The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

Spindle shape and volume differ in high- and low-quality metaphase II oocytes.

In brief: Optical coherence microscopy non-invasively visualizes metaphase II spindles allowing for quantitative analysis of their volume and shape, which may prove useful in the assessment of the oocyte quality. Using a mouse model, we showed also that analysis of spindle length combined with morphokinetics improves the evaluation of the resulting embryos. Abstract: The proper development of embryos strongly depends on the quality of oocytes, so the evaluation of oocytes may be a useful initial step in IVF procedures. Additionally, it enables embryologists to make more informed decisions regarding the treatments chosen for the patients and better manage patients' expectations. Optical coherence microscopy (OCM) allows for non-invasive 3D visualization of intracellular structures, such as spindles or nuclei, which have been linked to the success of embryonic development. Here, we applied a mouse model to examine whether OCM imaging could be used in the quality assessment of metaphase II (MII) oocytes. We showed that quantitative parameters describing the shape and volume of the MII spindle were associated with the quality of the resulting embryos, including the likelihood of blastocyst formation and the embryos' ability to differentiate the trophectoderm and primitive endoderm, but not the epiblast. We also created a multivariate linear regression model, combining OCM-based quantification of MII spindles with morphokinetic analysis of the embryos, that allowed for improved evaluation of the embryo quality. Finally, we proved that OCM does not interfere with the viability of the scanned cells, at least during the preimplantation development. Therefore, we believe that OCM-based quantitative assessment of MII spindles can improve the oocyte and embryo selection in IVF procedures.

Optical coherence microscopy allows for quality assessment of immature mouse oocytes.

In brief: Optical coherence microscopy is a label-free and non-invasive imaging technique capable of 3D subcellular structure visualization. Here we show that this method allows for quality assessment of immature mouse oocytes based on their chromatin conformation and can be a valuable addition to the toolkit used in assisted reproduction procedures. Abstract: The success of assisted reproductive technologies, and particularly in vitro maturation, is tightly linked to the quality of oocytes. Therefore, there is a need for robust, reliable, and easy-to-assess biomarkers of oocyte developmental competence. Microscopy techniques visualizing oocyte intracellular structure could provide such biomarkers. However, fluorescence imaging methods, applied frequently in biology and allowing for detailed structural and dynamic studies of single cells, require fluorescent tags to visualize cellular architecture and may cause short- and long-term photo-damage. On the other hand, traditional light microscopy, although relatively non-invasive, does not provide detailed structural information. Optical coherence microscopy (OCM) is a promising alternative, as it does not require sample pre-processing or labelling and can provide 3D images of intracellular structures. Here we applied OCM to assess the chromatin conformation of immature mouse oocytes, a feature that corresponds with their transcriptional status and developmental competence and cannot be examined by traditional light microscopy. We showed that OCM distinguished oocytes with so-called non-surrounded nucleoli (NSN) and surrounded nucleoli (SN) chromatin conformation with very high sensitivity and specificity and that OCM scanning did not decrease the quality of oocytes. Finally, we cross-referenced OCM data with the oocyte ability to undergo normal nuclear and cytoplasmic maturation and proven that indeed oocytes scored with OCM as NSN mature less effectively than oocytes scored as SN. Our results suggest that OCM may be a valuable addition to the imaging toolkit used in assisted reproduction procedures.

Plastic ­ (not) fantastic? Impact of bisphenol A on functioning of mammalian oocytes and embryos / Plastic ­ (nie)fantastic? O wplywie bisfenolu A na funkcjonowanie oocytów i zarodków ssaków.

Bisphenol A is a monomeric organic compound belonging to phenols. It is widely used in the production of resins, polycarbonates and plastics. Mass production of this compound contributed to its widespread presence in the environment, and thus - in the organisms of animals and humans. BPA belongs to xenoestrogens, synthetic compounds exerting an estrogen-like effect on cells. BPA can therefore disrupt the functioning of animal (including human) organisms. This article focuses on the impact of BPA on selected aspects of mammalian fertility. Recent literature data indicate that BPA disturbs several processes in oocytes and embryos, including epigenetic modifications, energy metabolism and spindle assembly, and as a result, decreases their developmental competence. We discuss the latest data on the influence of BPA on cellular processes taking place in oocytes and early embryos and describe molecular mechanisms responsible for this effect. We also discuss the significance of the results obtained from experiments conducted in vitro and/or on animal models in the context of BPA impact on fertility of women.

Age-related alterations in fertilization-induced Ca2+ oscillations depend on the genetic background of mouse oocytes†.

Maternal aging affects various aspects of oocytes' physiology, including the functionality of their nuclear apparatus and mitochondria. In the present paper, we wished to investigate whether advanced reproductive age impacts oocytes' ability to generate proper Ca2+ oscillations in response to monospermic fertilization. We examined three different mouse strains/crosses: inbred C57BL/6Tar, outbred Tar:SWISS, and hybrid F1 (C57BL/6Tar × CBA/Tar). The females were either 2-4 months old (young) or 13-16 months old (aged). We observed that the Ca2+ oscillatory pattern is altered in a strain-dependent manner and changes were more profound in aged C57BL/6Tar and F1 than in aged Tar:SWISS oocytes. We also showed that maternal aging differently affects the size of Ca2+ store and expression of Itpr1, Atp2a2, Erp44, and Pdia3 genes involved in Ca2+ homeostasis in oocytes of C57BL/6Tar, Tar:SWISS, and F1 genetic background, which may explain partially the differences in the extent of age-dependent changes in the Ca2+ oscillations in those oocytes. Maternal aging did not have any visible impact on the distribution of the ER cisterns in oocytes of all three genetic types. Finally, we showed that maternal aging alters the timing of the first embryonic interphase onset and that this timing correlates in C57BL/6Tar and Tar:SWISS oocytes with the frequency of fertilization-induced Ca2+ oscillations. Our results indicate that extreme caution is required when conclusions about oocyte/embryo physiological response to aging are made and complement an increasing amount of evidence that mammalian (including human) susceptibility to aging differs greatly depending on the genetic background of the individual.

Correction to: From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford.

The authors of the article Ajduk & Duncan 2019 sincerely apologize for specifying the incorrect institutional affiliation for Professor Ali Brivanlou and also the incorrect spelling of Professor Brivanlou's surname in the text of the article.

From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford.

The British Society for Developmental Biology Autumn Meeting, held in Oxford in September 2018, was the third in a series of international workshops which have been focussed on development at the extraembryonic-embryonic interface. This workshop, entitled "Embryonic-Extraembryonic Interactions: from Genetics to Environment" built on the two previous workshops held in 2011 (Leuven, Belgium) and 2015 (Göttingen, Germany). This workshop brought together researchers utilising a diverse range of organisms (including both vertebrate and invertebrate species) and a range of experimental approaches to answer core questions in developmental biology. This meeting report highlights some of the major themes emerging from the workshop including an evolutionary perspective as well as recent advances that have been made through the adoption of emerging techniques and technologies.

Transcriptional status of mouse oocytes corresponds with their ability to generate Ca2+ release.

In fully grown ovarian follicles both transcriptionally active (NSN) and inactive (SN) oocytes are present. NSN oocytes have been shown to display lower developmental potential. It is possible that oocytes that have not completed transcription before meiosis resumption accumulate less RNA and proteins required for their further development, including those responsible for regulation of Ca2+ homeostasis. Oscillations of the cytoplasmic concentration of free Ca2+ ions ([Ca2+]i) are triggered in oocytes by a fertilizing spermatozoon and are crucial for inducing and regulating further embryonic development. We showed that NSN-derived oocytes express less inositol 1,4,5-triphosphate receptor type 1 (IP3R1), store less Ca2+ ions and generate weaker spontaneous [Ca2+]i oscillations during maturation than SN oocytes. Consequently, NSN oocytes display aberrant [Ca2+]i oscillations at fertilization. We speculate that this defective regulation of Ca2+ homeostasis might be one of the factors responsible for the lower developmental potential of NSN oocytes.

Dynamics of cytoplasm and cleavage divisions correlates with preimplantation embryo development.

In vitro fertilization has become increasingly popular as an infertility treatment. In order to improve efficiency of this procedure, there is a strong need for a refinement of existing embryo assessment methods and development of novel, robust and non-invasive selection protocols. Studies conducted on animal models can be extremely helpful here, as they allow for more extensive research on the potential biomarkers of embryo quality. In the present paper, we subjected mouse embryos to non-invasive time-lapse imaging and combined the Particle Image Velocimetry analysis of cytoplasmic dynamics in freshly fertilized oocytes with the morphokinetic analysis of recordings covering 5 days of preimplantation development. Our results indicate that parameters describing cytoplasmic dynamics and cleavage divisions independently correspond to mouse embryo's capacity to form a high-quality blastocyst. We also showed for the first time that these parameters are associated with the percentage of abnormal embryonic cells with fragmented nuclei and with embryo's ability to form primitive endoderm, one of the cell lineages differentiated during preimplantation development. Finally, we present a model that links selected cytoplasmic and morphokinetic parameters reflecting frequency of fertilization-induced Ca2+-oscillations and timing of 4-cell stage and compaction with viability of the embryo assessed as the total number of cells at the end of its preimplantation development. Our results indicate that a combined analysis of cytoplasmic dynamics and morphokinetics may facilitate the assessment of embryo's ability to form high-quality blastocysts.

Time-lapse imaging of cleavage divisions in embryo quality assessment.

In vitro fertilization (IVF) is one of the most important procedures for treating infertility. As several embryos are usually produced in a single IVF cycle, it is crucial to select only the most viable ones for transfer to the patient. Morphokinetics, i.e. analysis of the dynamics of cleavage divisions and processes such as compaction and cavitation, has provided both biologists and clinicians with a new set of data regarding embryonic behaviour during preimplantation development and its association with embryo quality. In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome. We also explain the statistics behind the predictive algorithms and discuss the future perspectives of morphokinetics.

Polarity and cell division orientation in the cleavage embryo: from worm to human.

Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells-blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos.

The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo.

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most - but not all - cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo.

How great thou ART: biomechanical properties of oocytes and embryos as indicators of quality in assisted reproductive technologies.

Assisted Reproductive Technologies (ART) have revolutionized infertility treatment and animal breeding, but their success largely depends on selecting high-quality oocytes for fertilization and embryos for transfer. During preimplantation development, embryos undergo complex morphogenetic processes, such as compaction and cavitation, driven by cellular forces dependent on cytoskeletal dynamics and cell-cell interactions. These processes are pivotal in dictating an embryo's capacity to implant and progress to full-term development. Hence, a comprehensive grasp of the biomechanical attributes characterizing healthy oocytes and embryos is essential for selecting those with higher developmental potential. Various noninvasive techniques have emerged as valuable tools for assessing biomechanical properties without disturbing the oocyte or embryo physiological state, including morphokinetics, analysis of cytoplasmic movement velocity, or quantification of cortical tension and elasticity using microaspiration. By shedding light on the cytoskeletal processes involved in chromosome segregation, cytokinesis, cellular trafficking, and cell adhesion, underlying oogenesis, and embryonic development, this review explores the significance of embryo biomechanics in ART and its potential implications for improving clinical IVF outcomes, offering valuable insights and research directions to enhance oocyte and embryo selection procedures.

Mitochondrial activity and redox status in oocytes from old mice: The interplay between maternal and postovulatory aging.

Maternal aging has been reported to reduce oocyte quality and, in turn, lower the developmental potential of the resulting embryos. Here, we show that maternally aged oocytes display two strikingly different phenotypes: some have normal morphology, whereas others have significantly shrunk cytoplasm. The latter phenotype usually prevails in aged females. Our objective was to characterize both types of maternally aged oocytes and investigate the origins of this diversity. Importantly, our experiments indicate that shrunk maternally aged oocytes are severely compromised in terms of mitochondrial functionality as compared to their young or morphologically normal maternally aged counterparts: they display significantly decreased mitochondrial activity and lower amounts of ROS. In contrast, morphologically normal maternally aged oocytes had the same mitochondrial activity as young ones, while their ROS levels were higher. Surprisingly, the shrunk phenotype was completely absent in maternally aged oocytes that matured in vitro, suggesting that it is not caused inherently by maternal aging, but may be related to other factors, like postovulatory aging. Indeed, an additional culture of in vitro matured young and old oocytes (i.e., in vitro postovulatory aging) significantly decreased their mitochondrial activity and led to cytoplasm shrinkage. In vivo postovulatory aging had a similar effect on oocytes from both young and old females. Finally, we examined the developmental potential of oocytes obtained from aged females. Shrunk (i.e., most likely postovulatory aged) oocytes failed to become fertilized, whereas morphologically normal ones (i.e., most likely not subjected to postovulatory aging) underwent fertilization and subsequent cleavage divisions, although they achieved the 2-cell stage less frequently than morphologically normal oocytes from young females. Importantly, the quality of blastocysts as well as the live birth rate for morphologically normal oocytes from old and young females were similar. In summary, our data clearly indicate that two pools of oocytes present in oviducts of aged females differ significantly in their quality and developmental potential and that the more severely affected phenotype results most likely from a synergistic action of maternal and postovulatory aging.

Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of ß-cell identity and α-cell expansion in the mature pancreas.

Abnormalities that characterize the pathophysiology of type 2 diabetes (T2D) include deficiencies of ß-cells and the expansion of α-cells in pancreatic islets, manifested by lower insulin release and glucagon oversecretion. The molecular mechanisms that determine intra-islet interactions between pancreatic α- and ß-cells are still not fully understood. The present study showed that stearoyl-coenzyme A (CoA) desaturase 1 (SCD1), an enzyme that is implicated in fatty acid metabolism, serves as a checkpoint in the control of endocrine cell equilibrium in pancreatic islets. Our data showed that SCD1 activity is essential for proper α-cell and ß-cell lineage determination during morphogenesis of the pancreas and the maintenance of mature ß-cell identity. The inhibition of SCD1 expression/activity led to both a decrease in the expression of ß-cell signature genes (e.g., Pdx1, Nkx6.1, MafA, and Neurod1, among others) and induction of the expression of the dedifferentiation marker Sox9 in mature pancreatic islets. The transcriptional repression of Pdx1 and MafA in SCD1-deficient ß-cells was related to the excessive methylation of promoter regions of these transcription factors. In contrast, SCD1 ablation favored the formation of α-cells over ß-cells throughout pancreas organogenesis and did not compromise α-cell identity in adult pancreatic islets. Such molecular changes that were caused by SCD1 downregulation resulted in the mislocalization of α-cells within the core of islets and increased the ratio of pancreatic α- to ß-cell mass. This was followed by islet dysfunction, including impairments in glucose-stimulated insulin release, simultaneously with elevations of basal glucagon secretion. Altogether, these findings provide additional mechanistic insights into the role of SCD1 in the pathogenesis of T2D.

p38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice.

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

Dynamic shapes of the zygote and two-cell mouse and human.

Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder-distorted-rounder shapes were revealed, as were changing patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis. During cell division, two vortices developed in each nascent cell and they rotated in opposite directions at each end of the cell, a pattern that sometimes persisted for up to 10 h. Exchange with the environment may have been promoted by these shape and area cycles and persisting circulation in the cytoplasm may have a similar function between a cell's interior and periphery. Some of these movements were sporadically also seen in human zygotes with abnormal numbers of pronuclei and the two-cell stages that developed from these compromised human zygotes.

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice.

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.

Light microscopy of mammalian gametes and embryos: methods and applications.

In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.