Preimplantation Embryogenesis Research Articles

Temporal variability and cell mechanics control robustness in mammalian embryogenesis.

How living systems achieve precision in form and function despite their intrinsic stochasticity is a fundamental yet ongoing question in biology. We generated morphomaps of preimplantation embryogenesis in mouse, rabbit, and monkey embryos, and these morphomaps revealed that although blastomere divisions desynchronized passively, 8-cell embryos converged toward robust three-dimensional shapes. Using topological analysis and genetic perturbations, we found that embryos progressively changed their cellular connectivity to a preferred topology, which could be predicted by a physical model in which actomyosin contractility and noise facilitate topological transitions, lowering surface energy. This mechanism favored regular embryo packing and promoted a higher number of inner cells in the 16-cell embryo. Synchronized division reduced embryo packing and generated substantially more misallocated cells and fewer inner-cell-mass cells. These findings suggest that stochasticity in division timing contributes to robust patterning.

O-171 Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

Abstract Assessing the morphological appearance of an embryo as it progresses from zygote to blastocyst is the primary method used by embryologists to evaluate embryo viability and developmental potential. Standard embryo grading techniques, using time-lapse microscopy, analyse morphological characteristics such as size, shape, number, and appearance of blastomeres. The aim of this study was to determine how individual cells of the embryo function on a subcellular level to build a healthy embryo. RNA localisation plays indispensable roles in establishing asymmetries and coordinating cell fate decisions during early embryogenesis across various non-mammalian species. To direct the spatiotemporal distribution of RNA within the cells of an embryo, the microtubule cytoskeleton provides highly sophisticated trafficking pathways. Yet, it remains unknown whether subcellular heterogeneities exist during mammalian preimplantation development and how they contribute to cell fate determination. Using advanced live imaging, we visualised global RNA transcripts at high spatiotemporal resolution from fertilisation to the blastocyst stage in living preimplantation mouse embryos. We discovered apicobasal RNA asymmetries specific to outer cells of the 16-cell stage mouse embryo, coinciding with cell fate decisions and the emergence of the pluripotent inner cell mass. Highly clustered RNAs accumulated proximal to the basal membrane, while more dispersed RNA foci were identified apically as the embryo reaches the late 16-cell stage. The targeted distribution of membrane-less RNA molecules was facilitated by the microtubule cytoskeleton, associated with lysosomes which serve as RNA transport vehicles. We discovered that apically located RNA foci were more dynamic and accompanied an enrichment of translation components. Furthermore, we uncovered that the established RNA asymmetries enhanced translation capacity in apical regions compared to basal regions. These spatiotemporal RNA heterogeneities were unevenly inherited by outer and inner daughter cells during subsequent cell divisions. Notably, embryos that were unable to establish RNA asymmetries failed to allocate cells to the trophectoderm and inner cell mass. Our study provides insights into a subcellular mechanism driving asymmetric RNA localisation and compartmentalised translational regulation in outer cells of the 16-cell stage embryo, which may contribute to cell fate decisions during mammalian preimplantation embryogenesis. We envision that when paired with classical morphological assessment criteria of the embryo, these findings may assist with selecting the embryo with the highest developmental potential.

Increased ROS levels in mitochondrial outer membrane protein Mul1-deficient oocytes result in abnormal preimplantation embryogenesis.

Reactive oxygen species (ROS) are associated with oocyte maturation inhibition, and N-acetyl-l-cysteine (NAC) partially reduces their harmful effects. Mitochondrial E3 ubiquitin ligase 1 (Mul1) localizes to the mitochondrial outer membrane. We found that female Mul1-deficient mice are infertile, andtheir oocytes contain high ROS concentrations. After fertilization, Mul1-deficient embryos showed a DNA damage response (DDR) and abnormal preimplantation embryogenesis, which was rescued by NAC addition and ROS depletion. These observations clearly demonstrate that loss of Mul1 in oocytes increases ROS concentrations and triggers DDR, resulting in abnormal preimplantation embryogenesis. We conclude that manipulating the mitochondrial ROS levels in oocytes may be a potential therapeutic approach to target infertility.

Dynamics of Mitochondrial DNA Copy Number and Membrane Potential in Mouse Pre-Implantation Embryos: Responses to Diverse Types of Oxidative Stress.

Mitochondria undergo a myriad of changes during pre-implantation embryo development, including shifts in activity levels and mitochondrial DNA (mtDNA) replication. However, how these distinct aspects of mitochondrial function are linked and their responsiveness to diverse stressors is not well understood. Here, we show that mtDNA content increased between 8-cell embryos and the blastocyst stage, with similar copy numbers per cell in the inner cell mass (ICM) and trophectoderm (TE). In contrast, mitochondrial membrane potential (MMP) was higher in TE than ICM. Culture in ambient oxygen (20% O2) altered both aspects of mitochondrial function: the mtDNA copy number was upregulated in ICM, while MMP was diminished in TE. Embryos cultured in 20% O2 also exhibited delayed development kinetics, impaired implantation, and reduced mtDNA levels in E18 fetal liver. A model of oocyte mitochondrial stress using rotenone showed only a modest effect on on-time development and did not alter the mtDNA copy number in ICM; however, following embryo transfer, mtDNA was higher in the fetal heart. Lastly, endogenous mitochondrial dysfunction, induced by maternal age and obesity, altered the blastocyst mtDNA copy number, but not within the ICM. These results demonstrate that mitochondrial activity and mtDNA content exhibit cell-specific changes and are differentially responsive to diverse types of oxidative stress during pre-implantation embryogenesis.

Insights into embryonic chromosomal instability: mechanisms of DNA elimination during mammalian preimplantation development.

Mammalian preimplantation embryos often contend with aneuploidy that arose either by the inheritance of meiotic errors from the gametes, or from mitotic mis-segregation events that occurred following fertilization. Regardless of the origin, mis-segregated chromosomes become encapsulated in micronuclei (MN) that are spatially isolated from the main nucleus. Much of our knowledge of MN formation comes from dividing somatic cells during tumorigenesis, but the error-prone cleavage-stage of early embryogenesis is fundamentally different. One unique aspect is that cellular fragmentation (CF), whereby small subcellular bodies pinch off embryonic blastomeres, is frequently observed. CF has been detected in both in vitro and in vivo-derived embryos and likely represents a response to chromosome mis-segregation since it only appears after MN formation. There are multiple fates for MN, including sequestration into CFs, but the molecular mechanism(s) by which this occurs remains unclear. Due to nuclear envelope rupture, the chromosomal material contained within MN and CFs becomes susceptible to double stranded-DNA breaks. Despite this damage, embryos may still progress to the blastocyst stage and exclude chromosome-containing CFs, as well as non-dividing aneuploid blastomeres, from participating in further development. Whether these are attempts to rectify MN formation or eliminate embryos with poor implantation potential is unknown and this review will discuss the potential implications of DNA removal by CF/blastomere exclusion. We will also extrapolate what is known about the intracellular pathways mediating MN formation and rupture in somatic cells to preimplantation embryogenesis and how nuclear budding and DNA release into the cytoplasm may impact overall development.

Single-cell RNA-seq and single-cell bisulfite-sequencing reveal insights into yak preimplantation embryogenesis

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

TET Family Members Are Integral to Porcine Oocyte Maturation and Parthenogenetic Pre-Implantation Embryogenesis.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.

Early developmental plasticity enables the induction of an intermediate extraembryonic cell state

Two fundamental elements of pre-implantation embryogenesis are cells' intrinsic self-organization program and their developmental plasticity, which allows embryos to compensate for alterations in cell position and number; yet, these elements are still poorly understood. To be able to decipher these features, we established culture conditions that enable the two fates of blastocysts' extraembryonic lineages-the primitive endoderm and the trophectoderm-to coexist. This plasticity emerges following the mechanisms of the first lineage segregation in the mouse embryo, and it manifests as an extended potential for extraembryonic chimerism during the pre-implantation embryogenesis. Moreover, this shared state enables robust assembly into higher-order blastocyst-like structures, thus combining both the cell fate plasticity and self-organization features of the early extraembryonic lineages.

Molecular diversity and phenotypic pleiotropy of ancient genomic regulatory loci derived from human endogenous retrovirus type H (HERVH) promoter LTR7 and HERVK promoter LTR5_Hs and their contemporary impacts on pathophysiology of Modern Humans

Timelines of population-level effects of viruses on humans varied from the evolutionary scale of million years to contemporary spread of viral infections. Correspondingly, these events are exemplified by: (i) emergence of human endogenous retroviruses (HERVs) from ancient germline infections leading to stable integration of viral genomes into human chromosomes; and (ii) wide-spread viral infections reaching a global pandemic state such as the COVID-19 pandemic. Despite significant efforts, understanding of HERV’s roles in governance of genomic regulatory networks, their impacts on primate evolution and development of human-specific physiological and pathological phenotypic traits remains limited. Remarkably, present analyses revealed that expression of a dominant majority of genes (1696 of 1944 genes; 87%) constituting high-confidence down-steam regulatory targets of defined HERV loci was significantly altered in cells infected with the SARS-CoV-2 coronavirus, a pathogen causing the global COVID-19 pandemic. This study focused on defined sub-sets of DNA sequences derived from HERVs that are expressed at specific stages of human preimplantation embryogenesis and exert regulatory actions essential for self-renewal and pluripotency. Evolutionary histories of LTR7/HERVH and LTR5_Hs/HERVK were charted based on evidence of the earliest presence and expansion of highly conserved (HC) LTR sequences. Sequence conservation analyses of most recent releases 17 primate species’ genomes revealed that LTR7/HERVH have entered germlines of primates in Africa after the separation of the New World Monkey lineage, while LTR5_Hs/HERVK successfully colonized primates’ germlines after the segregation of Gibbons’ species. Subsequently, both LTR7 and LTR5_Hs undergo a marked ~ fourfold–fivefold expansion in genomes of Great Apes. Timelines of quantitative expansion of both LTR7 and LTR5_Hs loci during evolution of Great Apes appear to replicate the consensus evolutionary sequence of increasing cognitive and behavioral complexities of non-human primates, which seems particularly striking for LTR7 loci and 11 distinct LTR7 subfamilies. Consistent with previous reports, identified in this study, 351 human-specific (HS) insertions of LTR7 (175 loci) and LTR5_Hs (176 loci) regulatory sequences have been linked to genes implicated in establishment and maintenance of naïve and primed pluripotent states and preimplantation embryogenesis phenotypes. Unexpectedly, HS-LTRs manifest regulatory connectivity to genes encoding markers of 12 distinct cells’ populations of fetal gonads, as well as genes implicated in physiology and pathology of human spermatogenesis, including Y-linked spermatogenic failure, oligo- and azoospermia. Granular interrogations of genes linked with 11 distinct LTR7 subfamilies revealed that mammalian offspring survival (MOS) genes seem to remain one of consistent regulatory targets throughout ~ 30 MYA of the divergent evolution of LTR7 loci. Differential GSEA of MOS versus non-MOS genes identified clearly discernable dominant enrichment patterns of phenotypic traits affected by MOS genes linked with LTR7 (562 MOS genes) and LTR5_Hs (126 MOS genes) regulatory loci across the large panel of genomics and proteomics databases reflecting a broad spectrum of human physiological and pathological traits. GSEA of LTR7-linked MOS genes identified more than 2200 significantly enriched records of human common and rare diseases and gene signatures of 466 significantly enriched records of Human Phenotype Ontology traits, including Autosomal Dominant (92 genes) and Autosomal Recessive (93 genes) Inheritance. LTR7 regulatory elements appear linked with genes implicated in functional and morphological features of central nervous system, including synaptic transmission and protein–protein interactions at synapses, as well as gene signatures differentially regulated in cells of distinct neurodevelopmental stages and morphologically diverse cell types residing and functioning in human brain. These include Neural Stem/Precursor cells, Radial Glia cells, Bergman Glia cells, Pyramidal cells, Tanycytes, Immature neurons, Interneurons, Trigeminal neurons, GABAergic neurons, and Glutamatergic neurons. GSEA of LTR7-linked genes identified significantly enriched gene sets encoding markers of more than 80 specialized types of neurons and markers of 521 human brain regions, most prominently, subiculum and dentate gyrus. Identification and characterization of 1944 genes comprising high-confidence down-steam regulatory targets of LTR7 and/or LTR5_Hs loci validated and extended these observations by documenting marked enrichments for genes implicated in neoplasm metastasis, intellectual disability, autism, multiple cancer types, Alzheimer’s, schizophrenia, and other brain disorders. Overall, genes representing down-stream regulatory targets of ancient retroviral LTRs exert the apparently cooperative and exceedingly broad phenotypic impacts on human physiology and pathology. This is exemplified by altered expression of 93% high-confidence LTR targets in cells infected by contemporary viruses, revealing a convergence of virus-inflicted aberrations on genomic regulatory circuitry governed by ancient retroviral LTR elements and interference with human cells’ differentiation programs.

Redox Status and Hematological Variables in Melatonin-Treated Ewes during Early Pregnancy under Heat Stress.

Simple SummaryHeat stress induces oxidative stress that negatively affects the fertility rate in farm animals, as it may disrupt normal follicular development and preimplantation embryogenesis. Although indigenous sheep breeds may exhibit adaptation to a certain level in high summer temperatures, their reproductive competence is still compromised under extreme severe heat stress conditions. Thus, the administration of melatonin is suggested, as an antioxidant regime, for improving the fertility rate and redox balance in heat-stressed ewes. The influence of environmental thermal stress in conjunction with melatonin treatment on hematological variables and cortisol secretion was also investigated. In this respect, melatonin seems to enhance the progressive adaptation of indigenous-breed ewes to environmental heat stress.The preovulatory follicles and preimplantation stage embryos are found to be rather sensitive to heat stress due to their low potential for scavenging reactive oxygen species (ROS). The aim of the present study was to assess the impact of melatonin administration on redox status and hematological variables during the preovulatory period and early stages of embryogenesis in heat-stressed ewes in vivo. Forty Karagouniko-breed ewes were divided in two groups, the melatonin (M, n = 20) group and control (C, n = 20) one. All animals were subjected to heat stress throughout the study, which lasted forty days (D0 to D40). In M group, melatonin implants were administered on D0. Then, oestrous synchronization was applied (D19-D33). On D34, six rams were introduced into the ewe flock for mating. Ultrasonographic examination was conducted on D73 for pregnancy diagnosis. The temperature humidity index (THI), the rectal temperature (RT), and the number of breaths per minute (BR) were evaluated twice daily. Redox biomarkers, namely total antioxidant capacity (TAC), reduced glutathione (GSH), and thiobarbituric acid reactive substances (TBARS), were assayed in blood samples collected on D0, D33, and D40. In addition, packed cell volume (PCV), white blood cells (WBCs), leukocyte differential count, and cortisol assessment were conducted in blood samples on D33 and D40. The results indicated improved fertility rate and mean number of lambs born per ewe due to improved redox status (p < 0.05) in ewes that received melatonin implants 34 days approximately before the onset of oestrus. The PCV decreased in both groups between the two time-points (p < 0.05). However, the NEU/LYMPH ratio decreased (p < 0.05) only in group M. The low cortisol levels and the decreased NEU/LYMPH ratio in both groups support the hypothesis that ewes of the indigenous Karagouniko breed may exhibit adaptation to environmental thermal stress. The administration of melatonin as an antioxidant regime may improve the reproductive competence of heat stressed ewes and may also enhance their ability to adapt at high ambient temperatures.

Changes in OCT4 expression play a crucial role in the lineage specification and proliferation of preimplantation porcine blastocysts

Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes. In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.

Evaluation of Sperm Selection Technique Using Hyaluronic Acid Binding During Icsi; a Randomized Controlled Trial

In-vitro sperm selection for intracytoplasmic sperm injection (Physiological ICSI or PICSI) is crucial as it has a direct influence on the paternal contribution of preimplantation embryogenesis. Men with oligozoospermia who require ICSI often demonstrate compromised DNA integrity and increased chromosomal aberrations of the sperms in their semen. Studies have shown that embryos resulting from ICSI in those men have higher risk of sex chromosome disomies, chromosomal aneuploidies, de novo numerical chromosomal aberrations and cytogenetically detectable structural chromosomal aberrations. Although the primary candidates for ICSI are oligozoospermic men, there is an increase in the use of ICSI; therefore, more couples are potentially exposed to those potential risks.

Micronucleus formation during early cleavage division is a potential hallmark of preimplantation embryonic loss in cattle

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Allele-specific H3K9me3 and DNA methylation co-marked CpG-rich regions serve as potential imprinting control regions in pre-implantation embryo.

Parental DNA methylation and histone modifications undergo distinct global reprogramming in mammalian pre-implantation embryos, but the landscape of epigenetic crosstalk and its effects on embryogenesis are largely unknown. Here we comprehensively analyse the association between DNA methylation and H3K9me3 reprogramming in mouse pre-implantation embryos and reveal that CpG-rich genomic loci with high H3K9me3 signal and DNA methylation level (CHM) are hotspots of DNA methylation maintenance during pre-implantation embryogenesis. We further profile the allele-specific epigenetic map with unprecedented resolution in gynogenetic and androgenetic embryos, respectively, and identify 1,279 allele-specific CHMs, including 19 known imprinting control regions (ICRs). Our study suggests that 22 ICR-like regions (ICRLRs) may regulate allele-specific transcription similarly to known ICRs, and five of them are confirmed to be important for mouse embryo development. Taken together, our study reveals the widespread existence of allele-specific CHMs and largely extends the scope of allele-specific regulation in mammalian pre-implantation embryos.

Differential regulation of Hippo signaling pathway components between 8-cell and blastocyst stages of bovine preimplantation embryogenesis.

The Hippo signaling pathway is an important regulator of lineage segregation (trophectoderm and inner cell mass) during blastocyst formation in the mouse embryos. However, the role and regulation of Hippo signaling pathway components during bovine embryonic development is not completely understood. This study was thus designed to interpret the roles of Hippo cell signaling pathway components using two different yet specific chemical inhibitors (Cerivastatin and XMU-MP-1). A significant decrease in the blastocyst rates were observed on treatment with Cerivastatin and XMU-MP-1 inhibitors for the treatment groups, in comparison to the control groups. At the 8-cell stage, a significant decrease was observed in the gene expression and nuclear protein localization of YAP1 (Yes Associated Protein 1) and pYAP1 components of Hippo signaling pathway. However, no such effect of Cerivastatin treatment was observed on the localization of TAZ at this cell stage. On the contrary, during bovine blastocyst formation a significant decrease in the gene expression and nuclear localization of both YAP1 and TAZ suggest differences in the regulation of these components at 8-cell and blastocyst stages of embryonic development. Furthermore, XMU-MP-1 mediated chemical inhibition of Mst1 at the blastocyst stage also suggests differences in the regulation of Yap1 and Taz components of Hippo signaling pathway. Overall, this study indicates novel differences in the regulation of Hippo signaling transcript levels and protein localization between the 8-cell and blastocyst stages of bovine preimplantation embryonic development.

Evaluation of Sperm Selection Technique Using Hyaluronic Acid Binding During ICSI; A Randomized Controlled Trial

Objective: To compare between Hyaluronan binding system for sperm selection for ICSI procedures (PICSI) and conventional morphology sperm selection. In vitro sperm selection for ICSI is important as it has a direct influence on the paternal contribution of preimplantation embryogenesis. Various laboratory tests were developed to assess the functions of the spermatozoa. Among them, only the tests for sperm DNA fragmentation, hyperactivation of the spermatozoa and the hyaluronan-binding ability are simple and fast. Study Design: A randomized controlled trial where three-hundred cases with male infertility were divided into 3 equal groups according to semen abnormality (oligo, astheno and teratozoospermia) then each group was randomly divided into 2 equal subgroups one underwent PICSI and the other underwent conventional ICSI. The outcomes were fertilization,high quality embryos and clinical pregnancy rate. Results: A significant difference was found in favor of PICSI as regards both high-quality embryos and clinical pregnancy rate in all 3 groups. A significant difference was also found in fertilization rate in favor of PICSI group in both oligozoospermia and teratozoospermia groups but not in asthenozoospermia group. Conclusion: PICSI technique provides better high-quality embryos, fertilization, and clinical pregnancy rates in cases with male factor infertility; however, further studies are needed to define specific criteria for performing PICSI taking into consideration both the expenses and time of the technique.

Overlapping functions of RBBP4 and RBBP7 in regulating cell proliferation and histone H3.3 deposition during mouse preimplantation development

ABSTRACT Preimplantation development is critical for reproductive successes in mammals. Thus, it is important to understand how preimplantation embryogenesis is regulated. As a key event of preimplantation development, epigenetic reprogramming has been widely studied, yet how epigenetic complexes regulate preimplantation development remains largely unknown. Retinoblastoma binding protein 4 (RBBP4) and 7 (RBBP7) are integral components of epigenetic complexes including SIN3A, NuRD, and CoREST. Here, we demonstrate that double knockdown of Rbbp4 and 7, but not individually, causes embryonic lethality during the morula-to-blastocyst transition. Mechanistically, depletion of RBBP4 and 7 results in dysregulation of genes related to cell cycle, lineage development, and regulation of transcription, which is accompanied by cell cycle block, disrupted lineage specification and chromatin structure. Interestingly, RBBP4/7 depletion leads to a dramatic increase in H3.3 and H3K27ac abundance during morula-to-blastocyst transition. ChIP-seq analysis in early embryos and embryonic stem cells reveals enrichment of H3.3 at the promoter regions of RBBP4/7 target genes. In summary, our studies demonstrate the compensatory role of RBBP4/7 and reveal its potential mechanisms in preimplantation development. Summary sentence: RBBP4 and RBBP7 play a compensatory role in regulating cell proliferation, apoptosis, and histone H3.3 deposition during preimplantation development.

Genomics-Guided Drawing of Molecular and Pathophysiological Components of Malignant Regulatory Signatures Reveals a Pivotal Role in Human Diseases of Stem Cell-Associated Retroviral Sequences and Functionally-Active hESC Enhancers.

Repetitive DNA sequences (repeats) colonized two-third of human genome and a majority of repeats comprised of transposable genetic elements (TE). Evolutionary distinct categories of TE represent nucleic acid sequences that are repeatedly copied from and pasted into chromosomes at multiple genomic locations and acquired a multitude of regulatory functions. Here, genomics-guided maps of stemness regulatory signatures were drawn to dissect the contribution of TE to clinical manifestations of malignant phenotypes of human cancers. From patients’ and physicians’ perspectives, the clinical definition of a tumor’s malignant phenotype could be restricted to the early diagnosis of sub-types of malignancies with the increased risk of existing therapy failure and high likelihood of death from cancer. It is the viewpoint from which the understanding of stemness and malignant regulatory signatures is considered in this contribution. Genomics-guided analyses of experimental and clinical observations revealed the pivotal role of human stem cell-associated retroviral sequences (SCARS) in the origin and pathophysiology of clinically-lethal malignancies. SCARS were defined as the evolutionary- and biologically-related family of genomic regulatory sequences, the principal physiological function of which is to create and maintain the stemness phenotype during human preimplantation embryogenesis. For cell differentiation to occur, SCARS expression must be silenced and SCARS activity remains repressed in most terminally-differentiated human cells which are destined to perform specialized functions in the human body. Epigenetic reprogramming, de-repression, and sustained activity of SCARS results in various differentiation-defective phenotypes. One of the most prominent tissue- and organ-specific clinical manifestations of sustained SCARS activities is diagnosed as a pathological condition defined by a consensus of morphological, molecular, and genetic examinations as the malignant growth. Here, contemporary evidence are acquired, analyzed, and reported defining both novel diagnostic tools and druggable molecular targets readily amenable for diagnosis and efficient therapeutic management of clinically-lethal malignancies. These diagnostic and therapeutic approaches are based on monitoring of high-fidelity molecular signals of continuing SCARS activities in conjunction with genomic regulatory networks of thousands’ functionally-active embryonic enhancers affecting down-stream phenotype-altering genetic loci. Collectively, reported herein observations support a model of SCARS-activation triggered singular source code facilitating the intracellular propagation and intercellular (systemic) dissemination of disease states in the human body.

A Comparative Analysis of Hippo Signaling Pathway Components during Murine and Bovine Early Mammalian Embryogenesis.

The time required for successful blastocyst formation varies among multiple species. The formation of a blastocyst is governed by numerous molecular cell signaling pathways, such as the Hippo signaling pathway. The Hippo signaling pathway is initiated by increased cell–cell contact and via apical polarity proteins (AMOT, PARD6, and NF2) during the period of preimplantation embryogenesis. Cell–cell contact and cell polarity activate (phosphorylates) the core cascade components of the pathway (mammalian sterile twenty like 1 and 2 (MST1/2) and large tumor suppressor 1 and 2 (LATS1/2)), which in turn phosphorylate the downstream effectors of the pathway (YAP1/TAZ). The Hippo pathway remains inactive with YAP1 (Yes Associated protein 1) present inside the nucleus in the trophectoderm (TE) cells (polar blastomeres) of the mouse blastocyst. In the inner cell mass (ICM) cells (apolar blastomeres), the pathway is activated with p-YAP1 present in the cytoplasm. On the contrary, during bovine embryogenesis, p-YAP1 is exclusively present in the nucleus in both TE and ICM cells. Contrary to mouse embryos, transcription co activator with PDZ-binding motif (TAZ) (also known as WWTR1) is also predominantly present in the cytoplasm in all the blastomeres during bovine embryogenesis. This review outlines the major differences in the localization and function of Hippo signaling pathway components of murine and bovine preimplantation embryos, suggesting significant differences in the regulation of this pathway in between the two species. The variance observed in the Hippo signaling pathway between murine and bovine embryos confirms that both of these early embryonic models are quite distinct. Moreover, based on the similarity of the Hippo signaling pathway between bovine and human early embryo development, bovine embryos could be an alternate model for understanding the regulation of the Hippo signaling pathway in human embryos.