Poissonian Cellular Potts Models Reveal Nonequilibrium Kinetics of Cell Sorting.

Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as ad hoc dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations.

Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Shape of the first mitotic spindles impacts multinucleation in human embryos.

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

Differential proliferation regulates multi-tissue morphogenesis during embryonic axial extension: integrating viscous modeling and experimental approaches.

A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.

Embryonic development of a centralised brain in coleoid cephalopods.

The last common ancestor of cephalopods and vertebrates lived about 580 million years ago, yet coleoid cephalopods, comprising squid, cuttlefish and octopus, have evolved an extraordinary behavioural repertoire that includes learned behaviour and tool utilization. These animals also developed innovative advanced defence mechanisms such as camouflage and ink release. They have evolved unique life cycles and possess the largest invertebrate nervous systems. Thus, studying coleoid cephalopods provides a unique opportunity to gain insights into the evolution and development of large centralised nervous systems. As non-model species, molecular and genetic tools are still limited. However, significant insights have already been gained to deconvolve embryonic brain development. Even though coleoid cephalopods possess a typical molluscan circumesophageal bauplan for their central nervous system, aspects of its development are reminiscent of processes observed in vertebrates as well, such as long-distance neuronal migration. This review provides an overview of embryonic coleoid cephalopod research focusing on the cellular and molecular aspects of neurogenesis, migration and patterning. Additionally, we summarize recent work on neural cell type diversity in embryonic and hatchling cephalopod brains. We conclude by highlighting gaps in our knowledge and routes for future research.

Dependency of mitochondrial quantity on blastocyst timeline obscures its actual effect to pregnancy outcomes.

Objectives: To explore the correlation between mitochondrial quantity and the blastocyst development timeline as well as their respective contributions to early pregnancy. Methods: A retrospective study was conducted using a dataset comprising 2,633 embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2023. The study was divided into three subsets to address distinct aspects: the representativeness of a single trophectoderm (TE) biopsy for mitochondrial quantity (n=43), the correlation between morphokinetic features and mitochondrial quantity (n=307), and the association analysis among mitochondrial quantity, blastocyst timeline factor, and reproductive outcomes (n=2,283). Distribution assessment of mitochondrial quantity across an individual blastocyst involved the identification within multiple biopsies and spent culture media. Timeline evaluation included correlating mitochondrial quantity with time-lapse datasets. Finally, multivariate logistic regression models, incorporating potential effectors alongside mitochondrial quantity, were employed to analyze their respective contributions to early pregnancy endpoints. Results: Of distribution assessment, mitochondrial quantity exhibited an even distribution across the entire trophectoderm (Spearman's ρ=0.82), while no detectable mtDNAs in the corresponding spent culture media. Then the timeline correlation study revealed significant association between mitochondrial quantity and blastocyst features of both the day of expanded blastocyst formation (95% Confidence intervals, CIs: 0.27~4.89, p=0.03) and the timing of expanded blastocyst formation (tEB) (95% CIs: -0.24~-0.01, p=0.04) in the regression model, indicating a strong dependency between mitochondrial quantity and the blastocyst development timeline. For the contribution to early pregnancy, multivariate logistic regression models showed that the day of expanded blastocyst formation contributed to four endpoints persistently: positive for HCG (odd ratio, OR: 0.71, p=0.006), gestational sac (OR: 0.78, p=0.04), fetal heartbeat (OR: 0.71, p=0.004), and progression to 14 weeks (OR: 0.69, p=0.002). Contrastingly, no notable correlation was observed between the mitochondrial quantity and these endpoints. Conclusions: Strong interaction was observed between mitochondrial quantity and the blastocyst timeline, particularly the timing of expanded blastocyst formation. It suggests that the primary determinant influencing pregnancy outcomes lies in the time-dependent parameter of blastocyst rather than in the specific mitochondrial quantity.

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

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.

Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

[Progress on the involvement of HDAC11 in the regulation of mammalian oocyte maturation and early embryonic development].

Oocyte maturation and early embryonic development are key steps in the reproductive physiology of female mammals, and any error in this process can adversely affect reproductive development. Recent studies have shown that epigenetic modifications of histones play important roles in the regulation of oocyte meiosis and quality assurance of early embryonic development. Histone deacetylase 11 (HDAC11) is the smallest known member of the histone deacetylases (HDACs) family, and inhibition of HDAC11 activity significantly suppresses the rate of oocyte maturation, as well as the development of 8-cell and blastocyst embryos at the embryonic stage. This paper focuses on recent progress on the important role of HDAC11 in the regulation of mammalian oocyte maturation and early embryonic development, hoping to gain insights into the key roles played by epitope-modifying proteins represented by HDAC11 in the regulation of mammalian reproduction and their molecular mechanisms.

An aggregated mitochondrial distribution in preimplantation embryos disrupts nuclear morphology, function, and developmental potential.

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.

The beginning of becoming a human.

According to birth certificates, the life of a child begins once their body comes out of the mother's womb. But when does their organismal life begin? Science holds a palette of answers-depending on how one defines a human life. In 1984, a commission on the regulatory framework for human embryo experimentation opted not to answer this question, instead setting a boundary, 14 days post-fertilization, beyond which any experiments were forbidden. Recently, as the reproductive technologies developed and the demand for experimentation grew stronger, this boundary may be set aside leaving the ultimate decision to local oversight committees. While science has not come closer to setting a zero point for human life, there has been significant progress in our understanding of early mammalian embryogenesis. It has become clear that the 14-day stage does in fact possess features, which make it a foundational time point for a developing human. Importantly, this stage defines the separation of soma from the germline and marks the boundary between rejuvenation and aging. We explore how different levels of life organization emerge during human development and suggest a new meaning for the 14-day stage in organismal life that is grounded in recent mechanistic advances and insights from aging studies.

The Clock and Wavefront Self-Organizing model recreates the dynamics of mouse somitogenesis in vivo and in vitro.

During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro.

Mechanically guided cell fate determination in early development.

Cell fate determination, a vital process in early development and adulthood, has been the focal point of intensive investigation over the past decades. Its importance lies in its critical role in shaping various and diverse cell types during embryonic development and beyond. Exploration of cell fate determination started with molecular and genetic investigations unveiling central signaling pathways and molecular regulatory networks. The molecular studies into cell fate determination yielded an overwhelming amount of information invoking the notion of the complexity of cell fate determination. However, recent advances in the framework of biomechanics have introduced a paradigm shift in our understanding of this intricate process. The physical forces and biochemical interplay, known as mechanotransduction, have been identified as a pivotal drive influencing cell fate decisions. Certainly, the integration of biomechanics into the process of cell fate pushed our understanding of the developmental process and potentially holds promise for therapeutic applications. This integration was achieved by identifying physical forces like hydrostatic pressure, fluid dynamics, tissue stiffness, and topography, among others, and examining their interplay with biochemical signals. This review focuses on recent advances investigating the relationship between physical cues and biochemical signals that control cell fate determination during early embryonic development.

A novel method for gas mixing and distribution in multi-chamber embryo incubators.

BACKGROUND: High-quality control of the gas environment in incubators is crucial for in vitro embryo development, which requires high accuracy, fast recovery, and low gas consumption. OBJECTIVE: In this study, we propose a novel gas mixing and distribution system and method as an alternative solution for multi-chamber embryo incubators. METHODS: The system-based embryo incubator enables a controllable gas circulation process and a quantitative supply of CO2 and N2. To determine the optimal parameters for the mixing time and flow rate of the circulated gases, we conducted contrast experiments on the system-based incubator. To evaluate the performance of the gas system in the incubator, we conducted tests under four different initial conditions, simulating various practical application scenarios. Furthermore, we performed a mouse embryo assay to assess the system's effectiveness. RESULTS: The results show that the system achieved a gas concentration accuracy of ± 0.2% (volume fraction) after stabilization, a minimum recovery time of 5 minutes, an average consumption of 8.9 L/d for N2 and 0.83 L/d for CO2 during routine operation, and a blastocyst rate exceeding 90% observed after 96 hours of culture in the incubator. CONCLUSION: The system and method demonstrate a significant advantage in terms of low gas consumption compared to existing incubators, while still maintaining high accuracy and fast recovery.

Paraspeckles / CARM1 mediates the regulation of OEVs on cell differentiation during in vitro embryonic development of yak.

Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of blastocysts, the low ratio of the number of inner cell mass cells to the number of trophoblastic cells, and the high apoptosis rate of blastocysts, resulting in low embryo implantation rate. Therefore, optimizing in vitro culture conditions has become a key technology to im-prove the quality of preimplantation embryos. Oviduct Epithelial cells exosomes (OEVs) can be absorbed and internalized by embryos to improve the blastocyst rate and blastocyst quality of embryos in vitro. As a special nuclear structure, Paraspeckles are involved in the fate determination of mammalian early embryonic mammalian cells. However, the regulation of embryonic cell differentiation by OEVs remains unknown. We aimed to investigate the effects of OEVs on paraspeckle formation and cell fate determination in yak in vitro fertilization (IVF) of em-bryos. To simulate the in vivo oviduct environment after ovulation, we used follicular fluid exosomes (FEVs) to stimulate yak oviduct epithelial cells and collect OEVs. OEVs were added to the yak IVF embryo culture system. Paraspeckle formation, cell differentiation, and blastocyst quality in yak embryos were determined. Our results show that, development of yak embryos is unique compared to other bovine species, and OEVs can be used as a supplement to the in vitro culture system of yak embryos to improve embryonic development and blas-tocyst quality. And also Paraspeckles/CARM1 mediated the regulation of OEVs on cell differentiation during in vitro yak embryo production. These results provide new insights into the study of yak embryonic development and the role of OEVs in embryonic development.

The first lineage determination in mammals.

This review describes in detail the morphological, cytoskeletal and gene expression events leading to the gene regulatory network bifurcation point of trophoblast and inner cell mass cells in a variety of mammalian preimplantation embryos. The interrelated processes of compaction and polarity establishment are discussed in terms of how they affect YAP/WWTR activity and the location and fate of cells. Comparisons between mouse, human, cattle, pig and rabbit embryos suggest a conserved role for YAP/WWTR signalling in trophoblast induction in eutherian animals though the mechanisms for, and timing of, YAP/WWTR activation differs among species. Downstream targets show further differences, with the trophoblast marker GATA3 being a direct target in all examined mammals, while CDX2-positive and SOX2-negative regulation varies.

Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway.

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

Expression of miRNA from spent pre-implantation embryos culture media.

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome.