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

281 ATTEMPTS FOR ESTABLISHMENT OF PORCINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2009 ◽  
Vol 21 (1) ◽  
pp. 237
Author(s):  
H. M. Kim ◽  
J. K. Park ◽  
S. G. Lee ◽  
C. H. Park ◽  
S. W. Yoon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Control Group ◽  
Es Cell ◽  
Cell Stage

The porcine embryonic stem (ES) cells could be a useful tool for the production of transgenic animals and the study of developmental gene regulation. Even though the efficiency of establishment of ES cells from in vivo blastocysts is relatively high, especially in mice, it is difficult and expensive to obtain in vivo embryos in domestic animals. Recent development of techniques in the production of embryos in vitro could be a useful source for the establishment of ES cells. However, the morphology and cell quality of in vitro-produced embryos are inferior to those of their in vivo counterparts. Although many attempts have been made to establish ES cells from in vitro-produced embryos, the overall efficiency is extremely low because of the poor embryo quality. However, aggregation of in vitro-produced embryos was developed to increase the number of cells in the inner cell mass (ICM) of blastocysts and could be useful in the application to ES cell establishment. Therefore, in this study, we attempted to derive porcine ES cells by using aggregation of in vitro-produced embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Embryos at the 4-cell stage were produced by culturing embryos for 2 days after IVF and SCNT. After removal of the zona pellucida with acid Tyrode’s solution, three 4-cell-stage embryos (IVF3X) from IVF and two 4-cell-stage embryos (NT2X) from SCNT were aggregated by co-culturing them in an aggregation plate followed by culturing to the blastocyst stage. Embryos from IVF (IVF control) and SCNT (NT control) were also cultured to the blastocyst stage. All blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Two primary colonies were formed in the IVF control group (3.9%), whereas four primary colonies were formed in the IVF3X group (12.5%). One primary colony was formed in the NT2X group (20%), although no colony was formed in the NT control group. One of the IVF3X lines gradually disappeared after sub-passing, and the NT2X line also disappeared. Two ES-like cell lines derived from the IVF control were maintained up to 14 passages, and three ES-like lines from IVF3X were also maintained for more than 14 passages. These cells morphologically resembled human ES cells (flat and single layered) and expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, Oct-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. These results indicated that a porcine ES cell line could be established from in vitro-produced aggregated blastocysts. Further research is required to establish ES cell lines from SCNT embryos and characterize the differentiation and developmental abilities of these porcine ES-like cells. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 540-548 ◽  
Author(s):  
Li-Bing Ma ◽  
Xiao-Ying He ◽  
Feng-Mei Wang ◽  
Jun-Wei Cao ◽  
Teng Cheng
Keyword(s):  
Nuclear Transfer ◽  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Pluripotency Genes

SummarySomatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep–sheep intra-species cloned embryos, goat–sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals Controlled spermatozoa–oocyte interaction improves embryo quality in sheep

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Debora Agata Anzalone ◽  
Luca Palazzese ◽  
Marta Czernik ◽  
Annalaura Sabatucci ◽  
Luca Valbonetti ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Time Frame ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Free Oxygen Radicals ◽  
High Concentration ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Set Up

AbstractThe current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

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.

225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 210 ◽  
Author(s):  
S. W. Yoon ◽  
C. H. Park ◽  
S. G. Lee ◽  
H. M. Kim ◽  
J. K. Park ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Mrna Levels ◽  
Cell Stage ◽  
Significant Difference ◽  
Apoptotic Effect

Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P < 0.05). The total cell number of blastocysts also increased approximately threefold (P < 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P < 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 125-137 ◽  
Author(s):  
A.V. Makarevich ◽  
P. Chrenek ◽  
N. Žilka ◽  
J. Pivko ◽  
J. Bulla
Keyword(s):  
Culture Medium ◽  
Detrimental Effect ◽  
Transgenic Animals ◽  
Cell Number ◽  
High Rate ◽  
Cleavage Stage ◽  
Thin Sections

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19–20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94–96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p<0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi-and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p<0.001) and lower total cell number (p<0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

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