Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay.

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals.

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

Need for choosing the ideal pH value for IVF culture media.

PURPOSE: Monitoring the pH of IVF culture media is a good practice, but the required pH levels have been "arbitrarily" set. Assisted reproductive technology centers around the world are spending time and money on pH monitoring without any consensus to date. The objective of this narrative review was to evaluate the importance of pH monitoring during IVF, discover how the oocyte and embryo regulate their intracellular pH and try to determine the optimal pH to be applied. METHODS: A narrative literature review was performed on publications in the PubMed database reporting on the impact of pH on cellular function, oocyte and embryo development, IVF outcomes and pathophysiology, or on physiological pH in the female reproductive tract. RESULTS: Intracellular pH regulates many cellular processes such as meiotic spindle stability of the oocyte, cell division and differentiation, embryo enzymatic activities, and blastocoel formation. The internal pH of the human embryo is maintained by regulatory mechanisms (mainly Na+/H+ and HCO3-/Cl- exchangers) that can be exceeded, particularly in the oocyte and early-stage embryos. The opinion that the optimal pH for embryo culture is physiological pH is not correct since several physicochemical parameters specific to IVF culture conditions (temperature, medium composition, duration of culture, or implication of CO2) can modify the intracellular pH of the embryo and change its needs and adaptability. CONCLUSIONS: Because correct and stable extracellular pH is essential to embryo health and development, monitoring pH is imperative. However, there is a lack of clinical data on choosing the ideal pH for human IVF culture media.

Master control: transcriptional regulation of mammalian Myod.

MYOD is a master regulator of the skeletal myogenic program. But what regulates expression of Myod? More than 20 years ago, studies established that Myod expression is largely controlled by just two enhancer regions located within a region 24 kb upstream of the transcription start site in mammals, which regulate Myod expression in the embryo, fetus and adult. Despite this apparently simple arrangement, Myod regulation is complex, with different combinations of transcription factors acting on these enhancers in different muscle progenitor cells and phases of differentiation. A range of epigenetic modifications in the Myod upstream region also play a part in activating and repressing Myod expression during development and regeneration. Here the evidence for this binding at Myod control regions is summarized, giving an overview of our current understanding of Myod expression regulation in mammals.

Primordial germ cells: the first cell lineage or the last cells standing?

Embryos of many animal models express germ line determinants that suppress transcription and mediate early germ line commitment, which occurs before the somatic cell lineages are established. However, not all animals segregate their germ line in this manner. The 'last cell standing' model describes primordial germ cell (PGC) development in axolotls, in which PGCs are maintained by an extracellular signalling niche, and germ line commitment occurs after gastrulation. Here, we propose that this 'stochastic' mode of PGC specification is conserved in vertebrates, including non-rodent mammals. We postulate that early germ line segregation liberates genetic regulatory networks for somatic development to evolve, and that it therefore emerged repeatedly in the animal kingdom in response to natural selection.

High Potassium Concentration during Culturing of Early Mammalian Embryos: Normal or Extreme Situation?

Analysis of the element composition of oviduct and uterine fluid in mammals showed high potassium concentrations in the early embryo microenvironment in vivo. The results of early embryogenesis of mammals in vitro in the presence of high potassium concentrations are discussed. The data are summarized in accordance with the conditions of experimentally modeled pre-implantation development. Comparative assessment of the quality of embryo development until the blastocyst stage in vitro proved the embryos more successfully developed at potassium concentrations close to those registered in the oviductal fluid.

Cell fate in animal and human blastocysts and the determination of viability.

Understanding the mechanisms underlying the first cell differentiation events in human preimplantation development is fundamental for defining the optimal conditions for IVF techniques and selecting the most viable embryos for further development. However, our comprehension of the very early events in development is still very limited. Moreover, our knowledge on early lineage specification comes primarily from studying the mouse model. It is important to recognize that although mammalian embryos share similar morphological landmarks, the timing and molecular control of developmental events may vary substantially between species. Mammalian blastocysts comprise three cell types that arise through two sequential rounds of binary cell fate decisions. During the first decision, cells located on the outside of the developing embryo form a precursor lineage for the embryonic part of the placenta: the trophectoderm and cells positioned inside the embryo become the inner cell mass (ICM). Subsequently, ICM cells differentiate into embryonic lineages that give rise to a variety of tissues in the developing foetus: either the epiblast or extraembryonic primitive endoderm. Successful formation of all three lineages is a prerequisite for implantation and development to term. A comprehensive understanding of the lineage specification processes in mammals is therefore necessary to shed light on the causes of early miscarriages and early pregnancy pathologies in humans.

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.

Erythroid development in the mammalian embryo.

Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

Mechanisms for sperm mitochondrial removal in embryos.

BACKGROUND: Different animal species have different characteristics regarding the transmission of mitochondrial DNA. While some species have biparental mitochondrial inheritance, others have developed pathways to remove paternal mtDNA. These pathways guarantee the uniparental mitochondrial inheritance, so far well known in mammals, avoiding heteroplasmy, which may have the potential to cause certain mitochondrial diseases in the offspring. SCOPE OF REVIEW: This review aims to address the main mechanisms that involve mitochondrial degradation in different animal species, as well as to describe what is present in the literature on the mechanisms involved in mitochondrial inheritance. MAJOR CONCLUSIONS: Two theories are proposed to explain the uniparental inheritance of mtDNA: (i) active degradation, where mechanisms for paternal mitochondrial DNA elimination involve mitochondrial degradation pathway by autophagy and, in some species, may also involve the endocytic degradation pathway; and (ii) passive dilution, where the paternal mitochondria are diluted in the cells of the embryo according to cell division, until becoming undetectable. GENERAL SIGNIFICANCE: This work brings a wide review of the already published evidence on mitochondrial inheritance in the animal kingdom and the possible mechanisms to mtDNA transmission already described in literature.

The Effect of Polymer Dots During Mammalian Early Embryo Development and Their Biocompatibility on Maternal Health.

Conjugated polymer dots have excellent fluorescence properties in terms of their structural diversity and functional design, showing broad application prospects in the fields of biological imaging and biosensing. Polymer dots contain no heavy metals and are thought to be of low toxicity and good biocompatibility. Therefore, systematic studies on their potential toxicity are needed. Herein, the biocompatibility of poly[(9,9-dioctylfluorenyl-2,7diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)],10% benzothiadiazole(y) (PFBT) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) polymer dots on early embryo development as well as maternal health is studied in detail. The results show that prepared polymer dots are dose-dependently toxic to preimplantation embryos, and low-dose polymer dots can be used for cell labeling of early embryos without affecting the normal development of embryos into blastocysts. In addition, the in vivo distribution data show that the polymer dots accumulate mainly in the maternal liver, spleen, kidney, placenta, ovary, and lymph nodes of the pregnant mice. Histopathological examination and blood biochemical tests demonstrate that exposure of the maternal body to polymer dots at a dosage of 14 µg g-1 does not affect the normal function of the maternal organs and early fetal development. The research provides a safe basis for the wide application of polymer dots.

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning.

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.

The Regulative Nature of Mammalian Embryos.

The striking developmental plasticity of early mammalian embryos has been known since the classical experiments performed in the 1950s and 1960s. There are many lines of evidence that the mammalian embryo is able to continue normal development even when exposed to severe experimental manipulations of the number and position of cells within the embryo. These observations have raised the question about the mechanisms involved in emergence, maintenance, and progressive restriction of this plasticity. Only recently, we have begun to understand these mechanisms. In this review, in order to explain the molecular and cellular events underlying the remarkable plasticity of the early mammalian embryo, we discuss results of classical experiments demonstrating developmental potential of mammalian embryos and link them with the novel data provided by contemporary experimental approaches. We also show how developmental flexibility of mammalian embryos is manifested in nature, and discuss its implications for basic research and medicine.

Human embryos cultured in vitro to 14 days.

We know a great deal about the development of the mammalian embryo until the time that the blastocyst implants into the uterus. With model organisms such as the mouse, we have also developed a considerable understanding of development immediately around gastrulation as embryos can be recovered at this stage for short-term in vitro culture. However, the intervening period of development remained a 'black box' because it takes place as the blastocyst is implanting into the uterus. Over the past 6 years, techniques pioneered and developed in Magdalena Zernicka-Goetz's laboratory for the in vitro culture of embryos through these implantation stages have opened up this box, affording the first glimpse of embryonic development through these previously hidden stages. Remarkably, the techniques developed with mouse embryos are equally applicable to human embryos, ushering the very first opportunities for studying our own development throughout this time. Here, I outline how the culture methods were developed, paving the way to culture of the human embryo to the point of gastrulation, an accomplishment recognized as the People's Choice for the Scientific Breakthrough of 2016 in Science magazine. I also discuss the new ethical challenges raised by the possibility of extending the time limits for human embryo culture.

Pluripotency Surveillance by Myc-Driven Competitive Elimination of Differentiating Cells.

The mammalian epiblast is formed by pluripotent cells able to differentiate into all tissues of the new individual. In their progression to differentiation, epiblast cells and their in vitro counterparts, embryonic stem cells (ESCs), transit from naive pluripotency through a differentiation-primed pluripotent state. During these events, epiblast cells and ESCs are prone to death, driven by competition between Myc-high cells (winners) and Myc-low cells (losers). Using live tracking of Myc levels, we show that Myc-high ESCs approach the naive pluripotency state, whereas Myc-low ESCs are closer to the differentiation-primed state. In ESC colonies, naive cells eliminate differentiating cells by cell competition, which is determined by a limitation in the time losers are able to survive persistent contact with winners. In the mouse embryo, cell competition promotes pluripotency maintenance by elimination of primed lineages before gastrulation. The mechanism described here is relevant to mammalian embryo development and induced pluripotency.

Deformidad del principio de autonomía para sustentar actos éticamente ilícitos / Distortion of the Principle of Autonomy to Support Ethically Illicit Acts / Deformação do princípio da autonomia para justificar atos eticamente ilícitos

Resumen Ensayo que busca profundizar en conceptos claves de la ética y la moral para dar cuenta de la deformidad en la utilización del principio de autonomía para avalar actos éticamente ilícitos como el aborto. El origen de la reflexión está en la experiencia de la autora en las aulas de clase donde captó el fracaso dialéctico en la enseñanza de la ética médica: muchos conceptos fueron impartidos desde una ideología global que lleva a una práctica clínica basada en convicciones individuales, que distan de la verdad ética y de lo filosóficamente demostrable y obedece a las necesidades «poco éticas¼ del mundo actual. Se hace una aproximación a lo que es la autonomía; se examina si el aborto puede calificarse como un acto moral, partiendo de la realidad biológica de lo que es un embrión humano. Se concluye que el aborto es una práctica en la que no puede primar el principio de autonomía porque no contiene una acción que sea puramente moral: no es posible afirmar que el aborto sea éticamente lícito. La sociedad actual pretende fundamentar legalmente los actos ilícitos antes de revisar los conceptos que conciernen a la ética y a la filosofía; parte del problema es que no se enseñan adecuadamente esos contenidos en las aulas de clase.

Abstract This essay aims to delve into fundamental concepts of ethics and morality to account for shortcomings in using the principle of autonomy to endorse ethically illicit acts such as abortion. The reflection originates from the author's experience in the classroom, where she grasped a dialectical failure in teaching medical ethics: Many concepts were taught from a global ideology that results in a clinical practice based on individual convictions. These convictions are far from the ethical truth and what is philosophically demonstrable and arise from the "unethical" needs of today's world. An approach is made to what autonomy is; the essay examines whether abortion can be qualified as a moral act based on the biological reality of what a human embryo is. In brief, abortion is a practice in which the principle of autonomy cannot prevail because it does not hold a purely moral action: It is impossible to affirm that abortion is ethically licit. Today's society intends to provide legal grounds for illicit acts before reviewing the concepts that concern ethics and philosophy; part of the problem is that these contents are not adequately taught in the classroom.

Resumo Ensaio que pretende aprofundar conceitos-chave da ética e da moral para evidenciar a deformação na utilização do princípio da autonomia para avaliar atos eticamente ilícitos, como o aborto. A origem da reflexão está na experiência da autora nas salas de aula onde captou o fracasso dialético no ensino da ética médica: muitos conceitos foram ministrados a partir de uma ideologia global que leva a uma prática clínica baseada em convicções individuais, que se afastam da verdade ética e do filosoficamente demonstrável, e obedece às necessidades "pouco éticas" do mundo atual. Faz-se uma abordagem sobre o que é autonomia; analisa-se se o aborto pode ser qualificado como um ato moral, partindo da realidade biológica do que é um embrião humano. Conclui-se que o aborto é uma prática na qual não pode primar o princípio da autonomia porque não contém uma ação que seja puramente moral: não é possível afirmar que o aborto seja eticamente lícito. A sociedade atual pretende fundamentar legalmente os atos ilícitos antes de averiguar os conceitos que concernem à ética e à filosofia; parte do problema é que esses conteúdos não são ensinados adequadamente no ambiente acadêmico.

Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.

Dynamics of histone H3 acetylation in the nucleosome core during mouse pre-implantation development.

In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.

The Role of Maternal-Effect Genes in Mammalian Development: Are Mammalian Embryos Really an Exception?

The essential contribution of multiple maternal factors to early mammalian development is rapidly altering the view that mammals have a unique pattern of development compared to other species. Currently, over 60 maternal-effect mutations have been described in mammalian systems, including critical determinants of pluripotency. This data, combined with the evidence for lineage bias and differential gene expression in early blastomeres, strongly suggests that mammalian development is to some extent mosaic from the four-cell stage onward.