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.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P &lt; 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P &lt; 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P &lt; 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P &lt; 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P &lt; 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P &lt; 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

89 ZYGOTICALLY ACTIVATED GENES ARE SUPPRESSED IN MOUSE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 162
Author(s):  
T. Suzuki ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Stage ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Gene Activation ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Cell Nuclei ◽  
Cell Stage

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the mouse, major zygotic gene activation (ZGA) occurs at the 2-cell stage after fertilization and leads to the transition of gene regulation from maternal control to embryonic control. Suppression of the ZGA by a transcription inhibitor was shown to decrease the viability of embryos, and causes developmental arrest at the 2-cell stage. An abnormal ZGA may therefore affect the viability of NT embryos and cause further abnormalities in later embryonic development. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. The developmental rate of NT embryos to blastocysts (32.9%) was significantly lower than that of IVF (92.7%) or PA (92.8%). In addition, the cell numbers of NT embryos at the blastocyst stage (39.5 � 2.6; n = 19) were less than those of IVF (66.8 � 2.1; n = 30) or PA embryos (48.2 � 2.1; n = 30). Using these embryos, we first identified 4 genes that were differentially expressed between NT and IVF embryos at the 2-cell stage. Among the identified genes, Inpp5b and Chst12 were up-regulated, and MuERV-L and Dnaja2 were down-regulated in the NT embryos compared with IVF embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-γ, Dub-1, Spz1, DD2106, and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of pre-implantation development as a failure of nuclear reprogramming.

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.

scholarly journals Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Jared M. Campbell ◽  
Michelle Lane ◽  
Ivan Vassiliev ◽  
Mark B. Nottle
Keyword(s):  
Embryonic Stem ◽  
Cell Number ◽  
Poor Quality ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Hesc Derivation

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage.

84 PORCINE EMBRYOS UTILIZE SMALL AMOUNTS OF PYRUVATE, LACTATE, AND GLUCOSE IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
B. R. Redel ◽  
L. D. Spate ◽  
B. Elliott ◽  
M. Paczkowski ◽  
R. L. Krisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Culture ◽  
Glucose Transporters ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Energy Substrates ◽  
Stage Embryo

Porcine embryo culture systems are suboptimal to the in vivo environment, and significant effort has been made to improve development to the blastocyst stage in vitro. Since metabolism of the early embryo has many similarities to the Warburg effect, our goal was to determine the role of glucose on development, gene expression, and metabolism of other energy substrates in the blastocyst stage embryo. Pig embryos were in vitro produced and cultured in MU1 containing pyruvate, lactate, amino acids, and either 0, 7.5, 15, or 250 µM glucose, N = 1164, 4 replications. There was no difference in blastocyst percentage between the 0 µM and 7.5 µM glucose (34% ± 6.5 v. 29% ± 8.2), but there was a decrease in development in response to 15 and 250 µM compared with 0 µM glucose (25% ± 8.5, 23% ± 8.7 v. 34% ± 6.5; P ≤ 0.01). Glucose transporters (SLC2A1 and SLC2A2) and hexokinases (HK1 and HK2) were analysed by qPCR to detect differences in gene expression, 3 replicates containing 10 blastocyst pools. The abundance of both HK1 and HK2 was decreased in blastocysts cultured with 7.5 µM glucose compared with 0 µM (P ≤ 0.04). Glucose transporters were not affected by glucose supplementation (P ≥ 0.5). Metabolic data were collected to determine if embryos were adjusting their energy substrate use in response to glucose. Two assays were completed to determine lactate and pyruvate consumption or release into the media by embryos, in comparison with media without embryos. In vitro-produced embryos were cultured in MU1 with 0 or 7.5 µM glucose N = 360, 4 replications. Both treatments consumed lactate, but there were no differences between treatments (6.8 ± 9.4 pmol/blastocyst/h v. 12.5 ± 1.6 pmol/blastocyst/h; P = 0.6). Blastocysts cultured in 7.5 µM glucose consumed pyruvate, whereas blastocysts without glucose produced pyruvate (–0.34 ± 0.3 pmol/blastocyst/h v. 0.73 ± 0.2 pmol/blastocyst/h; P < 0.01). It has been suggested that fructose is a more efficient replacement for glucose in pig embryo culture. Therefore, we produced pig embryos in vitro and cultured these embryos in MU1, MU1 + 2 mM glucose, or MU1 + 2 mM fructose to the blastocyst stage, 4 replications, N = 389. Again, there was a decrease in embryos that developed to the blastocyst stage in 2 mM glucose compared with MU1 control blastocysts (26% ± 5.8 v. 11% ± 2.5; P = 0.001), but there was only a trend for a decrease in development in response to 2 mM fructose (17 ± 2.3%; P = 0.06). There was no difference in total cell number between MU1, 2 mM glucose, and 2 mM fructose (30.6 ± 2.2, 30.5 ± 3.7, and 32.6 ± 3.0, respectively; P ≥ 0.9) 3 replications, N = 32. Because there is very little consumption of lactate and very low levels of pyruvate are being consumed when glucose is present, it does not appear that any of these energy substrates are major players for the developing pig embryo. Future experiments should be conducted to determine other means of energy production and metabolism in these embryos. The research was funded by Food for the 21st Century.

Development of four-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu

1990 ◽  
Vol 2 (1) ◽  
pp. 1 ◽  
Author(s):  
H Monis ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Co2 Concentration ◽  
Inhibitory Effect ◽  
Defined Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Of Development

Constituents of the culture milieu known to influence development of hamster 2-cell and 8-cell embryos were examined for effects on the 4-cell stage. Embryos were collected at the mid 4-cell stage (approx. 45-46 h after egg activation) from superovulated females and cultured for 24 h in a chemically defined medium (TLP-PVA). As with the 2-cell stage, inorganic phosphate (Pi) strongly inhibited development of 4-cell embryos, although some (14%) were able to reach the 8-cell stage or further in the presence of Pi. However, unlike 2-cell embryos, no significant inhibitory effect of glucose on development of 4-cell embryos was found. In the absence of glucose and Pi, development of 4-cell embryos was sensitive to amino acids in the medium: the mean cell number was increased using 21 amino acids compared with 4 amino acids, similarly to the 2-cell stage; however, late blastocyst development (blastocele formation) from 4-cell embryos was reduced using 21 compared with 4 amino acids, as with 8-cell embryos. Similarly to the 2-cell and 8-cell stages, raising the CO2 concentration from 5% to 10% in the gas atmosphere for culture increased the percentage of total blastocysts developing from the 4-cell stage, but did not affect the proportions of late-stage blastocysts. These data show that 4-cell-stage hamster embryos are somewhat similar to 2-cell embryos with respect to the regulation of development by constituents of the culture milieu, but, to some extent, the 4-cell embryo is a transitional stage of development.

scholarly journals Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner
Keyword(s):  
Ethylene Glycol ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Initial Increase ◽  
Cell Stage ◽  
Free Treatment ◽  
Zona Hardening ◽  
Free Media ◽  
Oocyte Freezing

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

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