scholarly journals Advanced maternal age perturbs mouse embryo development and alters the phenotype of derived embryonic stem cells

2021 ◽  
pp. 1-11
Author(s):  
Pooja Khurana ◽  
Neil R. Smyth ◽  
Bhavwanti Sheth ◽  
Miguel A. Velazquez ◽  
Judith J. Eckert ◽  
...  
Keyword(s):  
Maternal Age ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Advanced Maternal Age ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Ki 67 ◽  
Altered Expression ◽  
Developmental Potential ◽  
Developmental Mechanisms

Abstract Advanced maternal age (AMA) is known to reduce fertility, increases aneuploidy in oocytes and early embryos and leads to adverse developmental consequences which may associate with offspring lifetime health risks. However, investigating underlying effects of AMA on embryo developmental potential is confounded by the inherent senescence present in maternal body systems further affecting reproductive success. Here, we describe a new model for the analysis of early developmental mechanisms underlying AMA by the derivation and characterisation of mouse embryonic stem cell (mESC-like) lines from naturally conceived embryos. Young (7–8 weeks) and Old (7–8 months) C57BL/6 female mice were mated with young males. Preimplantation embryos from Old dams displayed developmental retardation in blastocyst morphogenesis. mESC lines established from these blastocysts using conventional techniques revealed differences in genetic, cellular and molecular criteria conserved over several passages in the standardised medium. mESCs from embryos from AMA dams displayed increased incidence of aneuploidy following Giemsa karyotyping compared with those from Young dams. Moreover, AMA caused an altered pattern of expression of pluripotency markers (Sox2, OCT4) in mESCs. AMA further diminished mESC survival and proliferation and reduced the expression of cell proliferation marker, Ki-67. These changes coincided with altered expression of the epigenetic marker, Dnmt3a and other developmental regulators in a sex-dependent manner. Collectively, our data demonstrate the feasibility to utilise mESCs to reveal developmental mechanisms underlying AMA in the absence of maternal senescence and with reduced animal use.

The Influence of Thyroid Hormone on Ca2+ Signaling Pathways during Embryonal Development

2021 ◽  
Vol 21 ◽  
Author(s):  
Joachim Krebs
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Brain Development ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Fetal Brain ◽  
Mouse Embryonic Stem Cell ◽  
Dependent Manner ◽  
Transcription Activator

: Thyroid hormones influence brain development through regulation of gene expression. Ca2+-dependent gene expression is a major pathway controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) which in turn is induced by the thyroid hormone T3 as also demonstrated in a mouse embryonic stem cell line. In addition, T3 is controlling the expression of neurexin, synaptotagmin2 (SYT2), synaptotagmin-related gene1 (SRG1) and a number of other genes, involved in neurotransmitter release in a Ca2+-dependent manner. It has been noticed that the development of dopaminergic neurons by evoking significant calcium entry occurs through TRPC calcium channels. It also was demonstrated that the T3-mediated development of an early neuronal network is characteristic for depolarizing GABAergic neurons concomitant with intracellular calcium transients. An important aspect of T3-dependent regulation of gene expression in the developing brain is its modulation by the transcription activator COUP-TF1. Regulation of alternative splicing by CaMKIV is another important aspect for embryonal neural development since it can lead to the expression of PMCA1a, the neuronal specific isoform of the plasma membrane calcium pump. Maternal hypothyroidism or CaMKIV deficiency can have a severe influence on fetal brain development.

scholarly journals E-cadherin regulates the stability and transcriptional activity of β-catenin in embryonic stem cells

2021 ◽  
Author(s):  
Sinjini Bhattacharyya ◽  
Ridim D Mote ◽  
Jacob Freimer ◽  
Surya Bansi Singh ◽  
Sandhya Arumugam ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Altered Expression ◽  
Cell State ◽  
State Dependent ◽  
Exact Function ◽  
A Cell ◽  
E Cadherin

E-CADHERIN is abundantly expressed in embryonic stem cells (ESCs) and plays an important role in the maintenance of cell-cell adhesions. However, the exact function of this molecule beyond cell adhesion, in the context of cell fate decisions is largely unknown. Using mouse ESCs (mESCs), we demonstrate that E-CADHERIN and β-CATENIN interact at the membrane and continue to do so upon internalization within the cell. Knockout of the gene encoding E-CADHERIN, Cdh1, in mESCs resulted in a failure to form tight colonies, accompanied by altered expression of differentiation markers, and retention of pluripotency factor expression during differentiation. Interestingly, Cdh1-/- mESCs showed a dramatic reduction in β-CATENIN levels. Transcriptional profiling of Cdh1-/- mESCs displayed a significant alteration in the expression of a subset of β-CATENIN targets, in a cell-state dependent manner. While treatment with a pharmacological inhibitor against GSK3β could rescue levels of β-CATENIN in Cdh1-/- mESCs, expression of downstream targets were altered in a context-dependent manner, indicating an additional layer of regulation within this subset. Together, our results reveal the existence of a cell-state-dependent regulation of β-CATENIN and its transcriptional targets in an E-CADHERIN dependent manner. Our findings hint at hitherto unknown roles played by E-CADHERIN in regulating the activity of β-CATENIN in ESCs.

scholarly journals Deciphering the Nature of Trp73 Isoforms in Mouse Embryonic Stem Cell Models: Generation of Isoform-Specific Deficient Cell Lines Using the CRISPR/Cas9 Gene Editing System

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3182
Author(s):  
Lorena López-Ferreras ◽  
Nicole Martínez-García ◽  
Laura Maeso-Alonso ◽  
Marta Martín-López ◽  
Ángela Díez-Matilla ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Dependent Manner ◽  
P53 Family ◽  
Dual Nature ◽  
Cellular Models

The p53 family has been widely studied for its role in various physiological and pathological processes. Imbalance of p53 family proteins may contribute to developmental abnormalities and pathologies in humans. This family exerts its functions through a profusion of isoforms that are generated by different promoter usage and alternative splicing in a cell type dependent manner. In particular, the Trp73 gene gives rise to TA and DN-p73 isoforms that confer p73 a dual nature. The biological relevance of p73 does not only rely on its tumor suppression effects, but on its pivotal role in several developmental processes. Therefore, the generation of cellular models that allow the study of the individual isoforms in a physiological context is of great biomedical relevance. We generated specific TA and DN-p73-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 gene editing system and validated them as physiological bona fide p73-isoform knockout models. Global gene expression analysis revealed isoform-specific alterations of distinctive transcriptional networks. Elimination of TA or DN-p73 is compatible with pluripotency but prompts naïve pluripotent stem cell transition into the primed state, compromising adequate lineage differentiation, thus suggesting that differential expression of p73 isoforms acts as a rheostat during early cell fate determination.

scholarly journals Establishment of bovine expanded potential stem cells

2021 ◽  
Vol 118 (15) ◽  
pp. e2018505118
Author(s):  
Lixia Zhao ◽  
Xuefei Gao ◽  
Yuxuan Zheng ◽  
Zixin Wang ◽  
Gaoping Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Adult Individual ◽  
Developmental Potential ◽  
Cell Lineages

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

scholarly journals Perturbations in imprinted methylation from assisted reproductive technologies but not advanced maternal age in mouse preimplantation embryos

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Audrey J. Kindsfather ◽  
Megan A. Czekalski ◽  
Catherine A. Pressimone ◽  
Margaret P. Erisman ◽  
Mellissa R. W. Mann
Keyword(s):  
Embryo Culture ◽  
Maternal Age ◽  
Assisted Reproductive Technologies ◽  
Advanced Maternal Age ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Clinical Methods ◽  
First Time

Abstract Background Over the last several decades, the average age of first-time mothers has risen steadily. With increasing maternal age comes a decrease in fertility, which in turn has led to an increase in the use of assisted reproductive technologies by these women. Assisted reproductive technologies (ARTs), including superovulation and embryo culture, have been shown separately to alter imprinted DNA methylation maintenance in blastocysts. However, there has been little investigation on the effects of advanced maternal age, with or without ARTs, on genomic imprinting. We hypothesized that ARTs and advanced maternal age, separately and together, alter imprinted methylation in mouse preimplantation embryos. For this study, we examined imprinted methylation at three genes, Snrpn, Kcnq1ot1, and H19, which in humans are linked to ART-associated methylation errors that lead to imprinting disorders. Results Our data showed that imprinted methylation acquisition in oocytes was unaffected by increasing maternal age. Furthermore, imprinted methylation was normally acquired when advanced maternal age was combined with superovulation. Analysis of blastocyst-stage embryos revealed that imprinted methylation maintenance was also not affected by increasing maternal age. In a comparison of ARTs, we observed that the frequency of blastocysts with imprinted methylation loss was similar between the superovulation only and the embryo culture only groups, while the combination of superovulation and embryo culture resulted in a higher frequency of mouse blastocysts with maternal imprinted methylation perturbations than superovulation alone. Finally, the combination of increasing maternal age with ARTs had no additional effect on the frequency of imprinted methylation errors. Conclusion Collectively, increasing maternal age with or without superovulation had no effect of imprinted methylation acquisition at Snrpn, Kcnq1ot1, and H19 in oocytes. Furthermore, during preimplantation development, while ARTs generated perturbations in imprinted methylation maintenance in blastocysts, advanced maternal age did not increase the burden of imprinted methylation errors at Snrpn, Kcnq1ot1, and H19 when combined with ARTs. These results provide cautious optimism that advanced maternal age is not a contributing factor to imprinted methylation errors in embryos produced in the clinic. Furthermore, our data on the effects of ARTs strengthen the need to advance clinical methods to reduce imprinted methylation errors in in vitro-produced embryos.

scholarly journals SETDB1 prevents TET2-dependent activation of IAP retroelements in naïve embryonic stem cells

2017 ◽  
Author(s):  
Özgen Deniz ◽  
Lorenzo de la Rica ◽  
Kevin C. L. Cheng ◽  
Dominik Spensberger ◽  
Miguel R. Branco
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Endogenous Retroviruses ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Epigenetic Mechanisms

BackgroundEndogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET- dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos.ResultsWe used naïve ESCs to investigate the role of SETDB1 in ERV regulation and, in particular, its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We found that, in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, however, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these transposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming.ConclusionsOur results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation.

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