Early Stage Embryos Research Articles

The yin and yang of self-regulation in developing vertebrates.

All living organisms are thermodynamic open systems constantly exchanging energy with the environment to maintain organization and structure. In a state of non-equilibrium they undergo a back and forth pattern of self-regulation and dysregulation in energy exchange. This state of dynamic non-equilibrium can be observed during vertebrate development in which high phenotypic variation and plasticity exists, especially in the presence of stressors. While investigations of the effects of stressors on mechanisms of self-regulation are usually measured by systemic changes (e.g. metabolic rate) between baseline (control) and stimulus (or stressor), real world stressors do not switch on and off in predictable patterns, rarely occur alone, and can be acute (short-term) or chronic (long-term). In this short review, application of two processes underlying self-regulation and dysregulation are explored, 1) allostasis, stability through change or the processes underlying self-regulation and, 2) metastasis, instability through change or the processes underlying dysregulation, to understand the effects of environmental stressors on the energetics of fish early life history stages (embryos and larvae). In mammalian physiology, allostasis theory was developed to maximize the probability of survival under stress, while reducing or limiting somatic damage. Yet, allostatic responses have energetic costs. Multiple stress responses over time result in systemic somatic damage accompanied by a loss of resilience due to an inability to self-regulate. Allostatic costs and their systemic effects on neuroendocrine, metabolic, cardiovascular and immune systems are cumulative and understood for adults but not yet for earlier stages. Developing stages with greater challenges for allostatic self-regulation than older stages because of limited resources have higher growth rates, smaller aerobic scopes, elevated metabolic costs, tight energy budgets, and employ compensatory versus additive energy budgets. Along with high mortality, early stages are expected to exhibit decreased physiological resilience and increased vulnerability in response to stressors, but little examination of energetic strategies to preserve functional stability in the face of stressors exists. While dysregulation/metastatic processes are more difficult to document in development, they increase our understanding of how organisms exposed to chronic/multiple stressors may reach allostatic overload leading to either, 1) systemic dysfunction and/or death or possibly, 2) novel physiological adaptive states. Evaluation of factors driving the yin and yang of self-regulation and dysregulation will provide knowledge of factors triggering or retarding metastatic processes, thus identifying those that prevent or reduce their affects, important in a world undergoing rapid global change.

When is warmer better? Disentangling within‐ and between‐generation effects of thermal history on early survival

Abstract Understanding the fitness consequences of thermal history is necessary to predict organismal responses to global warming. This is especially challenging for ectotherms with complex life cycles, where distinct life stages can differ in thermal sensitivity, acclimate to different thermal environments and accrue responses to acclimation within and between generations. Although acclimation is often hypothesized to benefit organisms by helping them (or their offspring) to compensate for negative impacts of environmental change, mixed support for this hypothesis highlights the need to assess alternatives. Assessments that explicitly dissect responses across life stages and generations, however, remain limited. We assess alternative hypotheses of acclimation (none, beneficial, colder‐is‐better and warmer‐is‐better) within and between generations of a marine tubeworm whose vulnerability to warming rests on survival at early planktonic stages (gametes, embryos and larvae). First, we acclimate parents, gametes and embryos to ambient and projected warmer temperatures (17°C and 22°C) factorially by life stage. Next, we rear offspring with differing acclimation histories to the end of larval development at test temperatures from 10°C to 28°C (lower and upper survival limits respectively). Last, we estimate thermal survival curves for development, and compare them among thermal histories. We show that survival curves are most responsive to parental acclimation followed by acclimation at embryogenesis, but are buffered against acclimation at fertilization. Moreover, curves respond independently to acclimation within and between generations, and respond largely as predicted by the warmer‐is‐better hypothesis, despite the semblance of beneficial acclimation after successive doses of warmer temperature. Our study demonstrates the varied nature of thermal acclimation and the importance of considering how responses aggregate across complex life cycles when predicting vulnerability to warming. Read the free Plain Language Summary for this article on the Journal blog.

The effect of the egg-predator Carcinonemertes conanobrieni on the reproductive performance of the Caribbean spiny lobster Panulirus argus

BackgroundThe Caribbean spiny lobster Panulirus argus is heavily fished throughout its Greater Caribbean and Gulf of Mexico distribution, suggesting a heightened susceptibility to a fisheries collapse. In 2017, a nemertean worm, Carcinonemertes conanobrieni was described from ovigerous females of P. argus in Florida, USA. A year later, the presence of the same egg predator was recorded along the southern Caribbean coast (Colombia). The effect of this egg predator on the reproductive performance, including fecundity, embryo mortality, and reproductive output, of its host is unknown. This study tested whether C. conanobrieni affects embryo mortality, fecundity, and reproductive output in brooding females of P. argus.ResultsArtisan fishers caught 90 ovigerous lobsters near Pueblo Viejo, Magdalena, Colombia. Each ovigerous female was examined for the presence/absence of the egg predator. Lobster egg mortality (%), fecundity (nº eggs female−1), and reproductive output (%) were estimated. Prevalence of C. conanobrieni in the studied population was 87.78%. The mean intensity of C. conanobrieni (all life stages) in the population was 11.68 (± 1.98) egg predators per brood mass sample. Infected females brooding late-stage embryos exhibited lower fecundity, lower reproductive performance values, and higher embryo mortality compared to infected females brooding early-stage embryos. Embryo stage and worm infection level negatively impacted fecundity and reproductive output. Worm infection level and the number of adult nemertean worms also negatively affected embryo mortality.ConclusionsThese results demonstrate an adverse effect of C. conanobrieni on the reproductive performance of P. argus. The interactive impact of this egg predator, natural stressors, and anthropogenic stressors on individual P. argus reproductive performance could facilitate losses at large-scale fisheries levels.

P-136 An embryo evaluation algorithm based on deep learning correlates strongly with cell number and degree of fragmentation on day 2 and 3

Abstract Study question Does the embryo evaluation model iDAScore v2.0 correlate with manually assessed cell numbers and fragmentation grade in early-stage embryos? Summary answer iDAScore v2.0 significantly correlated with morphological assessment of preimplantation embryos on days 2 and 3. Correlation between cell numbers and iDAScore decreased with increasing fragmentation. What is known already Time-lapse technology, including continuous documentation of embryo development, has enabled the implementation of more automated scoring systems, based on images and video sequences. iDAScore (ranging from 1-9.9) is a fully automated annotation-free scoring system based on deep learning and has been shown to discriminate between implanting and non-implanting embryos to a similar extent as KIDScore, which required manual annotations. iDAScore v2.0 also demonstrated a good agreement between predicted probabilities and observed implantation rates. However, not much is known regarding the correlation between specific morphological day 2 and day 3 variables and iDAScore v2.0. Study design, size, duration Retrospective observational study from two ART centers, together performing > 2000 OPU per year. The study includes all normally fertilized embryos (n = 5378) from 1792 treatments between 2016 and 2021 where fresh single embryo transfer was performed on day 2 or day 3 post fertilization. The association of iDAScore v2.0 with manual assessment of cell numbers and grade of fragmentation on day 2 (treatments = 762, embryos = 2170) and day 3 (treatments = 1030, embryos = 3208) was analysed using video sequences. Participants/materials, setting, methods Inclusion criteria were treatment cycles with own (autologous) oocytes and embryo transfer on day 2 or 3. Exclusion criteria were cycles with double embryo transfers or PGT cycles. Clinical and laboratory processes were performed according to standard procedures. Time-lapse culture and embryo annotations were performed using the Embryoscope+® with GTL® culture medium (Vitrolife, Gothenburg, Sweden). Wilcoxon rank sum test was used to compare iDAScores between groups. Main results and the role of chance Day 2: For the number of cells, iDAScore differed between embryos having < = 3 cells compared to > = 4 cells (medians: 2.3 vs. 4.0, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 4.7; 3.9; 2.6 and 2.1, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When cell numbers and fragmentation grade were combined, it was seen that having < = 3 cells versus > = 4 was significantly correlated to iDAScore when the fragmentation grade was scored as 0%, but this association decreased as the fragmentation grade increased. Day 3: A similar pattern was seen for day 3 morphology analyses. It was found that having < = 5 cells vs. > = 6 cells correlated to a significant difference in iDAScore (medians: 2.5 vs. 4.4, p < 0.001). The median iDAScores for fragmentation grade 0%; <20%; <50%; > 50% were 5.6; 3.8; 2.6 and 2.3, respectively. Furthermore, 0% was significantly different from each of the other groups (p < 0.001). When combining cell numbers and fragmentation grade in the same analysis, the association of cell numbers to iDAScore decreased as fragmentation increased. Limitations, reasons for caution The present study is of retrospective design, with analysis of data from two clinics, and the results may not apply in a wider context. In addition, only two morphological variables, cell numbers and fragmentation, were included. Wider implications of the findings Our results show that there is a strong correlation between iDAScore v2.0 and the number of cells and the fragmentation grade of the embryo, both for each individual variable and when combined. This indicates that iDAScore v2.0 identifies some of the well-established morphological evaluations for cleavage stage transfers. Trial registration number Not applicable

ITRAQ proteome analysis of insight into TLR20.2 functions through IFN1 signaling exerts regulatory effects on GCRV replication

Maternal immunization is an efficacious way to protect offspring from pathogen infections. To clarify the immune-enhancing mechanism of maternal immunization in grass carp, the iTRAQ assay was applied to screen differentially expressed proteins (DEPs) among eggs produced from maternal-immunized and non-immunized female grass carp. A total of 138 DEPs were obtained and TLR20.2 was the sole identified consensus DEP among the maternal-immunized and non-immunized groups. In general, the gene and protein expressions of TLR20.2 in the egg cell and early developmental stage embryos could be enhanced by maternal-immunized treatment. The mRNA levels of tlr20.2 were up-regulated in the liver, head kidney, and spleen in response to grass carp reovirus (GCRV) infection. Mechanistically, TLR20.2 functions through the IFN1 signaling and displayed inhibitory functions on GCRV replication by remarkably down-regulation of vp2 and vp7 expressions. The HSP90 was identified to be one of the proteins that interacted with TLR20.2, which coexpressed with TLR20.2 and mainly concentrated around the central veins. HSP90 promotes GCRV replication by inhibiting the irf3, irf7, and ifn1 expressions, which resulted in remarkably increased vp2 and vp7 gene expressions. The HMGB1a and HMGB1b could bind to the TLR20–2 promoter, and showed positive effects on tlr20.2 expression by significantly promoting its promoter activity (P < 0.01). Moreover, HMGB1a and HMGB1b exerted negative effects on HSP90 expression and thus the GCRV replication. Overall, this study provided molecular evidence that maternal immunization treatment protect against GCRV infection through the enhancement of TLR20.2 expression, and the HMGB1-TLR20.2-HSP90-IFN axis confers regulatory effects on GCRV replication. The results are novel findings to help elucidate the resistance-enhancing mechanism in fish species.

Refinements in embryo manipulation applied to CRISPR technology in livestock

The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2–8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.

High-quality total RNA extraction from early-stage lamprey embryos.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density(OD)260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminantsand improved RNA integrity. Egg membrane sources were suspected tocause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

EFFECTIVENESS OF OVINE FOLLICULAR FLUID AND EWE AGE ON IN VITRO NUCLEAR MATURATION AND EMBRYO EVOLUTION OF AWASSI EWES

Two experiments were conducted to study the efficiency of the ovine follicular fluid (OFF) and the age of ewe on in vitro nuclear maturation of Awassi sheep oocytes (Experiment 1) and the subsequent stages of early embryos and their quality (Experiment 2). The OFF has been added to the basic maturation medium (TCM-199) with three different levels (A: 0% (Control group), B:15%, C:25% and D:50%) while the ages of the ewes were determined into two main groups (&gt; 2 and 1-2 year). In the first experiment, across the two age groups, matured oocytes in C solution achieved the highest (p=0.006) rates of metaphase-II (85.70 and 82.35 % respectively). In the second experiment, oocytes belonging to &gt; 2 and 1-2-year ewe age groups (C Solution) achieved the highest (p=0.001) rates of fertilization (23.17 and 30.73% respectively). Similar trend applies to the cleavage stage, the rates reached 35.14 and 41.27% respectively (p=0.01). No significant differences were noticed at the different stages of cleavage (2-4 cell, morula, and blastocyst). The resulting embryos of B (&gt; 2-year ewe age group) and C solution (1-2-year age group) achieved the highest (p=0.04) rates of Type1 embryos (69.56 and 76.92% respectively). A significant difference was noticed in type2 embryos (p=0.02), embryos of the first and second age groups (Solution D) achieved the highest rates (46.15 and 33.33% respectively). It can be concluded that the adding of 25% OFF to the maturation media led to significant improvement in the rates of maturation, cleavage and type1 embryos of sheep oocytes.

Abstract 1293: GPRC5A drives an implantation-like phenotype in early-stage lung disseminated cancer cells of esophageal squamous carcinoma

Abstract Esophageal squamous carcinoma (ESCC) is one of the leading malignancies worldwide, especially in Asian countries, largely threatening human life. The 5-year survival rate of ESCC is less than 20%, and patients with distant metastases have dismal survival with &amp;lt;5% of five-year survival rate. Traditional therapies e.g., surgery, chemotherapy, radiotherapy demonstrate poor clinical outcomes for ESCC patients. Metastases are commonly seen in ESCC patients, and lung metastases approximately account for 5% of all ESCC metastases. The underlying mechanism of ESCC lung metastasis remains unsettled. In our study, we established ESCC lung metastasis mice model via intravenous injection of ESCC cells in multiple timepoints. Lung disseminated cancer cell (DCCs) of different timepoints were isolated and thrown into single cell RNA sequencing (scRNA-seq). Via bioinformatic analysis, we found the upregulated genes in early-stage lung DCCs were highly enriched in embryo development, placenta development, pre-implantation embryos processes, neither in late-stage lung DCCs. It highly suggested that ESCC lung metastasis process might be similar to embryo implantation. Based on above findings, 5 intersected genes (GPRC5A, FSTL3, WNT7A, TM4SF1, NT5E) of early-stage lung DCCs and embryo implantation were figured out to construct an implantation-like signature. Enrichment analysis results implied that ESCCs subgroups with implantation-like phenotype showed higher ability to migration, focal adhesion and response to stress. Preliminary data indicated that the representative gene GPRC5A was upregulated in ESCC tumors compared with normal esophageal epithelium in clinical samples and public databases. GPRC5A overexpression promoted tumor growth, invasion, migration and sphere formation both in vivo and in vitro, as the same as other two signature genes, WNT7A and FSTL3. Moreover, GPRC5A-overexpresssing ESCC cells displayed high ability to develop resistance to stress and generated less reactive oxygen species (ROS) in vitro. In vivo, GPRC5A overexpression increased the quantalities of early-stage lung DCCs in ESCC lung metastasis mice model and disruption of GPRC5A expression abolished its pro-metastatic effect. Together, our preliminary findings identify an implantation-like signature in early-stage ESCC lung metastasis and disrupting this phenotype helps to prevent ESCC tumorigenesis and metastasis. Citation Format: Hongyu Zhou, Beilei Liu, Ching Ngar Wong, Xinyuan Guan. GPRC5A drives an implantation-like phenotype in early-stage lung disseminated cancer cells of esophageal squamous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1293.

Maslinic Acid Supplementation during the In Vitro Culture Period Ameliorates Early Embryonic Development of Porcine Embryos by Regulating Oxidative Stress

As a pentacyclic triterpene, MA exhibits effective free radical scavenging capabilities. The purpose of this study was to explore the effects of MA on porcine early-stage embryonic development, oxidation resistance and mitochondrial function. Our results showed that 1 μM was the optimal concentration of MA, which resulted in dramatically increased blastocyst formation rates and improvement of blastocyst quality of in vitro-derived embryos from parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Further analysis indicated that MA supplementation not only significantly decreased the abundance of intracellular reactive oxygen species (ROS) and dramatically increased the abundance of intracellular reductive glutathione (GSH) in porcine early-stage embryos, but also clearly attenuated mitochondrial dysfunction and inhibited apoptosis. Moreover, Western blotting showed that MA supplementation upregulated OCT4 (p < 0.01), SOD1 (p < 0.0001) and CAT (p < 0.05) protein expression in porcine early-stage embryos. Collectively, our data reveal that MA supplementation exerts helpful effects on porcine early embryo development competence via regulation of oxidative stress (OS) and amelioration of mitochondrial function and that MA may be useful for increasing the in vitro production (IVP) efficiency of porcine early-stage embryos.

STEEL enables high-resolution delineation of spatiotemporal transcriptomic data.

Advances in spatial transcriptomics enlarge the use of single cell technologies to unveil the expression landscape of the tissues with valuable spatial context. Here, we propose an unsupervised and manifold learning-based algorithm, Spatial Transcriptome based cEll typE cLustering (STEEL), which identifies domains from spatial transcriptome by clustering beads exhibiting both highly similar gene expression profiles and close spatial distance in the manner of graphs. Comprehensive evaluation of STEEL on spatial transcriptomic datasets from 10X Visium platform demonstrates that it not only achieves a high resolution to characterize fine structures of mouse brain but also enables the integration of multiple tissue slides individually analyzed into a larger one. STEEL outperforms previous methods to effectively distinguish different cell types/domains of various tissues on Slide-seq datasets, featuring in higher bead density but lower transcript detection efficiency. Application of STEEL on spatial transcriptomes of early-stage mouse embryos (E9.5-E12.5) successfully delineates a progressive development landscape of tissues from ectoderm, mesoderm and endoderm layers, and further profiles dynamic changes on cell differentiation in heart and other organs. With the advancement of spatial transcriptome technologies, our method will have great applicability on domain identification and gene expression atlas reconstruction.

P66Shc in sheep preimplantation embryos: Expression and regulation of oxidative stress through the manganese superoxide dismutase-reactive oxygen species metabolic pathway.

p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4- to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

Embryonic poly(A)-binding protein interacts with translation-related proteins and undergoes phosphorylation on the serine, threonine, and tyrosine residues in the mouse oocytes and early embryos.

Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.

AhR-mediated lipid peroxidation contributes to TCDD-induced cardiac defects in zebrafish

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant that activates the aryl hydrocarbon receptor (AhR) pathway, has been reported to cause cardiac damage. However, the mechanism underlying AhR-induced cardiac defects in response to TCDD exposure remains unclear. In this study, we characterized the impacts of TCDD exposure on heart morphology and cardiac function in zebrafish. TCDD exposure in the early developmental stage of zebrafish embryos led to morphological heart malformation and pericardial edema, concomitant with reduced cardiac function. These cardiac defects were attenuated by inhibiting AhR activity with CH223191. Transcriptome profiling showed that, along with an upregulation of the AhR signaling pathway by TCDD treatment, the expression of pro-ferroptotic genes was upregulated, while that of genes implicated in glutathione metabolism were downregulated. Moreover, lipid peroxidation, as indicated by malonaldehyde (MDA) production, was increased in TCDD-exposed cardiac tissue. Accordingly, inhibiting lipid peroxidation with liproxstatin-1 reversed the adverse cardiac effects induced by TCDD treatment. Taken together, our findings demonstrate that AhR-mediated lipid peroxidation contributes to cardiac defects in the early developmental stage in zebrafish embryos exposed to TCDD.

HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas.

Walleye Egg Survival Was Unaffected by Four Semen Inclusion Techniques during Artificial Fertilization

Improving the percent hatch during artificial spawning of walleye (Sander vitreus) could save agencies substantial resources. Semen inclusion techniques used to artificially fertilize walleye eggs may have a subsequent influence on the percent hatch. We compared walleye egg survival to the early embryo stage across four semen inclusion techniques used during fertilization to determine if one or more of the techniques would result in a higher percent hatch. Six mL of freshly collected (within 2 h) semen was presented to common pools of walleye eggs separated into 250 mL lots by either 1) pouring semen on top of the eggs before sperm activation, 2) placing 3 mL of semen below and above the eggs before sperm activation, 3) pouring 3 mL of semen on top of the eggs, activating sperm, and adding the remaining 3 mL of semen after 30 s, or 4) activating the sperm by adding semen to 1 L of water, mixing and pouring the solution on the eggs within 3 s. All treatments received 1 L of filtered lake water for sperm activation and fertilization. Mean egg survival was similar ranging from 49.5% to 56.7% among the four techniques and was not significantly different across methods. These results suggest that any of the semen inclusion techniques will likely provide a similar percent hatch for walleye eggs when 250 mL of eggs, 6 mL of semen, and 1 L of water are used during fertilization.

Bioplastic leachates characterization and impacts on early larval stages and adult mussel cellular, biochemical and physiological responses

Bioplastics are promoted as safer alternatives to tackle the long-term persistence of conventional plastics. However, information on the potential release of additives and non-intentionally added substances (NIAS) in the surrounding environment is limited, and biological effects of the leachates have been little studied. Leachates produced from three bioplastics, i.e. compostable bags (CB), bio-polyethylene terephthalate bottles (bioPET) and polylactic acid cups (PLA), and a control polymeric material, i.e. rubber tire (TR), were examined. The chemical nature of bioplastic polyesters PET, PLA and poly (butylene adipate-co-terephthalate) (PBAT) in CB, was confirmed by analytical pyrolysis. Fragments were incubated in artificial sea water for 14 days at 20 °C in darkness and leachate contents examined by GC-MS and HPLC-MS/MS. Catalysts and stabilizers represented the majority of chemicals in TR, while NIAS (e.g. 1,6-dioxacyclododecane-7,12-dione) were the main components of CB. Bisphenol A occurred in all leachates at a concentration range 0.3–4.8 μg/L. Trace metals at concentrations higher than control water were found in all leachates, albeit more represented in leachates from CB and TR. A dose response to 11 dilutions of leachates (in the range 0.6–100%) was tested for biological effects on early embryo stages of Mytilus galloprovincialis. Embryotoxicity was observed in the whole range of tested concentrations, the magnitude of effect depending on the polymers. The highest concentrations caused reduction of egg fertilization (CB, bioPET, TR) and of larvae motility (CB, PLA, TR). TR leachates also provoked larvae mortality in the range 10–100%. Effects on adult mussel physiology were evaluated after a 7-day in vivo exposure to the different leachates at 0.6% concentration. Nine biomarkers concerning lysosomal functionality, neurotransmission, antioxidant and immune responses were assessed. All lysosomal parameters were affected, and serum lysozyme activity inhibited. Harmonized chemical and biological approaches are recommended to assess bioplastic safety and support production of sustainable bioplastics.

TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.

Members of PARP family are responsible for poly(ADP-ribose) (pADPr) posttranslational modification synthesis. They are intensively studied proteins with more than 20,500 related papers in PubMed database search to date. PARG, the main enzyme that degrades pADPr, is unfairly attracted less attention, and 40 times less papers (a little more than 500) are related to its functioning. The difficulties to work with PARG knockout animals due to its early embryo lethality could be one reason for this huge difference. Mice PARG-specific antibodies are not available from any vendor, which also complicates the research process. There is one available for public PARG knockout mice line generated by KOMP project. It has LacZ cassette, which replaces three critical exons in PARG gene. Here, we present the method to genotype these mice with Taqman qPCR multiplex approach. It allows to work with a small amount of DNA material like early embryo stages and to separate maternal DNA contamination. The modification of this method is also applicable for studying PARG conditional knockouts and identifying the success of floxed PARG gene exon deletion by Cre-driven recombination.

Comparative Toxicity of 3-5 Ringed Polycyclic Aromatic Hydrocarbons to Skeletal Development in Zebrafish Embryos and the Possible Reason.

Polycyclic aromatic hydrocarbons (PAHs) are pervasive pollutants in the environment. To compare the developmental toxicity of PAHs with different ring numbers to fish embryos, benzo(a)pyrene (BaP), pyrene (Pyr) and phenanthrene (Phe) were selected as the representatives of 3, 4 and 5-ringed PAHs, and fertilized embryos of zebrafish (Danio rerio) were exposed to 5 nM PAHs for 72h. The PAH-treated embryos showed defects in craniofacial cartilage. The order of toxicity to the development of craniofacial cartilage was Phe > Pyr > BaP. The transcription of genes related to the development of craniofacial cartilage was downregulated. The GC-MS/MS detection showed that bioaccumulation of BaP in the exposed embryos was two orders of magnitude lower than that of Phe and Pyr. It is suggested that the more uptake and accumulation of Phe and Pyr could be one of the reasons for their greater toxicity to development in early stage embryos.

Oocyte maturity, oocyte fertilization and cleavage-stage embryo morphology are better in natural compared with high-dose gonadotrophin stimulated IVF cycles

Research questionDoes high-dose gonadotrophin stimulation have an effect on oocyte and early-stage embryo development? DesignThis was a retrospective study including 616 natural cycle IVF (NC-IVF) and 167 conventional IVF (cIVF) cycles. In total, 2110 oocytes were retrieved and analysed in fresh cycles. In NC-IVF, only human chorionic gonadotrophin was applied to trigger ovulation. In cIVF, antagonist protocols with daily 150–300 IU of human menopausal gonadotrophins were performed. The effect of gonadotrophins on oocyte and early-stage embryo development was analysed. Primary outcomes were the occurrence of mature (metaphase II) oocytes, zygotes and embryos with good morphology at the cleavage stage 2 days after oocyte retrieval. ResultsThe mature oocyte rate (number of mature oocytes/number of retrieved oocytes) was higher in NC-IVF than cIVF cycles (89% versus 82%, adjusted odds ratio [aOR] 1.79, P = 0.001), as was the zygote rate per oocyte retrieved (70% versus 58%, aOR 1.76, P = 0.001) and the zygote rate per mature oocyte (79% versus 71%, aOR 1.62, P = 0.001). The percentage of zygotes that developed into cleavage-stage embryos was no different. For the transferred embryos, the probability of having a good embryo morphology with four blastomeres and a fragmentation of <10% (score 0) in cleavage-stage embryos was found to be higher in NC-IVF (proportional aOR for four blastomeres 2.00, P < 0.001; aOR 1.87 for a fragmentation score of 0, P = 0.003). ConclusionsOocyte maturity, oocyte fertilization and morphology of the cleavage-stage embryo are affected by high-dose gonadotrophin stimulation in fresh IVF cycles.