scholarly journals Histone Arginine Methyltransferase CARM1-Mediated H3R26me2 Is Essential for Morula-to-Blastocyst Transition in Pigs

2021 ◽  
Vol 9 ◽  
Author(s):  
Zubing Cao ◽  
Xu Tong ◽  
Huiqun Yin ◽  
Naru Zhou ◽  
Xiangdong Zhang ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Sequencing Analysis ◽  
Blastocyst Development ◽  
Arginine Methyltransferase ◽  
Functional Studies ◽  
Early Embryos

Coactivator-associated arginine methyltransferase 1 (CARM1) is involved in both establishment of first pluripotent lineage and pluripotency maintenance of embryonic stem cells (ESCs) in mice. However, the histone substrates and role of CARM1 in early embryonic development remain largely unknown. Here, we show that CARM1 specifically catalyzes H3R26me2 to promote porcine blastocyst formation. The putative histone substrates of CARM1, including H3R2me2, H3R17me2, and H3R26me2, are present in pig early embryos. The changes of CARM1 mRNA during early embryogenesis parallel that of H3R26me2. Functional studies using a combinational approach of chemical inhibition and RNA interference (RNAi) showed that catalytic activity inhibition of CARM1 protein or knockdown (KD) of CARM1 mRNA did not alter the levels of both H3R2me2 and H3R17me2, but significantly reduced H3R26me2 levels in porcine embryos. Furthermore, CARM1 inhibition or KD did not affect embryo development to the 2-cell, 4-cell, 8-cell, and morula stages, but severely compromised blastocyst development. CARM1 knocked down embryos that developed to the blastocyst stage had fewer total cells, inner cell mass (ICM), and trophectoderm (TE) cells. Mechanistically, single embryo RNA-sequencing analysis revealed that CARM1 KD altered the transcriptome characterized by downregulation of key genes associated with Hippo and PI3K-AKT signaling pathways. Taken together, these results demonstrate that CARM1 specifically catalyzes H3R26me2 in porcine embryos and participates in blastocyst development.

scholarly journals Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

2021 ◽  
Author(s):  
Chuan Chen ◽  
Wenqiang Liu ◽  
Jiayin Guo ◽  
Yuanyuan Liu ◽  
Xuelian Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Rrna Synthesis ◽  
Binding Ability ◽  
Transcriptome Regulation ◽  
Early Embryos ◽  
Intrinsic Mechanism ◽  
Regulatory Rnas

AbstractN6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

scholarly journals CARM1 is heterogeneous in mouse four-cell embryo and important to blastocyst development

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 91-104
Author(s):  
Hongzheng Sun ◽  
Jianmin Su ◽  
Teng Wu ◽  
Fengyu Wang ◽  
Jian Kang ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Type I ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Arginine Methyltransferase ◽  
Heterogeneous Distribution ◽  
Arginine Residues

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I arginine methyltransferase that methylates the arginine residues of histone and nonhistone. Carm1 regulates various cellular processes, including transcriptional regulation, mRNA processing, cellular proliferation, and differentiation. Blastomeres with high Carm1 expression levels show cleavage tendency to inner cell mass (ICM) in mouse embryos. However, details about the factors for CARM1 distribution in mouse early embryos and the role of Carm1 in blastocyst development remain unclear. Here, the endonuclear distribution of CARM1 protein was heterogeneous between blastomeres from the late four-cell stage to the blastocyst stage. The heterogeneity of CARM1 distribution in blastomeres at the late four-cell stage was randomly obtained from two-cell stage embryos. From the four-cell stage to morula, CARM1 in individual blastomere remained heterogeneous. In the blastocyst stage, CARM1 protein level in ICM was much higher than that in trophoblast. We found that microRNA (miRNA) miR-181a is an important regulator for Carm1 distribution at the late four-cell stage. The ratio of heterogeneous embryos was reduced in all the embryos when miR-181a was inhibited. CARM1 inhibition reduced the level of symmetrical histone H3 arginine-26 dimethylation and impaired blastocyst development. Silencing Carm1 reduced cell number and increased cell apoptosis at the blastocyst stage. These results show a CARM1 heterogeneous distribution from the four-cell embryos to the blastocysts. miR-181a regulates the control of CARM1 heterogeneous distribution in the four-cell-stage embryos, and CARM1 is an important protein in regulating blastocyst development.

scholarly journals Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2232 ◽  
Author(s):  
Claudia Springer ◽  
Valeri Zakhartchenko ◽  
Eckhard Wolf ◽  
Kilian Simmet
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Model Organism ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Lineage Differentiation ◽  
Species Specific ◽  
And Control

The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another—so far unknown—pathway regulates HB differentiation.

scholarly journals Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts

Biology Open ◽  
2021 ◽  
Author(s):  
Yao Xiao ◽  
Froylan Sosa ◽  
Pablo J. Ross ◽  
Kenneth E. Diffenderfer ◽  
Peter J. Hansen
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Activin A ◽  
Blastocyst Development ◽  
Sox2 Expression ◽  
Nanog Expression ◽  
Canonical Wnt

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of inner cell mass and epiblast during embryonic development.

scholarly journals 45PRODUCTION OF CHIMERIC TRANSCHROMOSOMIC CALVES

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
P. Kasinathan ◽  
M.F. Nichols ◽  
J.E. Griffin ◽  
J.M. Robl
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Peripheral Blood Lymphocytes ◽  
Blastocyst Stage ◽  
Human Artificial Chromosome ◽  
Fish Analysis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Fetal Fibroblasts

Chimeras have been used for investigating fundamental aspects of early embryonic development, and differentiation, and for introducing foreign genes into mammals (Robertson et al., 1986 Nature 323, 445–448; Cibelli et al., 1998 Science 280, 1256–1258). The main objective of this study was to determine if the transfer of blastomeres from in vitro-produced (IVP) embryos into cloned, transchromosomic embryos improved the efficiency of producing transchromosomic calves. Cloned embryos were produced using in vitro-matured bovine oocytes and bovine fetal fibroblasts containing a human artificial chromosome (HAC) (Kuroiwa et al., 2002 Nat Biotechnol 20, 889–894). IVP embryos were produced using standard procedures and blastomeres were harvested at the 8–16 cell stage by removing the zona pellucida with protease. Cloned embryos were randomly divided on Day 4 into two groups. One group received 3–4 IVP blastomeres while a second group served as a control (nonmanipulated cloned embryos). After transferring the blastomeres, the chimeric and cloned embryos were placed in culture (Kasinathan et al., 2001 Biol. Reprod. 64, 1487–1493) and on Day 7 development to the blastocyst stage was evaluated. Grades 1 and 2 embryos were transferred; two each per synchronized recipient. Pregnancy maintenance, calving, and calf survival were evaluated in both groups. Presence of a HAC in live calves was evaluated in both fibroblasts and peripheral blood lymphocytes (PBLs) using FISH analysis. Embryo development to the blastocyst stage, maintenance of pregnancy and number of calves born were analyzed using Chi-square. There were no differences in the rate of blastocyst development at day 7 or establishment of pregnancy at 40d (P>0.05). However, pregnancy rate at 120d, and number of calves that developed to term and were alive at birth (chimera 14/54 and clone 4/90), and at 1 month of age (chimera 13/54 and clone 1/90) were lower (P<0.01) for cloned embryos. The proportion of cells containing an HAC in PBLs, was higher in cloned calves (100%) compared to chimeric calves (26%). The HAC retension rates in PBLs in HAC-positive chimeric and cloned calves were 84% and 95%, respectively. These data indicate that, although the proportion of calves retaining an HAC was lower in chimeras compared to clones, more HAC-positive calves were produced in the chimeric treatment from fewer cloned embryos. We speculate that higher rates of development in the chimeras may be related to the normality of the placenta. Future studies will be required to determine the contribution of the IVP blastomeres to both the inner cell mass and trophectoderm. Therefore, a chimeric approach may be useful for improving the efficiency of producing cloned transchromosomic calves.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

Production and analysis of ES cell aggregation chimeras

1999 ◽  
Author(s):  
Andras Nagy ◽  
Janet Rossant
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Aggregation ◽  
Blastocyst Stage ◽  
Original Method ◽  
Embryonic Cells ◽  
Es Cell ◽  
Cleavage Stage

Embryonic stem (ES) cells behave like normal embryonic cells when returned to the embryonic environment after injection into a host blastocyst or after aggregation with earlier blastomere stage embryos. In such chimeras, ES cells behave like primitive ectoderm or epiblast cells (1), in that they contribute to all lineages of the resulting fetus itself, as well as to extraembryonic tissues derived from the gastrulating embryo, namely the yolk sac mesoderm, the amnion, and the allantois. However, even when aggregated with preblastocyst stage embryos, ES cells do not contribute to derivatives of the first two lineages to arise in development, namely, the extraembryonic lineages: trophoblast and primitive endoderm (2). The pluripotency of ES cells within the embryonic lineages is critical to their use in introducing new genetic alterations into mice, because truly pluripotent ES cells can contribute to the germline of chimeras, as well as all somatic lineages. However, the ability of ES cells to co-mingle with host embryonic cells, specifically in the embryonic, but not the major extraembryonic lineages, opens up a variety of possibilities for analysing gene function by genetic mosaics rather than by germline mutant analysis alone (3). There are two basic methods for generating pre-implantation chimeras in mice, whether it be embryo ↔ embryo or ES cell ↔ embryo chimeras. Blastocyst injection, in which cells are introduced into the blastocoele cavity using microinjection pipettes and micromanipulators, has been the method of choice for most ES cell chimera work (see Chapter 4). However, the original method for generating chimeras in mice, embryo aggregation, is considerably simpler and cheaper to establish in the laboratory. Aggregation chimeras are made by aggregating cleavage stage embryos together, or inner cell mass (ICM) or ES cells with cleavage stage embryos, growing them in culture to the blastocyst stage, and then transferring them to the uterus of pseudopregnant recipients to complete development. This procedure can be performed very rapidly by hand under the dissecting microscope, thus making possible high throughput production with minimal technical skill (4). In this chapter we describe some of the uses of pre-implantation chimeras, whether made by aggregation or blastocyst injection, but focus on the technical aspects of aggregation chimera generation. We also discuss the advantages and disadvantages of aggregation versus blastocyst injection for chimera production.

scholarly journals Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

2010 ◽  
Vol 191 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Daniel Mesnard ◽  
Daniel B. Constam
Keyword(s):  
Cell Surface ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Axis Formation ◽  
Fluorescent Biosensor ◽  
Membrane Bound ◽  
Biosensor Cell

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

scholarly journals Aggregation recovers developmental plasticity in mouse polyploid embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 404 ◽  
Author(s):  
Hiroyuki Imai ◽  
Wataru Fujii ◽  
Ken Takeshi Kusakabe ◽  
Yasuo Kiso ◽  
Kiyoshi Kano
Keyword(s):  
Mammalian Cells ◽  
Developmental Plasticity ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mammalian Development ◽  
Cellular Functions ◽  
Developmental Potential ◽  
Early Mammalian Development

Tetraploid embryos normally develop into blastocysts and embryonic stem cells can be established from tetraploid blastocysts in mice. Thus, polyploidisation does not seem to be so harmful during preimplantation development. However, the mechanisms by which early mammalian development accepts polyploidisation are poorly understood. In this study, we aimed to elucidate the effect of polyploidisation on early mammalian development and to further comprehend its tolerance using hyperpolyploid embryos produced by repetitive whole genome duplication. We successfully established several types of polyploid embryos (tetraploid, octaploid and hexadecaploid) and studied their developmental potential invitro. We demonstrated that all types of these polyploid embryos maintained the ability to develop to the blastocyst stage, which implies that mammalian cells might have basic cellular functions in implanted embryos, despite polyploidisation. However, the inner cell mass was absent in hexadecaploid blastocysts. To complement the total number of cells in blastocysts, a fused hexadecaploid embryo was produced by aggregating several hexadecaploid embryos. The results indicated that the fused hexadecaploid embryo finally recovered pluripotent cells in the blastocyst. Thus, our findings suggest that early mammalian embryos may have the tolerance and higher plasticity to adapt to hyperpolyploidisation for blastocyst formation, despite intense alteration of the genome volume.

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