236.Differential expression of monocarboxylate cotransporter proteins in preimplantation embryos

During preimplantation development mouse embryos demonstrate a switch in substrate preference. Pyruvate consumption, high during the first few cleavage stages, declines as the morula develops to a blastocyst, when glucose becomes the preferred substrate. Whilst pyruvate utilisation has been well characterised, changes in the function and expression of pyruvate transporters during this crucial period remain unclear. Pyruvate, lactate and other monocarboxylates are transported across mammalian cell membranes via a specific H+-monocarboxylate cotransporter (MCT). Fourteen members of this family have been identified of which MCT1, MCT2 and MCT4 are well characterised. Although mRNA expression profiles are known during early mouse development (1,2), the specific roles of each protein isoform are unknown. In order to understand these, the expression pattern for each isoform and their cellular localisation during preimplantation development have been determined. Mouse embryos were freshly collected from superovulated Quackenbush mice at 24, 48, 72 and 96 h post-hCG and expression of MCT1, MCT2 and MCT4 analysed by confocal laser scanning immunohistochemistry. Our results confirm that all three MCT proteins are expressed in preimplantation embryos. Immunoreactivity for MCT1 and MCT2 appears diffuse throughout the cytoplasm of cleavage stage embryos. As development proceeds, MCT1 localised to the basolateral membranes of morulae and blastocysts, whilst stronger MCT2 expression was found on the apical trophectoderm as well as the inner cell mass. MCT4 immunoreactivity on the other hand is apparent at cell-cell contact sites in cleavage stage embryos and morulae, but it is not apparent in the blastocyst. The demonstration of different expression patterns for MCT1, MCT2 and MCT4 in mouse embryos implies specific functional roles for each in the critical regulation of H+, pyruvate and lactate transport during preimplantation development. (1) Harding EA, Day ML, Gibb CA, Johnson MH, Cook DI (1999) The activity of the H+-monocarboxylate cotransporter during pre-implantation development in the mouse. Eur. J. Physiol. 438, 397–404. (2) H�rubel F, El Mouatassim S, Gu�rin P, Frydman R, M�n�zo Y (2002) Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development. Zygote 10, 175–181.

Download Full-text

Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development

Development ◽

10.1242/dev.117.3.1145 ◽

1993 ◽

Vol 117 (3) ◽

pp. 1145-1151 ◽

Cited By ~ 3

Author(s):

Q. Javed ◽

T.P. Fleming ◽

M. Hay ◽

S. Citi

Keyword(s):

Tight Junction ◽

Cell Mass ◽

Preimplantation Embryos ◽

Cell Contact ◽

Mouse Development ◽

Early Cleavage ◽

Cell Stage ◽

Tight Junction Formation ◽

Junction Protein ◽

Junction Formation

The expression of the tight junction peripheral membrane protein, cingulin (140 × 10(3) M(r), was investigated in mouse eggs and staged preimplantation embryos by immunoblotting and immunoprecipitation. Polyclonal antibody to chicken brush cingulin detected a single 140 × 10(3) M(r) protein in immunoblots of unfertilised eggs and all preimplantation stages. Relative protein levels were high in eggs and early cleavage stages, declined during later cleavage and increased again in expanding blastocysts. Quantitative immunoprecipitation of metabolically labelled eggs and staged embryos also revealed a biphasic pattern for cingulin synthesis with relative net levels being high in unfertilised eggs, minimal during early cleavage, rising 2.3-fold specifically at the onset of compaction (8-cell stage, when tight junction formation begins), and increasing further at a linear rate during morula and blastocyst stages. Cingulin synthesis in eggs is not influenced by fertilisation (or aging, if unfertilised), but this level declines sharply after first cleavage. These results indicate that cingulin is expressed by both maternal and embryonic genomes. The turnover of maternal cingulin (unfertilised eggs) and embryonic cingulin at a stage before tight junction formation begins (4-cell stage) is higher (t1/2 approximately 4 hours) than cingulin synthesised after tight junction formation (blastocysts; t1/2 approximately 10 hours). This increase in cingulin stability is reversed in the absence of extracellular calcium. Cingulin synthesis is also tissue-specific in blastocysts, being up-regulated in trophectoderm and down-regulated in the inner cell mass. Taken together, the results suggest that (i) cingulin may have a role during oogenesis and (ii) cell-cell contact patterns regulate cingulin biosynthesis during early morphogenesis, contributing to lineage-specific epithelial maturation.

Download Full-text

Comparison of gene expression in fresh and frozen–thawed human preimplantation embryos

Reproduction ◽

10.1530/rep-12-0047 ◽

2012 ◽

Vol 144 (5) ◽

pp. 569-582 ◽

Cited By ~ 31

Author(s):

Lisa Shaw ◽

Sharon F Sneddon ◽

Daniel R Brison ◽

Susan J Kimber

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Expression Patterns ◽

Developmental Expression ◽

Preimplantation Embryos ◽

Human Embryos ◽

Molecular Events ◽

Reliable Source ◽

Human Preimplantation Embryos ◽

Early Human

Identification and characterisation of differentially regulated genes in preimplantation human embryonic development are required to improve embryo quality and pregnancy rates in IVF. In this study, we examined expression of a number of genes known to be critical for early development and compared expression profiles in individual preimplantation human embryos to establish any differences in gene expression in fresh compared to frozen–thawed embryos used routinely in IVF. We analysed expression of 19 genes by cDNA amplification followed by quantitative real-time PCR in a panel of 44 fresh and frozen–thawed human preimplantation embryos. Fresh embryos were obtained from surplus early cleavage stage embryos and frozen–thawed embryos from cryopreserved 2PN embryos. Our aim was to determine differences in gene expression between fresh and frozen–thawed human embryos, but we also identified differences in developmental expression patterns for particular genes. We show that overall gene expression among embryos of the same stage is highly variable and our results indicate that expression levels between groups did differ and differences in expression of individual genes was detected. Our results show that gene expression from frozen–thawed embryos is more consistent when compared with fresh, suggesting that cryopreserved embryos may represent a reliable source for studying the molecular events underpinning early human embryo development.

Download Full-text

Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

Molecular and Cellular Biology ◽

10.1128/mcb.01188-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3123-3130 ◽

Cited By ~ 26

Author(s):

Klaus Fortschegger ◽

Bettina Wagner ◽

Regina Voglauer ◽

Hermann Katinger ◽

Maria Sibilia ◽

...

Keyword(s):

Matrix Protein ◽

Replicative Senescence ◽

Inner Cell Mass ◽

Umbilical Vein ◽

Cell Mass ◽

Preimplantation Embryos ◽

Proliferative Potential ◽

Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

Download Full-text

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab171 ◽

2008 ◽

Vol 20 (1) ◽

pp. 165

Author(s):

X. S. Cui ◽

X. Y. Li ◽

T. Kim ◽

N.-H. Kim

Keyword(s):

Gene Expression ◽

Trichostatin A ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Mouse Embryos ◽

Differentially Expressed ◽

Gene Expression Patterns ◽

Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

Download Full-text

105 THE Oct4/Cdx2 EXPRESSION AND CELL FATE OF INDIVIDUAL TWO-CELL BLASTOMERES IN TWO MOUSE STRAINS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab105 ◽

2008 ◽

Vol 20 (1) ◽

pp. 133

Author(s):

M. Katayama ◽

R. M. Roberts

Keyword(s):

Cell Mass ◽

Strain Differences ◽

Lineage Tracing ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Mouse Strains ◽

Mouse Development ◽

Cell Stage ◽

Developmental Potential ◽

Nih Swiss

Fertile adults and occasionally twins have been derived from murine blastomeres at the 2-cell stage, indicating that such blastomeres may be equivalently totipotent, but there are conflicting reports that individual blastomeres from 2-cell stage murine conceptuses make different contributions to the embryonic and abembryonic regions of the blastocyst, implying that they differ in developmental potential. Here, we have re-examined this subject using 2 mouse strains, CF1 and NIH Swiss (SW), and 2 experimental approaches, random blastomere destruction at the 2-cell stage by repeated insertion of a needle into its nucleus and lineage tracing with the dye, DiI-CM. The manipulated conceptuses and untreated controls were cultured in KSOM-AA to morula and blastocyst stages (84 or 108 h pc, respectively), fixed, and immunostained for Oct4 and Cdx2. Antigen distribution, number of nuclei (stained by 42,6-diamidino-2-phenylindole), and cell progeny labeled with DiI-CM were examined by confocal laser scanning microscopy. Cell numbers are means � SD and were analyzed by a Student t-test. Cells positive for Cdx2 were assumed to represent trophectoderm or trophectoderm precursors, ones positive for Oct4 but negative for Cdx2 (Oct+Cdx–) inner cell mass. Ablation of a blastomere failed to prevent developmental progression in either strain, but the total number of cells at both morula (SW 11.4 � 3.3 v. 19.2 � 7.1; CF1 10.1 � 2.5 v. 22.1 � 6.4) and blastocyst (SW 48.6 � 7.4 v. 69.4 � 9.9; CF1 24.8 � 6.2 v. 53.8 � 13.5) was significantly reduced. In SW, the average fraction of Oct+Cdx– cells after blastomere ablation was significantly lower (P < 0.05) than in controls in morulae (0.47 � 0.2 v. 0.65 � 0.1) but not in blastocysts (0.33 � 0.1 and 0.34 � 0.1). In CF1, the fraction of Oct+Cdx– cells was lower (P < 0.05) than controls in both morulae and blastocysts (0.31 � 0.2 v. 0.58 � 0.2 and 0.18 � 0.1 v. 0.27 � 0.04, respectively). The CF1 morulae fell mainly into 2 groups, one low fraction (≤0.3, 54%) of Oct+Cdx– cells and the other with a more normal fraction (0.3 to 0.8, 43%) relative to controls. A majority of NIH Swiss morulae had an Oct+Cdx– cell fraction >0.4 and in this respect resembled controls. We then examined these strain differences by lineage tracing. The majority of SW blastocysts (65%, n = 34) demonstrated a random localization of DiI-labeled cell progeny (i.e., there was no preferential distribution of labeled cells to either the embryonic or abembryonic poles). By contrast, in CF1 (n = 38), 32% of blastocysts had labeled cells confined to their embryonic end and 42% with DiI-labeled, Cdx2-positive cells clustered at the abembryonic locale. A random localization was observed in 26% of blastocysts. In conclusion, these data confirm that there is plasticity in early mouse development but also suggest that in CF1, but not in SW conceptuses, blastomeres at the 2-cell stage differ in their abilities to contribute to the embryonic pole. Similar strain differences may explain the disagreements among studies on lineage tracing in early cleavage stage conceptuses.

Download Full-text

258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

Reproduction Fertility and Development ◽

10.1071/srb08abs258 ◽

2008 ◽

Vol 20 (9) ◽

pp. 58

Author(s):

G. R. Kafer ◽

SA Lehnert ◽

P. L. Kaye ◽

R. J. Moser

Keyword(s):

Messenger Rna ◽

Expression Profiles ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Histone Variants ◽

Histone Variant ◽

Preimplantation Embryos ◽

Es Cell

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.

Download Full-text

What can we learn from gene expression profiling of mouse oocytes?

Reproduction ◽

10.1530/rep-07-0430 ◽

2008 ◽

Vol 135 (5) ◽

pp. 581-592 ◽

Cited By ~ 22

Author(s):

Toshio Hamatani ◽

Mitsutoshi Yamada ◽

Hidenori Akutsu ◽

Naoaki Kuji ◽

Yoshiyuki Mochimaru ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Oocyte Quality ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Gene Expressions ◽

Oocyte Competence

Mammalian ooplasm supports the preimplantation development and reprograms the introduced nucleus transferred from a somatic cell to confer pluripotency in a cloning experiment. However, the underlying molecular mechanisms of oocyte competence remain unknown. Recent advances in microarray technologies have allowed gene expression profiling of such tiny specimens as oocytes and preimplantation embryos, generating a flood of information about gene expressions. So, what can we learn from it? Here, we review the initiative global gene expression studies of mouse and/or human oocytes, focusing on the lists of maternal transcripts and their expression patterns during oogenesis and preimplantation development. Especially, the genes expressed exclusively in oocytes should contribute to the uniqueness of oocyte competence, driving mammalian development systems of oocytes and preimplantation embryos. Furthermore, we discuss future directions for oocyte gene expression profiling, including discovering biomarkers of oocyte quality and exploiting the microarray data for ‘making oocytes’.

Download Full-text

Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽

10.1530/rep.1.01021 ◽

2006 ◽

Vol 131 (5) ◽

pp. 895-904 ◽

Cited By ~ 60

Author(s):

Hakan Sagirkaya ◽

Muge Misirlioglu ◽

Abdullah Kaya ◽

Neal L First ◽

John J Parrish ◽

...

Keyword(s):

Gene Expression ◽

Dna Methyltransferase ◽

Expression Profiles ◽

Expression Patterns ◽

Blastocyst Stage ◽

Culture Conditions ◽

Preimplantation Embryos ◽

Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

Download Full-text

Molecular characterization of the prostaglandin E receptor subtypes 2a and 4b and their expression patterns during embryogenesis in zebrafish

10.3897/arphapreprints.e62570 ◽

2020 ◽

Author(s):

Wu Hong ◽

Han Yongjun ◽

Chang Hongbo

Keyword(s):

Amino Acid ◽

Phylogenetic Trees ◽

Expression Profiles ◽

Expression Patterns ◽

Cell Mass ◽

Prostaglandin E ◽

Receptor Subtypes ◽

Intermediate Cell ◽

Variable Expression ◽

High Amino Acid

The molecular expression profiles of zebrafish ep2a and ep4b have not been defined to-date. Phylogenetic trees of EP2a and EP4b in zebrafish and other species revealed that human EP4 and zebrafish EP4b were more closely related than EP2a. Zebrafish EP2a is a 281 amino acid protein with high identity to that of human (43%), mouse (44%), rat (43%), dog (44%), cattle (41%), and chicken (41%). Zebrafish EP4b encoded a precursor of 497 amino acids with high amino acid identity to that of mammals, including human (57%), mouse (54%), rat (55%), dog (55%), cattle (56%), and chicken (54%). Whole-mount in situ hybridization revealed that ep2a was robustly expressed in the anterior four somites at the 10-somites stages, but was absent in the somites at 19 hpf. It was observed again in the pronephric duct at 24 hpf, in the intermediate cell mass located in the trunk, and in the rostral blood island at 30 hpf. Ep2a was also expressed in the notochord at 48 hpf. During somitogenesis, ep4b was highly expressed in the eyes, somites, and the trunk neural crest. From 30 to 48 hpf, ep4b could be detected in the posterior cardinal vein and the neighboring ICM. From these data, we conclude that ep2a and ep4b are conserved in vertebrates and that the presence of ep2a and ep4b transcripts during developmental stages infers their role during early zebrafish larval development. In addition, the variable expression of the two receptor isoforms was strongly suggestive of divergent roles of molecular regulation.

Download Full-text

Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively

Journal of Cell Science ◽

10.1242/jcs.109.1.191 ◽

1996 ◽

Vol 109 (1) ◽

pp. 191-197

Author(s):

E. Dahl ◽

E. Winterhager ◽

B. Reuss ◽

O. Traub ◽

A. Butterweck ◽

...

Keyword(s):

Mouse Embryo ◽

Inner Cell Mass ◽

Cell Mass ◽

Confocal Laser ◽

Mouse Development ◽

Confocal Laser Scan Microscopy ◽

Early Mouse ◽

Junction Proteins ◽

In Cells ◽

Extraembryonic Tissues

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

Download Full-text