scholarly journals Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation development

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 777-785 ◽  
Author(s):  
Pablo J Ross ◽  
Neli P Ragina ◽  
Ramon M Rodriguez ◽  
Amy E Iager ◽  
Kannika Siripattarapravat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone H3 ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Polycomb Group ◽  
Mrna Levels ◽  
Stages Of Development ◽  
Cell Stage ◽  
Polycomb Group Genes

Trimethylation of histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes and in vitro-produced embryos were collected at different stages of development. Polycomb gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts for EZH2, EED, and SUZ12 were detected at all stages analyzed, with EZH2 levels being the highest of the three at early stages of development. By the time the embryo reached the blastocyst stage, the level of PcG gene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression of EZH2 at all stages while nuclear localized EED and SUZ12 were only evident at the morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was the highest in immature oocytes, which steadily decreased after fertilization to reach a nadir at the eight-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development.

51 CHARACTERIZATION OF HISTONE H3 LYSINE 27 TRI-METHYLATION AND POLYCOMB GROUP GENE EXPRESSION IN BOVINE FERTILIZED, PARTHENOGENETIC, AND CLONED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 106
Author(s):  
P. J. Ross ◽  
N. Ragina ◽  
R. M. Rodriguez ◽  
K. Siripattarapravat ◽  
N. Lopez-Corrales ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone H3 ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Polycomb Group ◽  
External Control ◽  
Santa Cruz ◽  
Cell Stage ◽  
Fluorescent Intensity ◽  
Polycomb Group Genes

The low developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos has been attributed to aberrant or incomplete epigenetic reprogramming. Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes (Ezh2, Eed, and Suz12) and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes during bovine preimplantation development and to compare the dynamics of H3K27me3 after IVF, parthenogenetic activation (PG), and SCNT. Embryos were produced using our standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). MII oocytes and pronuclear-, 2-cell-, 4-cell-, 8-cell-, morula-, and blastocyst-stage embryos were collected for quantitative RT-PCR or immunofluorescence (IF). IF analysis of embryos was performed using primary antibodies against H3K27me3, Eed, Ezh2 (Abcam, Cambridge, MA, USA), and Suz12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and imaged by confocal microscopy. The average background-corrected nuclear fluorescence intensity for each embryo was analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Ezh2, Eed, Suz12, and control transcript levels from oocytes and IVF-derived embryos were determined by absolute quantification as described by Bettegowda et al. (2006 Mol. Reprod. Dev. 73, 267–278). RNA levels were normalized to the external control and analyzed by ANOVA (mixed model). A steady decrease in total transcripts was observed from the MII to the 8-cell stage (P < 0.05). During this period, the levels of polyadenylated Suz12 and Eed transcripts remained steady, while Ezh2 transcripts increased at the 2-cell stage (P < 0.05) and then decreased from the 2- to 4-cell stage (P = 0.10). After embryonic genome activation, all three genes displayed an increase in transcript abundance by the blastocyst stage (P < 0.05). Concurrent IF analysis revealed that Ezh2 localized to the nucleus throughout preimplantation development, Eed localized to the cytoplasm at the 2-, 4- and 8-cell stages (expression confirmed by westernblot) and to the nucleus at morula and blastocyst stages, and Suz12 was exclusively nuclear and detected only at morula and blastocyst stages. IF analysis of H3K27me3 revealed that, for all embryo types, nuclear fluorescent intensity was highest at the MII stage, steadily decreased to reach a nadir at the 8-cell stage (P < 0.05), and then significantly increased at the blastocyst stage (P < 0.05). H3K27me3 fluorescent intensity was significantly higher in SCNT blastocysts than in IVF and PG blastocysts (P < 0.05). We conclude that H3K27me3 is efficiently removed from the embryonic and somatic cell chromatin, but is abnormally reestablished in SCNT embryos.

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 168
Author(s):  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Gene Expression ◽  
Similar Pattern ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Developmental Expression ◽  
Fertilization In Vitro ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Parthenogenetic Embryos

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

51 AGGREGATION REDUCES THE VARIATION OF GENE EXPRESSION LEVELS IN BOVINE CLONES

2006 ◽  
Vol 18 (2) ◽  
pp. 134
Author(s):  
S. Kurosaka ◽  
N. A. Leu ◽  
K. J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Coefficient Of Variation ◽  
Blastocyst Stage ◽  
Average Level ◽  
Cdna Libraries ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Expression Levels ◽  
Gene Expression Levels

Mammalian somatic cell clones frequently exhibit abnormal gene expression that presumably results from errors in reprogramming of the transplanted genome. In the mouse, aggregation of 4-cell stage clones with each other improves reprogramming with respect to Oct-4 expression in blastocysts and an increase in term development (Boiani et al. 2003 EMBO J. 22, 5304-5312). To determine if clone-clone aggregation has a similar beneficial effect in the bovine, we aggregated 8-16 cell bovine clones with each other and profiled gene expression levels in bovine clones and clone-clone aggregates at the blastocyst stage. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. For aggregation of embryos, we first removed the zonae pepellucidae by treatment with 0.5% pronase at the 8-16 cell stage and then placed two zona-free embryos per well into deep microwells produced on the bottom of a culture dish by pressing a heated darning needle onto the surface. Seven to 10 microwells in close proximity were covered by a culture 50-�L drop of culture medium, and embryos were cultured until Day 7. Real-time RT-PCR analysis for Oct-4, DNA methyltransferase 1 (Dnmt1), Dnmt3, glucose transporter 1 (Glut1), Glut3, and Poly(A) polymerase (PolyA) was performed on reusable Dynabead Oligo (dT)25-cDNA libraries synthesized from individual blastocysts at Day 7. In vitro-fertilized embryos were used as controls. To compare the variation of gene expression in each embryo within the group, the coefficient of variation (COV; standard deviation/mean) was calculated. Although spatial distribution of Oct-4 transcript is normal in bovine blastocyst stage clones (Kurosaka et al. 2004 Reprod. Fertil. Dev. 16, 147), we detected disturbances in the level of Oct-4 expression in clones: 44.4% (8 of 18) of clones expressed Oct-4 within a range of 0.5- and 1.5-fold of the average level of expression in IVF embryos, compared to 81.8% (9 of 11) of IVF embryos. Only 22.2% (4 of 18) of clones expressed all genes examined within a range of 0.5- and 2.0-fold of the average level of IVF embryos, versus 45.5% (5 of 11) of IVF embryos. Clone-clone aggregation did not increase the proportion of clones with normal expression levels but did reduce the coefficient of variation of gene expression levels between individual clones for the genes Oct-4, Dnmt1, Dnmt3a and PolyA, but not for Glut1 and Glut3. Interestingly, bovine clone-clone aggregates (n = 25) had less variation between individual embryos compared to IVF aggregates (n = 11) for all genes except Glut1 and Glut3, although variation of single clones was larger than that of single IVF embryos. Analysis of Oct-4 and �-Actin transcripts in mouse clone blastocysts indicated a similar decrease in gene expression variation subsequent to aggregation of mouse clones. These results demonstrate that bovine pre-implantation stage clones exhibit a high degree of variation in gene expression levels and suggest that aggregation of clones is beneficial in reducing the variation in expression of some genes.

194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 176
Author(s):  
D. X. Zhang ◽  
X. H. Shen ◽  
X. S. Cui ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Expression Levels ◽  
Apoptotic Gene

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan� MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 � 10(8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 � 4.5% v. buffer 94.6 � 5.8%); however, it did induce a significant degree of apoptosis (P < 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 � 1223 v. 3098 � 592; miR-16, 196216 � 958 v. 133514 � 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 � 0.12), the pro-apoptotic gene Bax (1.418 � 0.12), and Caspase 3 (1.314 � 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H2O2 induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P < 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.

299. Sensitivity of embryos to an environmental stressor, ammonium, is dependent on stage of temporal exposure

2005 ◽  
Vol 17 (9) ◽  
pp. 127
Author(s):  
D. L. Zander ◽  
J. G. Thompson ◽  
M. Lane
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Early Stage ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Stages Of Development ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Embryo

Extended embryo culture in vitro may cause increased cellular perturbations resulting in poorer developmental outcomes. Exposure of embryos to ammonium throughout the entire pre-implantation period decreased cell number and ICM development, increased apoptosis and perturbs glucose metabolism. The aim of this study was to examine the relative susceptibility of the pre- and post-compaction stage embryo to these perturbations resulting from temporal exposure to ammonium. Mouse embryos (n = 350 per treatment) were collected from F1 female mice. Embryos were exposed to either control medium or medium with 300 μM ammonium for the entire culture period. Temporal treatments involved culture with or without ammonium, from the zygote to 2-cell stage, 2-cell to 8-cell stage, or the 8-cell to the blastocyst stage. At the blastocyst stage, ICM development, apoptosis, gene expression and glucose metabolism were assessed. Differences between treatments were determined using generalised linear modelling and LSD post-hoc tests. Exposure to ammonium at any stage did not affect blastocyst development. Exposure to ammonium pre-compaction significantly decreased both blastocyst and ICM cell number while these were unaffected when exposure occurred post-compaction. Levels of apoptosis were significantly increased when exposure to ammonium was continual to the blastocyst stage (6.5% compared to control 2.4%, P < 0.05) or from the zygote to the 2-cell stage (5.8%, P < 0.05). However, apoptosis was not altered during post-compaction exposure (2.8%). Glucose uptake was decreased by culture with ammonium at all stages of development (P < 0.001). Gene expression of GLUT1 in the blastocyst was not altered by ammonium while GLUT3 expression was significantly reduced by exposure at all stages of development (P < 0.01). The data presented suggests that the pre-compaction stage embryo is most susceptible to ammonium stress and the effects of this early stage exposure appear irreversible. Intriguingly, glucose uptake and GLUT3 expression at the blastocyst stage appear to be markers of ammonium exposure.

scholarly journals Fetal bovine serum influences apoptosis and apoptosis-related gene expression in porcine parthenotes developing in vitro

Reproduction ◽  
2004 ◽  
Vol 127 (1) ◽  
pp. 125-130 ◽  
Author(s):  
Xiang-Shun Cui ◽  
Yu-Jeong Jeong ◽  
Hwa-Young Lee ◽  
Sun-Hong Cheon ◽  
Nam-Hyung Kim
Keyword(s):  
Gene Expression ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Blastocyst Stage ◽  
Related Gene ◽  
Embryo Viability ◽  
Cell Stage ◽  
Fetal Bovine ◽  
Apoptosis Related Gene

This study was conducted to determine the effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, total cell number, apoptosis and Bcl-xL and Bak gene expression in porcine presumptive diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell or late 4-cell stage parthenotes to the blastocyst stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced the frequency of blastocysts developed from both 2-cell (P < 0.001) and late 4-cell (P < 0.05) embryos and increased the percentage of blastocysts undergoing apoptosis (P < 0.001). The relative abundance of Bcl-xL mRNA in presumptive diploid parthenotes in the control, PVA- and BSA-supplemented medium was similar to that of in vivo-derived embryos, but was significantly higher than in parthenotes cultured with FBS supplement (P < 0.05). Bak mRNA significantly increased at the blastocyst stage in FBS-supplemented cells (P < 0.01). These results suggest that apoptosis-related gene expression is significantly affected by FBS, and that this may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

scholarly journals Embryonic genotype and inbreeding affect preimplantation development in cattle

Reproduction ◽  
2011 ◽  
Vol 141 (5) ◽  
pp. 625-632 ◽  
Author(s):  
G Lazzari ◽  
S Colleoni ◽  
R Duchi ◽  
A Galli ◽  
F D Houghton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Brown Swiss ◽  
Molecular Features ◽  
Cattle Herds

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7–8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

scholarly journals 25 MATERNAL-TO-EMBRYONIC TRANSITION FOLLOWING NUCLEAR TRANSFER OR PARTHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 121
Author(s):  
T. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
I. Lagutina ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
De Novo ◽  
Gene Expression Pattern ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Parthenogenetic Activation

The successful development of embryos generated by somatic cell nuclear transfer (SCNT) requires the ooplasm to reprogram the nucleus. This establishes the gene expression pattern necessary for full development by mechanisms that are currently being clarified. The ooplasm action on somatic nuclei shows many common aspects to the process that leads to the creation of a functional embryonic genome from the differentiated sperm and egg genomes. In order to investigate this aspect we studied a critical phase of early embryonic development: the maternal to embryonic transition (MET). We compared the pattern and level of gene expression between bovine embryos derived from in vitro fertilization (IVF), from nuclear transfer of adult fibroblasts (NT), or from parthenogenetic activation (PG). The study was performed in cattle because MET, in this species, occurs over four cell cycles, making it easier to detect even small deviations. Oocytes, matured for 22 h and fertilized in vitro or after cumulus removal, were enucleated and fused to fibroblast cells. Nuclear transfer and Met II oocytes were activated at 24-26 h of maturation with ionomycin (5 �M) for 5 min and 6DMAP (2 mM) for 4 h and then cultured in mSOFaa. Embryos were harvested at the required time for analysis at the 2-, 4-, 8-, and 16-cell; morula; and blastocyst stages and stored snap-frozen in a minimal volume of medium in groups of 5-10 embryos. Semiquantitative RT-PCR was used to study the expression of Nanog, Oct-4, Zar-1, and Par-3, because these genes are directly involved in early embryo development and have a specific expression pattern during MET. Data were analyzed with one-way ANOVA followed by Student-Newman-Keuls All Pairwise Multiple Comparison. No difference in pre-implantation development was observed among the three groups. The Nanog expression pattern was unchanged in all three groups, becoming detectable from the 8-16-cell stage onward. Oct-4 mRNA was detected at all stages in every group, but only in NT embryos did a significant increase occur at the 16-cell stage, suggesting the onset of an anticipated embryonic transcription. the Zar-1 expression pattern, with the characteristic de-novo transcription peak at the 4-cell stage, was observed in both IVF and NT embryos but not in PG embryos. In this group, Zar-1 mRNA levels were significantly higher at the 2- and 4-cell stage than in all of the following stages. The Par-3 gene showed the biggest differences among groups: IVF embryos expressed this gene from the 8-cell stage onward, whereas NT embryos showed high levels of Par-3 mRNA already at the 2-cell stage. Surprisingly, PG embryos showed no detectable Par-3 levels at any stages. The results indicate that, although in vitro development was not affected, gene-specific expression differences during MET occurred among groups. Relating the specific functions exerted by each of these genes in early development to the changes observed following the different manipulations provides useful data toward a better understanding of the role of these genes and of the mechanisms of nuclear reprogramming. This work was supported by FIRB RBNE01HPMX, FIRST 2004, and ESF-EuroStells.

198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Before And After ◽  
Vitro Development ◽  
Bovine Oocytes

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.

Differential gene expression and developmental competence in in vitro produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 281-290 ◽  
Author(s):  
Paula Ripamonte ◽  
Lígia Garcia Mesquita ◽  
Sylvia Sanches Cortezzi ◽  
Júlio César de Carvalho Balieiro ◽  
Giovana Krempel Fonseca Merighe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Similar Proportion ◽  
Cell Stage ◽  
Cellular Survival ◽  
Activation Mechanisms ◽  
Gene Expression Studies

SummaryThe embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (S4) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

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