elevated during the planktonic larval stage compared to the fertilized egg stage and is constitutively expressed in various tissues of adult oysters, suggesting its critical role in both larval development and the physiological processes of adult oysters. Transcriptomic analysis revealed diverse expression patterns of GLTs in oyster gills after 7 days of high-temperature stress, with CgEAAT3 showing a significant upregulation. A KEGG pathway enrichment analysis identified glutathione metabolism and ferroptosis as prominently enriched pathways. At 48 h after high-temperature stress, the expression levels of Glutathione Peroxidase 4 (CgGPX4) and CgEAAT3, along with elevated Fe content in the gills, significantly increased. Moreover, the RNAi-mediated the inhibition of CgEAAT3 expression under high-temperature stress, resulting in a significant reduction in CgGPX4 expression and a further increase in Fe accumulation in oyster gills. These results indicate that CgEAAT3 contributes to the regulation of ferroptosis and redox homeostasis by modulating CgGPX4 expression. This study provides new insights into the adaptive mechanisms of bivalves to environmental stress.

Reduced NET1 adversely affects early embryonic development in mice

Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily-specific guanine/nucleotide-exchange factor that exhibits critical roles in diverse biological processes. However, the functions in mouse preimplantation embryonic development have not yet determined. In the present study we demonstrated that NET1 is a key factor in the outcome of early mouse embryonic development. Immunofluorescence detection showed that NET1 is principally localized to the nucleus during mouse pre-implantation embryonic development. Silencing Net1 at the zygote stage using a specific siRNA impaired the developmental competence of early mouse embryos, and Net1-knockdown (Net1-KD) induced mitotic spindle-assembly defects and chromosomal alignment abnormalities at the first embryonic cleavage. In addition, reduced NET1 exacerbated reactive oxygen species production and DNA lesions in two-cell stage embryos, further augmenting cellular apoptosis in the preimplantation blastocyst. In summary, our data display key roles for NET1 in mitotic spindle assembly, oxidative stress, and DNA damage during early mouse embryonic development.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development. However, they may not be suitable for large-scale studies. The aim of this study was to investigate if a zebrafish-based xenograft model can recapitulate MB growth and enable rapid drug testing. Nine different MB cell lines or patient-derived cells were transplanted into blastula-stage zebrafish embryos. Tumor development and migration were then monitored using live imaging. RNA sequencing was performed to investigate transcriptome changes after conditioning cells in neural stem cell-like medium. Furthermore, drug treatments were tested in a 96-well format. We demonstrate here that transplantation of MB cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing. Blastula-stage zebrafish MB xenografts present an alternative to current MB mouse xenograft models, enabling quick evaluation of tumor cell growth, neurotropism, and drug efficacy.

Transcriptome analysis reveals the dynamics of gene expression during early embryonic development of hybrid groupers

Distant hybridization is widely applied in genetic improvement of aquatic animals. Although this technique should be suitable for genetic improvement of grouper, few studies related to offspring produced via distant hybridization have been reported. There we studied the gene expression dynamics of early embryonic development of groupers from self-breeding, intraspecific breeding and intergeneric hybridization. We first obtained offspring from female brown-marbled grouper (Epinephelus fuscoguttatus) and male leopard coral grouper (Plectropomus leopardus) and described the morphological characteristics of different developmental stages of embryos. We then constructed the transcriptome maps during early embryonic development of hybrid groupers. In addition, we also found that the process of MZT occurred from high blastula to early gastrula stage in the embryo of hybrid grouper and that wnt signaling pathway play an important role in dorsal-ventral axial formation during embryonic development of grouper. These results provide a foundation for the hybrid breeding of leopard coral grouper.

The impact of maternal anti-inflammatory drugs on surgical anesthesia-induced neuroinflammation and cognitive impairment in offspring mice.

The impact of maternal surgery combined with general anesthesia on neuroinflammation and the development of learning and memory impairment in offspring remains unclear. This study utilized a pathogen-free laparotomy model to investigate these changes during the second trimester, as well as their response to anti-inflammatory therapy. C57BL/6 pregnant mice at the 14.5-day embryo stage (E 14.5) were either exposed to sevoflurane anesthesia alone or underwent laparotomy procedure. The neuroinflammatory response was evaluated at 7, 14, 21, and 28 days postnatal (P7, P14, P21, P28). Tau phosphorylation and cognitive ability were assessed at P28 and P30, respectively. The impact of perioperative administration of ibuprofen (60 mg/kg) on these aforementioned changes was subsequently evaluated. In the laparotomy group, levels of inflammatory factors (IL-4, IL-8, IL-17A, TGF-β, M-CSF, CCL2) in the brains of offspring mice, including the cerebral cortex and hippocampus, remained consistently elevated from P7 to P28. At P14, while the majority of inflammatory cytokine has no statistical difference, there was still a significant reactivation of inflammatory cytokines observed in the frontal cortex and hippocampus at P28. Furthermore, abnormal phosphorylation of tau and deficits in learning and memory were observed at P28 and P30. Administration of perioperative ibuprofen led to improvements in cognitive performance, reduction of systemic inflammation, and inhibiting abnormal phosphorylation of tau in the frontal cortex and hippocampus. Our findings indicate that cognitive dysfunction is correlated with elevated levels of inflammatory cytokines and tau phosphorylation. Cognitive impairment and tau phosphorylation after laparotomy can persist at least until P28. Anti-inflammatory medications have been shown to enhance cognitive function by rapidly reducing inflammation in the brain, while also impacting neurological changes. This discovery may have implications for the development of treatment strategies aimed at managing cognitive impairment in post-operative patients.