Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 909-917 ◽  
Author(s):  
Dong-Kyung Lee ◽  
Chi-Hun Park ◽  
Kwang-Hwan Choi ◽  
Yeon-ik Jeong ◽  
Kyung-Jun Uh ◽  
...  
Keyword(s):  
Histone Deacetylase Inhibitors ◽  
Embryo Quality ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Cell Number ◽  
Total Cell ◽  
Large Animal ◽  
Blastocyst Formation ◽  
Cell Stage

SummaryThe development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.

24 IMPROVEMENT OF PORCINE CLONED EMBRYONIC STEM-LIKE CELLS DERIVATION BY ZONA-MEDIATED EMBRYO AGGREGATION

2014 ◽  
Vol 26 (1) ◽  
pp. 126 ◽  
Author(s):  
D. K. Lee ◽  
C.-H. Park ◽  
Y.-I. Jeong ◽  
J. Y. Hwang ◽  
J.-N. Oh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Histone Deacetylase ◽  
Embryo Quality ◽  
Embryonic Stem ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Cell Stage

Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.

75 Culture of isolated blastomeres supplemented with L-ascorbic acid 2-phosphate in a well-of-the-well culture dish

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
Y. Hasiyada ◽  
H. Matsuda ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
T. Yamanouchi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage ◽  
Quality Grade

We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P&lt;0.05) and 500 µM (150v. 158 hpi; P&lt;0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.

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

67 Effects of phytohemagglutinin on the culture of isolated bovine blastomeres derived from the 8-cell stage invitro-produced embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 159
Author(s):  
Y. Hashiyada ◽  
Y. Aikawa ◽  
H. Matsuda ◽  
T. Yamanouchi
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Flat Surface ◽  
Formation Rate ◽  
Cell Number ◽  
The Other ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage ◽  
Tissue Culture Dish

Monozygotic twin embryos which can efficiently be produced by blastomere separation and aggregation of early cleavage stages of embryos using commercially provided well-of-the-well (WOW) culture dish. Phytohaemagglutinin (PHA) is a plant lectin that binds to and aggregates on the surface of animal cells, but also contains toxicity that causes food poisoning. The present study was conducted to evaluate the toxicity to embryos and the effect to development of isolated blastomeres on PHA-supplemented WOW culture. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and invitro culture (IVC). The tissue culture medium 199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10mgmL−1 bovine serum albumin, and CR1aa medium containing 5% CS were used for each culture step. For the evaluation of PHA toxicity, 89 embryos that developed to the 5-8-cell stage were obtained at Day 2 after insemination. Each embryo was cultured in a droplet of 5 µL/embryo IVC culture medium supplemented with or without PHA. For the evaluation of PHA to development of isolated blastomeres, 111 of 8-cell stage embryos were obtained 48-54h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into single blastomeres by gentle pipetting in IVC medium. Each four blastomeres were formed in the shape of a bunch inside the thin cylinder at the tip of the Pasteur pipette by gentle pipetting. Then, each mass of blastomeres in each 60 masses was cultured individually in 5-µL droplets of IVC medium supplemented with or without PHA on the flat surface of a tissue culture dish. On the other hand, each four blastomeres were introduced into a single conical micro-well each having a diameter and depth of ~287µm and 168µm (Dai Nippon Printing). This culture of blastomeres was performed covered with a droplet of 2.5µL well−1 IVC medium supplemented with or without PHA in each 50 or 52 wells. In all of investigations, PHA was used at 50µgmL−1 (Akagi et al. 2011 J. Reprod. Dev. 57). Statistical analysis was performed using Student's t-test and analysis of variance. The blastocyst formation rate (71.1±2.3% vs. 72.7±1.7%), total cell number (120 vs. 122), and inner cell mass cell number (47 vs. 51) at Day 7 after IVF did not differ between PHA-supplemented and PHA-free group in the toxicity test, respectively. In the blastomere culture, the blastocyst formation rate was very low (10.0±5.9% vs. 5.0±2.9%) regardless of the PHA supplementation in drops on the flat surface of a tissue culture dish. On the other hand, blastocyst formation was improved using the WOW culture dish (24.0±3.6% vs. 40.4±7.6%) but there was no difference with or without PHA supplementation. Although nontoxicity of PHA and efficacy of WOW culture for isolated-aggregated blastomeres were confirmed, no improvement of PHA supplementation on development was observed in this study. Subsequently, experiments on the optimum concentration of PHA for aggregation and development of blastomeres in WOW culture are required.

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Z. B. Cao ◽  
L. C. Sui ◽  
S. F. Ji ◽  
J. W. Chen ◽  
T. Gui ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Total Cell ◽  
High Oxygen ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Nuclear Maturation ◽  
Air Atmosphere ◽  
Significant Difference

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P < 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

scholarly journals Valproic Acid Improves Porcine Parthenogenetic Embryo Development Through Transient Remodeling of Histone Modifiers

2015 ◽  
Vol 37 (4) ◽  
pp. 1463-1473 ◽  
Author(s):  
Yongye Huang ◽  
Lin Yuan ◽  
Tianye Li ◽  
Anfeng Wang ◽  
Zhanjun Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Histone Deacetylase Inhibitors ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Cell Stage ◽  
Alternative Source ◽  
Histone Modifiers ◽  
Parthenogenetic Embryos

Background/Aims: Parthenogenetic embryos are useful in many applications, such as being an alternative source of embryonic stem cells that would avoid ethical problems. Aberrance in epigenetic reprogramming is considered the major reason for the developmental failure of parthenogenetic embryos. Many histone deacetylase inhibitors have been shown to improve the reprogramming of stem cells and embryos. Here, the relationship between histone modification and parthenogenetic embryonic development was explored. Methods: Valproic acid (VPA) treatment was applied during the culture of parthenogenetic embryos. The abundance of histone modifiers was examined by immunofluorescence and quantified by Image-pro software. Results: The acH3K9 level in in vitro fertilized embryos was significantly higher than parthenogenetic embryos. VPA treatment improved both the blastocyst formation rate and the acH3K9 level in parthenogenetic embryos. The signal intensities of acH4K5 and H3K4me2 were also enhanced in VPA treated embryos. The H3K27me2 level was decreased in the VPA treated embryos at the 2-cell stage. However, the enhancement in the acH3K9, acH4K5 and H3K4me2 level, or the decrease in the H3K27me2 level disappeared shortly after VPA withdrawal. Conclusion: Optimizing histone modifications for a short time following activation was sufficient to enhance the in vitro development of parthenogenetic embryos.

60 THE EFFECT OF L-ASCORBIC ACID DURING CULTURE, CRYOPRESERVATION, OR BOTH ON PORCINE EMBRYOS PRODUCED IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 177 ◽  
Author(s):  
M. Castillo-Martín ◽  
M. Yeste ◽  
R. Morató ◽  
T. Mogas ◽  
S. Bonet
Keyword(s):  
Ascorbic Acid ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Fertilisation ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Apoptosis Index ◽  
Number Of Cells

The benefits of adding l-ascorbic acid during the cryopreservation procedure have been reported before in mouse and bovine. In this study, the effects of l-ascorbic acid (AC) supplementation during culture, cryopreservation, or both procedures on the developmental ability and embryo quality of in vitro produced porcine blastocysts were examined. Embryo quality criteria consisted of total cell number, percentage of apoptosis, and cryotolerance. After in vitro fertilisation, presumptive zygotes were randomly assigned to 2 culture treatments in which the culture medium NCSU23 was supplemented with 100 µM AC (n = 1162) or nonsupplemented (n = 1163) for a 144-h period. On Day 6, blastocyst formation was assessed by stereomicroscopy, and a representative fraction of Grade I- and II-blastocysts of each culture treatment was evaluated using 4′,6-diamidino-2-phenylindole-TUNEL co-staining and considered as fresh-control. The remaining fraction of Grade I- and II-blastocysts was vitrified/warmed following the Cryotop® method. To determine the effect of AC supplementation during cryopreservation procedures, each culture treatment was divided into 2 groups: (1) embryos exposed to 100 µM AC, and (2) nonexposed embryos (vitrified-control). Survival was determined according to reexpansion rates after 24 h of recovery in NCSU23 medium. After 24 h, reexpanded blastocysts were co-stained using the 4′,6-diamidino-2-phenylindole-TUNEL technique, and total number of cells and apoptosis indexes were determined. Experiment was replicated 9 times for each group. Data were analyzed by t-test for independent variables and a 2-way ANOVA. Results are expressed as means ± SE, and the significant level was set at 5% (Table 1). After culture, supplementing NCSU23 medium with AC showed no significant differences in blastocyst formation (fresh-control 11.6 ± 7.8 v. AC 11.6 ± 7.7), in number of cells (fresh-control 36.7 ± 15.8 v. AC 36.1 ± 15.9), or in apoptosis index (fresh-control 2.9 ± 5.7 v. AC 3.5 ± 4.7). On the other hand, only when both culture and vitrified media were supplemented with AC was there a significant increase of blastocyst survival. In contrast, no significant differences in embryo survival were observed when only 1 of these 2 media (culture or vitrification) was supplemented. Supplementing culture media or cryopreservation solutions with AC did not affect the total cell number or apoptosis index in vitrified blastocysts. In conclusion, the addition of 100 µM l-ascorbic acid to the culture and cryopreservation solutions improves the cryotolerance of in vitro-produced porcine blastocysts. Table 1.Survival of blastocysts (24 h), total cell number, and percentage of apoptosis after vitrification/warming

Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos

Zygote ◽  
2010 ◽  
Vol 19 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Sol Ji Park ◽  
Ok Jae Koo ◽  
Dae Kee Kwon ◽  
Ma Ninia Limas Gomez ◽  
Jung Taek Kang ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Term Treatment ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Short Term ◽  
Cleavage Rate ◽  
Short Term Treatment ◽  
Total Cell Number ◽  
Long Term Treatment

SummaryTreatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 μg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

scholarly journals 173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 237 ◽  
Author(s):  
G. Lazzari ◽  
I. Lagutina ◽  
G. Crotti ◽  
P. Turini ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Es Cell ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

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