Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing in vitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing in vitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

Plasma-derived extracellular vesicles improve mice embryo development.

BACKGROUND: To investigate the effect of plasma-derived extracellular vesicles (EVs) or conventional medium in fertilization and early embryo development rate in mice. METHODS AND RESULTS: MII oocytes (matured in vivo or in vitro conditions) were obtained from female mice. The extracellular vesicles were isolated by ultracentrifugation of plasma and were analyzed and measured for size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM). By western blotting analysis, the EVs proteins markers such as CD82 protein and heat shock protein 90 (HSP90) were investigated. Incorporating DiI-labeled EVs within the oocyte cytoplasm was visible at 23 h in oocyte cytoplasm. Also, the effective proteins in the early reproductive process were determined in isolated EVs by western blotting. These EVs had a positive effect on the fertilization rate (P < 0.05). The early embryo development (8 cell, morula and blastocyst stages) was higher in groups supplemented with EVs (P < 0.01). CONCLUSION: Our findings showed that supplementing in vitro maturation media with EVs derived- plasma was beneficial for mice's embryo development.

A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment.

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Vascular development, remodeling and maturation.

The development of the vascular system is crucial in supporting the growth and health of all other organs in the body, and vascular system dysfunction is the major cause of human morbidity and mortality. This chapter discusses three successive processes that govern vascular system development, starting with the differentiation of the primitive vascular system in early embryonic development, followed by its remodeling into a functional circulatory system composed of arteries and veins, and its final maturation and acquisition of an organ specific semi-permeable barrier that controls nutrient uptake into tissues and hence controls organ physiology. Along these steps, endothelial cells forming the inner lining of all blood vessels acquire extensive heterogeneity in terms of gene expression patterns and function, that we are only beginning to understand. These advances contribute to overall knowledge of vascular biology and are predicted to unlock the unprecedented therapeutic potential of the endothelium as an avenue for treatment of diseases associated with dysfunctional vasculature.

The control of transitions along the main body axis.

Although vertebrates display a large variety of forms and sizes, the mechanisms controlling the layout of the basic body plan are substantially conserved throughout the clade. Following gastrulation, head, trunk, and tail are sequentially generated through the continuous addition of tissue at the caudal embryonic end. Development of each of these major embryonic regions is regulated by a distinct genetic network. The transitions from head-to-trunk and from trunk-to-tail development thus involve major changes in regulatory mechanisms, requiring proper coordination to guarantee smooth progression of embryonic development. In this review, we will discuss the key cellular and embryological events associated with those transitions giving particular attention to their regulation, aiming to provide a cohesive outlook of this important component of vertebrate development.

Segmentation of mature human oocytes provides interpretable and improved blastocyst outcome predictions by a machine learning model.

Within the medical field of human assisted reproductive technology, a method for interpretable, non-invasive, and objective oocyte evaluation is lacking. To address this clinical gap, a workflow utilizing machine learning techniques has been developed involving automatic multi-class segmentation of two-dimensional images, morphometric analysis, and prediction of developmental outcomes of mature denuded oocytes based on feature extraction and clinical variables. Two separate models have been developed for this purpose-a model to perform multiclass segmentation, and a classifier model to classify oocytes as likely or unlikely to develop into a blastocyst (Day 5-7 embryo). The segmentation model is highly accurate at segmenting the oocyte, ensuring high-quality segmented images (masks) are utilized as inputs for the classifier model (mask model). The mask model displayed an area under the curve (AUC) of 0.63, a sensitivity of 0.51, and a specificity of 0.66 on the test set. The AUC underwent a reduction to 0.57 when features extracted from the ooplasm were removed, suggesting the ooplasm holds the information most pertinent to oocyte developmental competence. The mask model was further compared to a deep learning model, which also utilized the segmented images as inputs. The performance of both models combined in an ensemble model was evaluated, showing an improvement (AUC 0.67) compared to either model alone. The results of this study indicate that direct assessments of the oocyte are warranted, providing the first objective insights into key features for developmental competence, a step above the current standard of care-solely utilizing oocyte age as a proxy for quality.

Embryonic development through in vitro fertilization using high-quality bovine sperm separated in a biomimetic cervix environment.

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

Oscillatory control of embryonic development.

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 µM CGA treatment during the in vitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

The effect of coenzyme Q10 on cryotolerance of in vivo-derived mouse embryos.

OBJECTIVE: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation. METHODS: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 µM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate. RESULTS: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 µM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 µM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 µM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05). CONCLUSIONS: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.

Association between embryo morphokinetic development and intracytoplasmic sperm injection with epididymal sperm via time-lapse imaging.

The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

The carryover effects of embryonic incubation temperature on subsequent growth and thermal tolerance in white sturgeon.

Environmental variation experienced during early periods of development can lead to persistent phenotypic alteration, known as carryover effects. Such effects increase concern for threatened or endangered species such as the white sturgeon (Acipenser transmontanus), particularly considering expected thermal changes due to climate change. We evaluated how temperature during embryonic development affects physiological parameters such as larval and early juvenile growth and thermal tolerance. Nechako River white sturgeon embryos were incubated at different environmental temperatures (Te) of 12 °C (the natural spawning temperature of this population), 15 °C (the hatchery incubation temperature), and 18 °C (representing potential increases in river temperatures given global climate change). After hatch, fish were reared at a common 15 °C for 80 days post-hatch (dph). Individuals from each temperature treatment were tested for thermal tolerance using the critical thermal maximum method (CTmax), euthanized, and measured. Fish were examined at regular intervals from 13 to 80 dph, which bridged the time from the start of exogenous feeding through the transition into early juveniles. We found carryover effects of high embryonic Te in the short term for both thermal tolerance and growth. Fish that developed at 18 °C had the lowest thermal tolerance during the start of exogenous feeding. However, differences in thermal tolerance were small for early juveniles and were unlikely to be ecologically relevant in the longer term. Fish that developed at 18 °C were smallest over the observation period, indicating a possible cost for survival from increasing environmental temperatures during embryonic development. This research represents a window into a critical period of development during which fish are particularly vulnerable to climatic variation, and shows that cooler temperatures (12 °C) during incubation are optimal for this population. The results can inform environmental managers on the best strategies to help conserve current white sturgeon populations across their range.

CPEB3 Maintains Developmental Competence of the Oocyte.

Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.

Individually Cultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in an Extremely Confined Environment.

The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Effect of cytoplasmic fragmentation on embryo development, quality, and pregnancy outcome: a systematic review of the literature.

The role of cytoplasmic fragmentation in human embryo development and reproductive potential is widely recognized, albeit without standard definition nor agreed upon implication. While fragmentation is best understood to be a natural process across species, the origin of fragmentation remains incompletely understood and likely multifactorial. Several factors including embryo culture condition, gamete quality, aneuploidy, and abnormal cytokinesis seem to have important role in the etiology of cytoplasmic fragmentation. Fragmentation reduces the volume of cytoplasm and depletes embryo of essential organelles and regulatory proteins, compromising the developmental potential of the embryo. While it has been shown that degree of fragmentation and embryo implantation potential are inversely proportional, the degree, pattern, and distribution of fragmentation as it relates to pregnancy outcome is debated in the literature. This review highlights some of the challenges in analysis of fragmentation, while revealing trends in our evolving knowledge of how fragmentation may relate to functional development of the human embryos, implantation, and pregnancy outcome.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.