104 EMBRYO MANIPULATION TECHNIQUES ALTER PRE-IMPLANTATION DEVELOPMENT AND GENE EXPRESSION IN MOUSE EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 182 ◽  
Author(s):  
D. Jones ◽  
M. Paczkowski ◽  
T. Kuehl
Keyword(s):  
Gene Expression ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Human Embryos ◽  
Assisted Hatching ◽  
Long Term Effects ◽  
Slow Cooling ◽  
Developmental Potential ◽  
Cell Counts

Micromanipulation techniques are commonly employed with human embryos; however, the long-term effects on growth and development of embryos are unknown. Subjective techniques, such as embryo grading, may not be sensitive enough to show differences in developmental potential and more objective assessments are needed. The objective of this experiment was to evaluate whether manipulation techniques and cryopreservation alter morphologic and gene expression endpoints of embryos. Two-cell mouse embryos were cultured to blastocyst stage and divided into 6 treatment groups: nonmanipulated control (UNMAN; n = 252), laser-assisted hatching (LAH; n = 124), LAH with slow cooling cryopreservation (LAHcryo; n = 78), simulated biopsy (BIOP; n = 90) performed by laser disruption of 1/8 of the embryo’s cellular volume, BIOP with slow cooling cryopreservation (BIOPcryo; n = 91), and BIOP with vitrification (BIOPvit; n = 66). After manipulation or thaw, embryos were cultured 28 h to blastocyst stage. Fully hatched and hatching blastocysts were collected for cell counts and quantitative PCR to assay absolute transcript abundance for Plac8, Glut1, Oct4, and Cox2, 18s rRNA, and Ppia using standard curves generated from serial dilutions of digested plasmids. Data were analysed using ANOVA with Duncan’s post hoc test and significance was defined as P < 0.05. Manipulation groups differed in developmental stage at 28 h postmanipulation (P = 0.03), with a larger percentage of embryos completely hatched in LAH (46%) and BIOP (45%) groups compared to UNMAN (11%). Cryopreservation reduced percentages of completely hatched embryos (LAHcryo = 26%; BIOPcryo = 31%; BIOPvit = 19%). The number of cells per embryo also varied (P < 0.001); UNMAN and LAH groups had similar numbers of blastomeres (UNMAN = 71; LAH = 74), whereas BIOP groups had reduced cell counts (BIOP = 53; BIOPcryo = 56; BIOPvit = 48). Patterns of gene expression varied between groups; the BIOP group had more cDNA per cell (UNMAN = 125 ng cell–1; BIOP = 202 ng cell–1; P = 0.004), whereas BIOPcryo group did not show this effect (BIOPcryo = 111 ng cell–1). Transcript abundance of Cox2, Oct4, and Glut1 per embryo was significantly decreased (P < 0.05) in biopsied embryos that were cryopreserved, either by slow-cooling or vitrification (Cox2: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268; Oct4: UNMAN = 266, BIOP = 181, BIOPcryo = 95, BIOPvit = 97; Glut1: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268). The data suggests that micromanipulation alters developmental timelines and gene expression of embryos. While manipulations differ in degree and nature of effect, our investigation implies all manipulations have some effect on the transcriptome of the developing embryo. Differences in cDNA quantity in the BIOP group may indicate a stress response or recovery attempt that becomes blunted in biopsied embryos which are subsequently cryopreserved. Thus, our study suggests that cryopreservation of biopsied embryos may come at a price and offers scientific support for only judiciously selected and medically indicated embryo biopsies.

scholarly journals Predictive value of bovine follicular components as markers of oocyte developmental potential

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
Author(s):  
Satoko Matoba ◽  
Katrin Bender ◽  
Alan G. Fahey ◽  
Solomon Mamo ◽  
Lorraine Brennan ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Principal Component ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Thecal Cells ◽  
Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 299
Author(s):  
S. Matoba ◽  
S. Mamo ◽  
E. Gallagher ◽  
A. G. Fahey ◽  
T. Fair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Target Genes ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Cell Gene Expression ◽  
The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
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.

scholarly journals 314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 276
Author(s):  
J.R. Herrick ◽  
E. Behboodi ◽  
E. Memili ◽  
S. Blash ◽  
Y Echelard ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Similar Proportion ◽  
Developmental Potential ◽  
Cell Counts

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P&lt;0.05 were considered significant. The use of BSA did not improve (P&gt;0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P&gt;0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions. Table 1 Development of goat oocytes following IVM with different macromolecules.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

scholarly journals Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential

2017 ◽  
Vol 29 (5) ◽  
pp. 876 ◽  
Author(s):  
Denise Laskowski ◽  
Ylva Sjunnesson ◽  
Patrice Humblot ◽  
Marc-André Sirard ◽  
Göran Andersson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Quantitative Polymerase Chain Reaction ◽  
Blastocyst Stage ◽  
Specific Gene ◽  
Bovine Oocyte ◽  
Developmental Potential ◽  
Metabolic Imbalance ◽  
Negative Effect

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL–1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

scholarly journals Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Luis Aguila ◽  
Joao Suzuki ◽  
Amanda B. T. Hill ◽  
Mónica García ◽  
Karine de Mattos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genomic Imprinting ◽  
X Chromosome ◽  
De Novo ◽  
Chromosomal Abnormalities ◽  
Methylation Status ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Androgenetic Embryos

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

scholarly journals The Association of Kinetic Variables with Blastocyst Development and Ploidy Status

2021 ◽  
Author(s):  
Francesca Pennetta ◽  
Cristina Lagalla ◽  
Raffaella Sciajno ◽  
Nicoletta Tarozzi ◽  
Marco Nadalini ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Human Embryos ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Logistic Regression Models ◽  
Kolmogorov Smirnov ◽  
Ploidy Changes ◽  
Study Hypothesis ◽  
Ploidy Status ◽  
Smirnov Test

Background: Despite a plethora of studies conducted so far, a debate is still unresolved as to whether TLM can identify predictive kinetic biomarkers or algorithms universally applicable. Therefore, this study aimed to elucidate if there is a relationship between kinetic variables and ploidy status of human embryos or blastocyst developmental potential. Methods: For conducting this retrospective cohort study, the normal distribution of data was verified using Kolmogorov-Smirnov test with the Lilliefors’ amendment and the Shapiro-Wilk test. Kinetic variables were expressed as median and quartiles (Q1, Q2, Q3, Q4). Mann-Whitney U-test was used to compare the median values of parameters. Univariate and multiple logistic regression models were used to assess relationship between blastocyst developmental potential or ploidy status and kinetics. Several confounding factors were also assessed. Results: Blastocyst developmental potential was positively correlated with the t4-t3 interval (s2) (OR=1.417, 95% CI of 1.288-1.560). s2 median value was significantly different between high- and low-quality blastocysts (0.50 and 1.33 hours post-insemination, hpi, respectively; p=0.003). In addition, timing of pronuclear appearance (tPNa) (OR=1.287; 95% CI of 1.131-1.463) had a significant relationship with ploidy changes. The median value of tPNa was statistically different (p=0.03) between euploid and aneuploid blastocysts (Euploid blastocysts=8.9 hpi; aneuploid blastocysts=10.3 hpi).  Conclusion: The present findings are in line with the study hypothesis that kinetic analysis may reveal associations between cleavage patterns and embryo development to the blastocyst stage and ploidy status.

scholarly journals Developmental potential of 2n/3n mixoploid mouse embryos produced by fusion of individual second polar bodies and blastomeres of 2-cell embryos

2016 ◽  
Vol 28 (12) ◽  
pp. 1982
Author(s):  
Toshiaki Hino ◽  
Hiroyuki Tateno
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Human Embryos ◽  
Production Mechanism ◽  
Early Cleavage ◽  
Developmental Potential ◽  
Polar Bodies ◽  
Green Fluorescent

Using 2n/3n mixoploid mouse embryos produced by fusion of individual second polar bodies (PB2s) with individual blastomeres of 2-cell embryos, the dynamics of PB2 nuclei in the host blastomeres during mitosis were examined and the fate of the 3n cell line in the mixoploid embryos was followed. Most of the PB2 nuclei were synchronised with the cell cycle of the host blastomeres and all chromosomes were incorporated into a single mitotic spindle. The majority of the mixoploid embryos developed to blastocysts with 3n cells. In conceptuses at Day 11.5 and Day 18.5 of gestation, 3n cells were recognised in both of the embryonic/fetal and placental tissues. When green fluorescent protein (GFP)-transgenic mice were used as a donor of PB2, GFP-positive 3n cells were found in more than 40% of morulae and blastocysts, indicating that the PB2 genome can be reactivated during the pre-implantation stage. GFP-positive 3n cells were non-randomly allocated in trophectoderm in blastocysts. These findings may explain the production mechanism of 2n/3n mixoploid human embryos, that is, a PB2 is incorporated into one daughter blastomere during the early cleavage period.

265 DEVELOPMENT AND DYNAMICS OF GENE EXPRESSION OF BOVINE EMBRYOS CULTURED IN VITRO IN THREE DIFFERENT MEDIA

2006 ◽  
Vol 18 (2) ◽  
pp. 240
Author(s):  
H. Sagirkaya ◽  
M. Misirlioglu ◽  
A. Kaya ◽  
H. Odaman ◽  
N. First ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Apoptotic Cells ◽  
Glucose Transporter 1 ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Glut 1

Dramatic reprogramming of gene expression occurs during embryonic genome activation (EGA), an essential event initiating as early as the 1-cell zygotic stage in the bovine and increasing gradually as embryonic development advances. It is this reprogramming of gene expression that sets the stage for later development. Expression of embryonic genes is altered in different culture conditions and this may influence developmental potential both during pre-implantation and during fetal development. The objective of this study was to define some most commonly used embryo culture media (KSOMaa, CR1aa, and SOFaa) based on their ability to support embryonic development to the blastocyst stage, mean cell number, percentages of apoptotic cells, and the expression patterns of a panel of developmentally important genes. Oocytes with several layers of cumulus cells obtained from an abattoir were matured in TCM 199 (supplemented with 0.25 mM pyruvate, 0.5 μg/mL FSH, 5 μg/mL LH, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FCS) for 24 h and in vitro-fertilized (Day 0) using frozen bull semen. Presumptive zygotes were transferred into three different media (KSOMaa, CR1aa, and SOFaa) 16–18 h post-insemination, supplemented with 10% FCS on Day 4, and cultured until Day 8 at which time they were fixed or frozen for further analysis. Mean cell numbers and percentages of apoptotic cells in blastocysts were determined using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Real-time quantitative PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70), interferon-tau (IF-tau), insulin-like growth factor II receptor (Igf-2r), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). Gene expression data were analyzed relative to transcripts of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh). In three separate trials, a total of 538, 518, and 503 oocytes were used for KSOMaa, CR1aa, and SOFaa groups, respectively. Cleavage rates were 79.2%, 77.5%, and 80.2%; and rates of development to the blastocyst stage were 22.2%, 23.4%, and 32.9% for KSOMaa, CR1aa, and SOFaa groups, respectively. The blastocyst rate of the SOFaa group was significantly higher than those of the KSOMaa and CR1aa groups (P < 0.05). Mean cell numbers were 109.3, 101.0, and 114.0; and the percentages of apoptotic cell numbers per blastocyst were 1.25, 1.91, and 1.87 for KSOMaa, CR1aa, and SOFaa groups, respectively. There was no difference among groups in terms of mean cell numbers and percentages of apoptotic cells per blastocyst. The expressions of Glut-1 and DcIII genes did not differ among the groups. However, expressions of Hsp70, IF-tau, and Dnmt3a genes were all significantly up-regulated in the CR1aa group as compared to the SOFaa and KSOMaa groups (P < 0.05). In conclusion, SOFaa supports higher development to the blastocyst stage than KSOMaa and CR1aa, and culture conditions influence gene expression.

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