scholarly journals 255EXPRESSION OF TRANSCRIPTION FACTORS PRIOR TO THE MATERNAL-TO-ZYGOTIC TRANSITION IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 247
Author(s):  
C. Vigneault ◽  
S. McGraw ◽  
G. Bujold ◽  
M.-A. Sirard
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Transcript Levels ◽  
Significant Difference ◽  
Maternal To Zygotic Transition ◽  
La Jolla ◽  
Triton X

During the first stages of bovine embryonic development, until the 8- to 16-cell stage, the zygote is maintained by the mRNA and proteins stored in the oocyte. New embryonic transcription is reported to begin only at the 8- to 16-cell stage even if some minor transcription is detected from the 2-cell stage. In order for this to occur, several factors are required to remodel the chromatin and activate the transcription machinery. Some regulating transcription factors are possibly present in the oocyte in their mRNA form, and their translation could enhance the maternal-to-zygotic transition (MZT). In our study, we observed the expression patterns of five transcription factors (ATF2, HMGN2, HMGB2, HUEL and MSY2) in bovine in vitro-produced embryos. Embryos were produced in vitro using selected cumulus-oocyte complexes from 3-5-mm follicles of slaughterhouse ovaries. Pooled GV or MII oocytes, and 2-, 4-, 8-cell and blastocyst-stage embryos (n=40/stage) were washed in PBS and frozen at −80°C. Each pool was spiked with 1 pg of GFP RNA containing a poly(A) tail. The RNA was extracted using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA), co-precipitated with linear acrylamide (Ambion, Austin, TX, USA) and reverse-transcribed with Omniscript (Quiagen). The quantitative amplification of the transcription factors was performed in triplicate using the equivalent of 1 oocyte or embryo per reaction on a Lightcycler (Roche, Indianapolis, IN, USA). Data were normalized with the GFP levels found in each pool and a Least-Significant-Difference method was used for statistical analysis. Immunocytochemistry studies were performed on oocytes and embryos fixed and permeabilized in a solution of paraformaldehyde and Triton X-100, and results were observed on a confocal microscope. Our results show that the transcripts of the transcription factors studied are found at higher levels in pre-MZT embryos and at lower levels in subsequent stages. For HMGN2 and MSY2, there is a decrease in mRNA during oocyte maturation. For both genes, the residual mRNA remains constant up to the 4-cell stage before another loss in transcript levels in the 8-cell stage. In the case of ATF2, HMGB2 and HUEL, the maternal transcript levels are maintained until the 4-cell stage, suggesting that the mRNA is protected from degradation until its possible translation at the MZT. These results, combined to immunolocalization of the proteins, suggest a possible implication of some of these factors in the bovine MZT.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 31-34 ◽  
Author(s):  
Roman Rybar ◽  
Jindra Horakova ◽  
Marie Machatkova ◽  
Katerina Hanzalova ◽  
Jiri Rubes
Keyword(s):  
De Novo ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Tween 20 ◽  
Dna Integrity ◽  
Cell Stage ◽  
Significant Difference ◽  
Painting Probes

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.

87 EVALUATION OF MITOCHONDRIAL FUNCTION IN SINGLE CLONED MINIATURE PIG EMBRYOS BY MEASURING OXYGEN CONSUMPTION

2007 ◽  
Vol 19 (1) ◽  
pp. 161
Author(s):  
S. Sugimura ◽  
M. Yokoo ◽  
K.-I. Yamanaka ◽  
T. Wakai ◽  
H. Abe ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Blastocyst Stage ◽  
Miniature Pig ◽  
Cell Stage ◽  
Significant Difference ◽  
Lower Oxygen ◽  
Energy Synthesis ◽  
Mitochondrial Uncoupler

Mitochondria are organelles that produce energy for embryogensis. Their function [oxidative phosphorylation (OXPHOS) and electron transport] is regulated by intercommunication with the nucleus. In somatic cell nuclear transfer (SCNT) embryos, incomplete reprogramming may lead to dysfunction of the intercommunication before or after embryonic activation, or both, although it is unknown whether reprogramming for energy synthesis is required. In the previous report (Abe et al. 2004 J. Mamm. Ova Rec. 21, 22), we developed a noninvasive method using a scanning electrochemical microscopy (SECM) for measurement of oxygen consumption that provides more direct information about mitochondrial function (Trimarch et al. 2000 Biol. Reprod. 62, 1866–1874). In the present study to evaluate mitochondrial function in individual miniature pig SCNT embryos, we measured oxygen consumption by SECM. Oocytes in pig ovaries collected from the local slaughterhouse were matured for 44 h in NCSU23 and used as recipient. After SCNT with fetal miniature pig fibroblasts, reconstructed embryos were cultured in vitro in NCSU23 or PZM-3. Oxygen consumption in single 2- and 4-cell-stage embryos, morulae, and blastocysts were measured, and the values were compared with those derived from IVF. All data were analyzed by ANOVA. In IVF embryos, oxygen consumption was lowest at the 2- and 4-cell stages, and reached a peak at the blastocyst stage on Day 5. However, there were significant differences (P &lt; 0.05) in blastocysts between NCSU23 and PZM-3: 0.61 � 0.14 vs. 0.83 � 0.18 at Day 5, 0.53 � 0.14 vs. 0.70 � 0.24 at Day 6, 0.47 � 0.11 vs. 0.73 � 0.20 � 10-14 mol s-1 at Day 7, respectively. In contrast, SCNT embryos showed no increase in oxygen consumption during pre-implantation stages in the 2 media, but there was a significant difference (P &lt; 0.05) at the 2-cell stage between NCSU23 and PZM-3 (0.35 � 0.09 vs. 0.43 � 0.10, respectively). Comparison of the Day 5 IVF and SCNT blastocysts cultured in PZM-3 showed no difference in total cell numbers but significantly (P &lt; 0.05) lower oxygen consumption in SCNT (0.83 � 0.18 vs. 0.40 � 0.13 � 10-14 mol s-1, respectively). After treatment with 1 �M CCCP (mitochondrial uncoupler) or 1 mM NaCN (mitochondrial electron transporter inhibitor), oxygen consumption in IVF and SCNT blastocysts at Day 5 increased (112 � 18 and 51 � 44%, respectively) or decreased (50 � 20 and 21 � 32%, respectively) compared with those of nontreated embryos. Sensitivity to these reagents differed significantly (P &lt; 0.05) between IVF and SCNT, indicating that the SCNT blastocysts had a lower OXPHOS capacity than those from IVF. These results suggest that reprogramming for sustaining mitochondrial function during pre-implantation development may be required in miniature pig SCNT embryos.

Postnatal weight of calves derived from in vitro matured and in vitro fertilized embryos developed under various oxygen concentrations

2000 ◽  
Vol 12 (8) ◽  
pp. 391 ◽  
Author(s):  
H. Iwata ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Oxygen Tension ◽  
Blastocyst Stage ◽  
High Oxygen ◽  
Gestation Length ◽  
Low Oxygen ◽  
Cell Stage ◽  
Fluid Medium ◽  
Significant Difference ◽  
Oxygen Conditions

In the present study, weights of calves (14 days after birth) derived from embryos of a homogeneous line (Tajima line) of Japanese Black Cow, cultured in vitro under various oxygen conditions was examined. In vitro matured and fertilized oocytes were incubated for 48 h in modified synthetic oviduct fluid medium under 5% CO 2in air and embryos reaching at least the 5-cell stage were selected for further culture under various gas conditions (high oxygen tension: 5% CO 2 in air; low oxygen tension: 5% O 2 , 5% CO 2 , 90% N 2 ) for 5 days. Embryos that developed to the blastocyst stage were transferred to Holstein cows or cryopreserved until transfer. When embryos were cultured under high oxygen tension and cryopreserved, the weights of male calves (at 14 days) were significantly heavier than in the other groups. However, there was no significant difference in gestation lengths of male calves. In female calves, no difference was observed in either the weight or gestation length of calves irrespective of oxygen tension during the culture period or embryo conditions (fresh or frozen). From the results of the present study, it is suggested that the oxygen concentration during culture and cryopreservation synergistically induced the production of overweight male calves without influencing gestation length.

scholarly journals Spatiotemporal expression of transcriptional regulators in concert with the maternal-to-embryonic transition during bovine in vitro embryogenesis

Reproduction ◽  
2009 ◽  
Vol 137 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Christian Vigneault ◽  
Serge McGraw ◽  
Marc-Andre Sirard
Keyword(s):  
Protein Expression ◽  
Embryo Development ◽  
Binding Protein ◽  
Bovine Genome ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Cell Stage ◽  
Protein Levels

Cleavage-stage bovine embryos are transcriptionally quiescent until they reach the 8- to 16-cell stage, and thus rely on the reserves provided by the stored maternal mRNAs and proteins found in the oocytes to achieve their first cell divisions. The objective of this study was to characterize the expression and localization of the transcriptional and translational regulators, Y box binding protein 2 (YBX2), TATA box-binding protein (TBP), and activating transcription factor 2 (ATF2), during bovine early embryo development. Germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, as well as 2-, 4-, 8-, 16-cell-stage embryos, morula, and blastocysts, producedin vitrowere analyzed for temporal and spatial protein expression. Using Q-PCR,ATF2mRNA expression was shown to remain constant from the GV-stage oocyte to the four-cell embryo, and then decreased through to the blastocyst stage. By contrast, the protein levels of ATF2 remained constant throughout embryo development and were found in both the cytoplasm and the nucleus. Both TBP and YBX2 showed opposite protein expression patterns, as YBX2 protein levels decreased throughout development, while TBP levels increased through to the blastocyst stage. Immunolocalization studies revealed that TBP protein was localized in the nucleus of 8- to 16-cell-stage embryos, whereas the translational regulator YBX2 was exclusively cytoplasmic and disappeared from the 16-cell stage onward. This study shows that YBX2, TBP, and ATF2 are differentially regulated through embryo development, and provides insight into the molecular events occurring during the activation of the bovine genome during embryo developmentin vitro.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

scholarly journals 244REAL TIME PCR EVALUATION OF EXPRESSION PROFILES OF SOME MATERNAL-SOURCED GENE TRANSCRIPTS IN IN VITRO PRODUCED PRE IMPLANTATION STAGE BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
S. Mamo ◽  
S. Ponsuksili ◽  
K. Wimmers ◽  
M. Gilles ◽  
K. Schellander
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Stage ◽  
Gene Transcripts

Gene expression profiling data collected in a time series and quality related parameters are important for understanding the developmental mechanisms carried out in a developing embryo, and are also a source to enrich the knowledge base of embryo development. However, such data are frequently constrained by limitation and handling of the sample as well as cost associated with generating such data. Cumulatively, these factors have contributed to the existing insufficient data compared to the large need stemming from a drive to control and guide optimum embryo development. In this ongoing study, with objectives to quantify and evaluate gene transcripts identified from certain developmental stages, expression profiles of two ESTs (C256 and C112), derived from an oocyte cDNA library, were analyzed from the above perspectives to understand the change in the level of these gene transcripts throughout the pre-implantation stage of embryo development. For this analysis, pools of oocytes and embryos were prepared by balancing the amount proportional to the number of cells present. mRNA was isolated separately from each pool of matured oocytes, 2-cell, 4-cell, 8-cell, and 16-cell stages, as well as morula and blastocyst stages by using Dynal beads Oligo (dt)25 (Dynabeads, Dynal Biotech, Oslo, Norway) following the manufacturer’s recommendations. These mRNAs were checked for DNA contamination and, when proved free, first-strand cDNA was synthesised by reverse transcribtion at 42°C for 2h following standard laboratory procedures. Transcript quantification was performed by real-time PCR using gene-specific primers, equal amounts of cDNA from each sample and SYBR Green universal master mix. Following this analysis, both transcripts were found to be expressed in a wave-like manner being highly expressed in mature oocytes, declining gradually as the development stage advanced, with the lowest level at the 16-cell stage, and then reviving in level thereafter until it reached blastocyst stage. Taking the 16-cell stage as calibrator for both, C256 was 26.4, 23.2, 8.5, 1.7. 2.4 and 2.7 times more expressed in oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively. Similarly, C112 was 110.7, 169.2, 9.8, 2.5, 4.1 and 7.3 times more expressed in oocyte, 2-cell, 8-cell, morula and blastocyst stages, respectively. These expression patterns suggest the probable origin of these transcripts initially to be maternal. C256 is strongly similar to human retinoid X receptor beta (RXRb) gene (NM_021976.3), which is involved in transcriptional functions and in increasing DNA binding, whereas C112 is strongly similar to TATA box-binding protein-associated factor gene (AY189986.1), which is also involved in transcriptional functions. As seen from their functions, these transcripts can be vital for developing the embryo and the variations at different developmental stages shows their most probable role as part of genes contributing to developmental competence in pre-implantation development stages.

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

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