Cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly- N-acetyllactosamine series

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 115-128
Author(s):  
S. Rastan ◽  
S. J. Thorpe ◽  
P. Scudder ◽  
S. Brown ◽  
H. C. Gooi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Medium ◽  
De Novo ◽  
Embryonic Cell ◽  
Preimplantation Embryos ◽  
Carbohydrate Binding ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Antigen Activity ◽  
Surface Carbohydrates

Roles of cell surface carbohydrates containing the 3-fucosyl-N-acetyllactosamine and poly-Nacetyllactosamine sequences (SSEA-1 and I antigens, respectively) in the compaction of mouse embryos have been investigated using the endo-β-galactosidase of Bacteroides fragilis to modify the surface of cleavage-stage embryos. Treatment with this enzyme abolished SSEA-1 activity and diminished I antigen activity on the embryonic cell surface. Embryos cultured in the presence of endo-β-galactosidase from the 2- to 4-cell stage onwards, or treated with the enzyme at the compacting 8-cell stage, continued to compact and proceeded to form blastocysts at the normal rate. However, when compacted 8- to 16-cell embryos were experimentally decompacted in calcium-free medium, treated for 1 h with endo-β-galactosidase and returned to normal culture medium, the time taken for 50 % of the embryos to recompact was prolonged five-fold. There was an even greater delay if these embryos were maintained in culture medium containing the enzyme. Blastocysts were eventually formed under both conditions. Thus, endo-β-galactosidase did not affect compaction unless the embryos were first decompacted. On the assumption that recompaction and de novo compaction occur by similar mechanisms, we propose that carbohydrate-binding molecules are involved which have high affinities for poly-Nacetyllactosamine structures and protect them from digestion by endo-β-galactosidase.

Tetraploidy and early development: effects on developmental timing and embryonic metabolism

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 91-108
Author(s):  
Martin A. Eglitis ◽  
Lynn M. Wiley
Keyword(s):  
Cell Surface ◽  
Surface Antigen ◽  
Gene Dosage ◽  
Genetic Material ◽  
Cell Number ◽  
Embryonic Cell ◽  
Cell Surface Antigen ◽  
Metabolic Effects ◽  
Cell Stage ◽  
Preimplantation Mouse

The effect of balanced gene dosage changes on the timing of cavitation and on the timing of appearance of a stage-specific embryonic cell surface antigen was studied in preimplantation mouse embryos. Gene dosage was increased by creating tetraploid embryos at the 4-cell stage, either by blastomere fusion with polyethylene glycol (PEG) or by incubation in cytochalasin B (cytB) to block cell division. Removal of the zona pellucida with Pronase from diploid embryos caused a 7 h delay in cavitation. Further manipulations, either with PEG or cytB to induce tetraploidy, did not produce a statistically significant additional delay in cavitation timing. Likewise, PEG-induced tetraploidy did not affect the timing of appearance or disappearance of the embryonic cell surface antigen as compared with diploid control embryos. In analysing the metabolic effects of tetraploidy, we found that in tetraploid embryos with cell number equivalent to intact diploid embryos, MDH activity did not double with the doubling of the genome, being only 50% greater than diploid levels in cytB-induced tetraploid embryos and only 20% greater than diploid levels in PEG-induced tetraploid embryos. However, in tetraploid embryos with one-half normal cell number, enzyme activity was equal to that in whole diploid embryos, suggesting that in such embryos, MDH activity increased in parallel with increases in gene dosage. Further studies showed that levels of RNA synthesis in PEGinduced etraploid embryos also did not increase in parallel with the doubling of the genome. Rather, these results suggested that in tetraploid embryos, compensation was made for at least part of the excess genetic material.

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

Permanence of de novo segmental aneuploidy in sequential embryo biopsies

2020 ◽  
Vol 35 (4) ◽  
pp. 759-769
Author(s):  
M C Magli ◽  
C Albanese ◽  
A Crippa ◽  
G Terzuoli ◽  
G La Sala ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Initial Diagnosis ◽  
Preimplantation Embryos ◽  
Comparative Genomic ◽  
Cleavage Stage ◽  
Preimplantation Genetic Testing ◽  
Initial Finding ◽  
Segmental Aneuploidy ◽  
Chromosome Imbalance

Abstract STUDY QUESTION Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance? SUMMARY ANSWER The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA. WHAT IS KNOWN ALREADY Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest. STUDY DESIGN, SIZE, DURATION This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization. MAIN RESULTS AND THE ROLE OF CHANCE The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P &lt; 0.001) and decreased to 11.2% in blastocysts (P &lt; 0.025 versus oocytes; P &lt; 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P &lt; 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes. LIMITATIONS, REASONS FOR CAUTION Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism. WIDER IMPLICATIONS OF THE FINDINGS The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained. There are no conflicts of interest.

Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system

Development ◽  
1996 ◽  
Vol 122 (3) ◽  
pp. 925-936
Author(s):  
M. Tiemeyer ◽  
C.S. Goodman
Keyword(s):  
Nervous System ◽  
Cell Surface ◽  
Neural Development ◽  
Small Subset ◽  
Carbohydrate Binding ◽  
Calculated Molecular Mass ◽  
Amino Terminal ◽  
Surface Carbohydrates ◽  
Drosophila Embryos ◽  
Cell Cell

Interactions between embryonic neural cells generate the specific patterns of connectivity observed in nervous systems. Cell surface carbohydrates have been proposed to function in cellular recognition events guiding such interactions. Carbohydrate-binding proteins (lectins) that recognize specific oligosaccharide ligands in embryonic neural tissue provide a molecular mechanism for carbohydrate-mediated cell-cell interactions in neural development. Therefore, we have screened an embryonic Drosophila melanogaster cDNA library, expressed in COS1 cells, for carbohydrate-binding activity. COS1 cells expressing putative Drosophila lectins were identified and recovered based on their adhesion to immobilized preparations of neutral and zwitterionic glycolipids extracted from Drosophila embryos. We have identified an endogenous lectin expressed during Drosophila embryogenesis. The cloned lectin, designated ‘gliolectin’, possesses a novel protein sequence with a calculated molecular mass of 24,993. When expressed in Drosophila S2 cells, the lectin mediates heterophilic cellular aggregation. In embryos, gliolectin is expressed by a subset of glial cells found at the midline of the developing nervous system. Expression is highest during the formation of the Drosophila embryonic axonal commissures, a process requiring midline glial cell funcion. Immunoprecipitation with a monoclonal antibody against gliolectin yields a protein of Mr=46,600 from Drosophila embryonic membranes, suggesting that post-translational modification of gliolectin is extensive. Epitope- tagged chimericproteins composed of the amino terminal one-half of gliolectin and the Fc region of human IgG bind a small subset of the total glycolipids extracted from Drosophila embryos, demonstrating that the lectin activity of gliolectin can discriminate between oligosaccharide structures. The presence of gliolectin in the developing Drosophila embryonic nervous system further supports a role for cell surface carbohydrates in cell-cell recognition and indicates that the molecular diversity of animal lectins is not yet completely defined.

De novo transcription of thyroid hormone receptors is essential for early bovine embryo development in vitro

2018 ◽  
Vol 30 (5) ◽  
pp. 779 ◽  
Author(s):  
N.-Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
G.-J. Rho ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Embryo Development ◽  
De Novo ◽  
Preimplantation Embryos ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Protein Levels ◽  
Development In Vitro

Thyroid hormone receptor (THR) α and THRβ mediate the genomic action of thyroid hormones (THs) that affect bovine embryo development. However, little is known about THRs in the preimplantation embryo. The aim of the present study was to investigate the importance of THRs in in vitro preimplantation bovine embryos. THR transcripts and protein levels were detected in developing preimplantation embryos up to the blastocyst stage. Embryonic transcription of THRs was inhibited by α-amanitin supplementation, and both maternal and embryonic transcription were knocked down by short interference (si) RNA microinjection. In the control group, mRNA and protein levels of THRs increased after fertilisation. In contrast, in both the transcription inhibition and knockdown groups there were significant (P < 0.05) decreases in mRNA expression of THRs from the 2-cell stage onwards. However, protein levels of THRs were not altered at 2-cell stage, although they did exhibit a significant (P < 0.05) decrease from the 4-cell stage. Moreover, inhibition of de novo transcripts of THRs using siRNA led to a significant (P < 0.01) decrease in the developmental rate and cell number, as well as inducing a change in embryo morphology. In conclusion, THRs are transcribed soon after fertilisation, before major activation of the embryonic genome, and they are essential for bovine embryo development in vitro.

Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 662-674 ◽  
Author(s):  
Kayla J. Perkel ◽  
Pavneesh Madan
Keyword(s):  
Culture Medium ◽  
Physiological State ◽  
Proton Nuclear Magnetic Resonance ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Isoleucine Leucine ◽  
H Nmr

SummarySpent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12–24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

scholarly journals Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Shiwen Ni ◽  
Teng Zhang ◽  
Chenmin Zhou ◽  
Min Long ◽  
Xuan Hou ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
De Novo ◽  
Biological Significance ◽  
Preimplantation Embryos ◽  
Inosine Monophosphate Dehydrogenase ◽  
Cell Stage ◽  
Simultaneous Formation

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed “cytoophidium” or “rod and ring” in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte–follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

scholarly journals Single-Cell Sequencing of Primate Preimplantation Embryos Reveals Chromosome Elimination Via Cellular Fragmentation and Blastomere Exclusion

2018 ◽  
Author(s):  
Brittany L. Daughtry ◽  
Jimi L. Rosenkrantz ◽  
Nathan H. Lazar ◽  
Suzanne S. Fei ◽  
Nash Redmayne ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Chromosome Instability ◽  
Complex Mixture ◽  
Chromosome Elimination ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cleavage Stage ◽  
Primate Model ◽  
Cell Stage

ABSTRACTAneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously demonstrated that human preimplantation embryos encapsulate mis-segregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a non-human primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N=50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the frequency of aneuploidy and micronucleation is conserved between humans and macaques and that cellular fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping demonstrated that these chromosome-containing cellular fragments (CCFs) can be either maternal or paternal in origin and display DNA damage via double-stranded breaks. Chromosome breakage and abnormal cytokinesis resulted in reciprocal losses/gains at the terminal ends of chromosome arms, uniparental genome segregation, and mixoploidy between blastomeres. Combining time-lapse imaging with scDNA-seq, we also determined that multipolar divisions at the zygote or 2-cell stage generated chaotic aneuploidy encompassing a complex mixture of maternal and paternal chromosomes. Despite frequent chromosomal mis-segregation at the cleavage-stage, we show that CCFs and non-dividing aneuploid blastomeres exhibiting extensive DNA damage are prevented from incorporation at the blastocyst stage. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination by cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

Nucleolus in apoptosis-induced mouse preimplantation embryos

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 271-283 ◽  
Author(s):  
V. Baran ◽  
D. Fabian ◽  
P. Rehak ◽  
J. Koppel
Keyword(s):  
Culture Medium ◽  
Actinomycin D ◽  
Cell Loss ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome ◽  
Early Embryos

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.

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