The first embryo, the origin of cancer and animal phylogeny. III. The totipotency as revealed by morphogenesis and the neoplasia controlled by cellular differentiation.

We have extensively described that the neoplastic process (NP) has deep evolutionary roots and we have made specific predictions about the connection between cancer and the formation of the first embryo, which allowed for the evolutionary radiation of metazoans. My main hypothesis is that the NP is at the heart of cellular mechanisms responsible for animal morphogenesis, and given its embryological basis, also at the center of cell differentiation-one of the most interesting and relevant aspects of embryogenesis. In this article, I take forward the idea of the role of physics in the modeling of the neoplastic functional module (NFM) and its contribution to morphogenesis to reveal the totipotency of the zygote. In my consideration of these arguments, I examine mechanical and biophysical clues and their intimate connection with cellular differentiation. I expound on how cancer biology is perfectly intertwined with embryonic differentiation and why it is considered a disease of cell differentiation. The neoplasia is controlled by textural gradients that lead to cell differentiation within the embryo. Thus, the embryo would be a benign tumor. Finally, inspired by evolutionary history and by what the nervous system represents for current biology and based on the impressive nervous system of ctenophores as seen in fossil records, I propose a hypothesis with physical foundations (mechanical morphogenesis) for the formation of a preneural pattern of the nervous system of the first animal embryo.

A mathematical framework for understanding the spontaneous emergence of complexity applicable to growing multicellular systems.

In embryonic development and organogenesis, cells sharing identical genetic codes acquire diverse gene expression states in a highly reproducible spatial distribution, crucial for multicellular formation and quantifiable through positional information. To understand the spontaneous growth of complexity, we constructed a one-dimensional division-decision model, simulating the growth of cells with identical genetic networks from a single cell. Our findings highlight the pivotal role of cell division in providing positional cues, escorting the system toward states rich in information. Moreover, we pinpointed lateral inhibition as a critical mechanism translating spatial contacts into gene expression. Our model demonstrates that the spatial arrangement resulting from cell division, combined with cell lineages, imparts positional information, specifying multiple cell states with increased complexity-illustrated through examples in C.elegans. This study constitutes a foundational step in comprehending developmental intricacies, paving the way for future quantitative formulations to construct synthetic multicellular patterns.

Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators.

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos.

The Effect of Cryopreserved Sperm on the Early Development, Survival, and Growth of Intergeneric Sterbel Hybrids (Acipenser ruthenus × Huso huso).

The aim of this study was to analyze the survival and growth of intergeneric (Acispenser ruthenus × Huso huso L.) sterbel hybrids obtained by fertilizing sterlet eggs with cryopreserved beluga semen. The rate of embryonic development did not differ between sterbel hybrids (experimental groups) and sterlets (control groups), and the hatching period was identical in all groups. The survival rate of hybrid larvae was higher in the experimental groups than in the control groups. Body weight and body length measurements revealed that sterbel hybrids grew at a faster rate than the control group sterlets. The hybrid origin of sterbels produced with the use of cryopreserved beluga semen was confirmed in a genetic analysis based on species-specific DNA fragments. To the best of the authors' knowledge, this is the first study to analyze the growth of sterbel hybrids derived from cryopreserved semen. The research findings indicate that this type of intergeneric hybridization delivers satisfactory results and can be applied in sturgeon aquaculture.

miRNAs in Follicular and Oviductal Fluids Support Global DNA Demethylation in Early-Stage Embryos.

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

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro.

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.

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.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Changes in the Transcription of Proliferation- and Apoptosis-Related Genes in Embryos in Women of Different Ages under the Influence of Extracellular Vesicles from Donor Follicular Fluid In Vitro.

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×1011 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years.

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.

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.

Histone Lactylation Is Involved in Mouse Oocyte Maturation and Embryo Development.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy.

Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

Heat-Stress Impacts on Developing Bovine Oocytes: Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Unveiling the dynamics of embryogenesis and immune genes expression pattern in the amur common carp (Cyprinus carpio haematopterus).

Amur common carp (Cyprinus carpio haematopterus), is a commercially important fish species that has been genetically improved over the years through selective breeding. Despite its significance in aquaculture, limited knowledge exists regarding its embryogenesis and immune genes associated with its early stages of life. This article represents a detailed study of the embryogenesis and innate immune gene expression analysis of the Amur common carp during its ontogenic developments. The entire embryonic developmental process of ∼44 h could be divided into eight periods, beginning with the formation of the zygote, followed by cleavage, morula, blastula, segmentation, pharyngula, and hatching. The segmentation period, which lasted for ∼ 6 h, exhibited the most significant changes, such as muscle contraction, rudimentary heart formation, increased somites number, and the initiation of blood circulation throughout the yolk. The expression of immune-related genes, namely toll-like receptor (TLR)4, nucleotide-binding oligomerization domain (NOD)1, NOD2 and interleukin (IL)-8 showed stage-specific patterns with varying levels of expression across the developmental stages. The TLR4 gene exhibited the highest expression during the neurella stage, while NOD1 and NOD2 peaked during hatching and IL-8 reached its maximum level during the gastrula stage. This is the first report of the innate immune gene expression during the embryogenesis of Amur common carp.