• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.163-170
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• 1995

Growth factors (GFs) produced by the embryo or by the maternal reproductive tract have been reported to regulate the embryonic development and differentiation. Among GFs, EGF as a mitogen plays a role in mitosis and functional differentiation of trophectoderm cells in mouse. The present study was carried out to investigate the effect of EGF on development of mouse embryos and to localize EGF in the mouse oocytes and embryos, which has been reported to be detected in the reproductive tract in mammals. To investigate the effect of EGF on the development of the embryo, mouse 2-cell embryos were cultured to blastocysts stage in Ham's F10 medium, treated with EGF(10-50 ng/ml) for 72 hrs. Immunocytochemistry was performed from oocyte to blastocyst stage with anti-EGF and anti-Mouse IgG, in order to determine the stage which EGF would be expressed in mouse. Exogenous EGF (more than 10 ng/ml) in the culture medium improved the developmental and hatching rates in the mouse embryos. As a result of immunocytochemistry, the embryonic EGF was expressed after the late 4-cell stage. EGF is thought to enhance preimplantation embryonic development and hatching. Exogenous EGF in the culture medium is thought to activate EGF receptor in the late 4-cell embryos and to enhance blastulation and hatching in mouse embryos. It is concluded that EGF enhances the developmental and hatching rates in the mouse embryos.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.19-24
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• 2008

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.5
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• pp.650-654
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• 2003

The accuracy of Polymerase chain reaction (PCR) assay in sexing of sheep embryos was assessed in this study. A total of 174 ovine embryos produced in vitro at different stages of development (2, 4-8 cell stages, morula and blastocyst) were sexed. The universal primers (P1-5EZ and P2-3EZ) used in this assay amplified ZFY/ZFX-specific sequences and yielded a 445 bp fragment in both sexes. Restriction enzyme analysis of ZFY/ZFX-amplified fragments with Sac I exhibited polymorphism between sexes, three and two fragments in males and in females, respectively. For verification of accuracy, blood samples of known sex were utilized as positive controls in each test. The mean percentages of sex identification by this method at 2 cell, 4-8 cell, morula and blastocyst were $73.00{\pm}5.72$, $89.77{\pm}3.79$, $3.33{\pm}8.08$ and $79.6{\pm}9.09$, espectively with the over all male to female ratio of 1:0.87. It is concluded that the ZFY/ZFX based method is highly reliable for the sexing of sheep embryos.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.129-135
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• 1990

This study was done to verify factors affecting viability after cryopreservation and pregnancy rate after frozen-thawed embryo transfer into uterus. Embryos were cryopreserved slow freezing and slow thawing and used DMSO as cryoprotectant. The results were to follows. 1. Viability of frozen-thawed embryos were 75.5% (94/105), which compared with viability of embryos according to cell stage, $2{\sim}5$ cell was 68.4% and $6{\sim}16$ cell 80.4% were significant differences (p<0.05). 2. No significant difference in duration of cryopreservation on effects affecting pregnancy rate was observed. 3. Number of embryo transfered into uterus was significant differences (p<0.05). 4. Four pregnancies resulted following replacement of 35 frozen-thawed.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.341-346
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• 1993

The development of isolated blastomeres from mammalian preimplantation embryos has been basically studied for the multiplication of embryos from superior animals. Therefore, this study was investigated the effect of co-culture with mouse fetal fibroblast cells(MFFC) on in vitro development of blastomeres from mouse preimplantation embryos. Mature female ICR mice were treated with hormone to induce superovulation and embryos were collected at each 2, 4, and 8-cell stage. Then, after removing zona pellucida with protease, blastomeres were isolated by micropipetting, or reconstituted with different stage blastomere, and incubated for 72 hrs either in T6 or TCM199 or on the monolayer of MFFC, which was prepared with fibroblast cells from 14∼14 day mouse fetus. After incubation, we examined their development rates every day and the nuclei numbers of each blastocyst by Hoechst-33342 staining. In the development rates of blastomeres, there were no significant differences between media but the higher rateswere found in the monolayer of MFFC, regardless of reconsititution. In addition, blastomeres cultured with MFFC had slightly greater number of nuclei than those cultured in single media. Generally, the higher development rates of blastomeres were found from earlier stage embryos than the later ones, regardless of culture conditions. Reconsitituted blastomeres had more nuclei but did not show the higher development rates, compared to the single blastomeres. Taken together, our results suggest that co-culture with MFFC have a beneficial effect on the in vitro development of blastomeres from mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.233-239
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• 1997

Embryos of most mammalian species grown in vitro would undergo developmental arrest at the approximate time of genomic activation. Stage-specific cell block and the resulting rapid loss of embryo viability in conventional culture media have limited the duration for which embryos may be cultured prior to transfer. As a result, embryos are usually transferred to the uterus at the 4-to 8-cell stage to avoid the loss of viability associated with long-term in vitro culture. Early transfer has led to asynchrony of the endometrium-trophectoderm interaction at the time of implantation and a resultant reduction in the rate of implantation. To overcome these problems, a variety of co-culture systems has been devised in which embryos can develop for a longer period prior to embryo transfer. Vero cells, derived from African green monkey kidney, share a common embryologic origin with cells from the genital tract. In addition, they are potentially safe to use, since they are highly controlled for viruses and other contaminants. Therefore, co-culture using Vero cells has been widely utilized to enhance embryo viability and development, although not without controversies. We thus designed a series of experiments to demonstrate whether Vero cells do indeed enhance mouse embryo development as well as to compare the efficacy of co-culturing mouse 1-cell embryos on Vero cell monolayer in both Ham's F-10 and human tubal fluid (HTF) culture media. 1-cell stage ICR mouse embryos were cultured either in the presence of Vero cells (Group A) or in conventional culture medium alone (Group B). In Ham's F-10 significantly more 3-to-8cell embryos developed in group A than group B (59.8 versus 10.0%; p<0.01). In contrast, there was no significant difference in embryonic development both group A and group B in HTF. However, significant differences were noted only in later embryonic stage (13 and 0%; p<0.05 of group A and B respectively, hatching or hatched). In Ham's F-10, we also could observe the beneficial effect of Vero cell on hatching process (70.7 and 42.1%; p<0.05 of group A and group B respectively).

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.189-203
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• 1999

To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.61-69
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• 1997

The present study was carried out to investigate the effects of EGF and anti-EGF on early embryonic development and hatching in mice. Developmental and hatching rates of mouse em-bryos from 2-cell to morular stage which were cultured in Ham's FlO medium supplemented with EGF (1-1,000 ng/ml) or anti-EGF (whole serum diluted from 1:10 to 1:1,000) were compared to those of control When mouse early 2-cell embryos were cultured in the EGF supplemented medium, blastulation was accelerated compared with control. Hatching rate was also significantly (p

• Korean Journal of Agricultural Science
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• v.22 no.1
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• pp.96-102
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• 1995

This study was conducted to investigate the effect of energy source on development of in vitro matured and fertilized porcine 2-cell embryos. The effects of glucose and phosphate for in vitro development of porcine 2-cell embryos were determined using modified BMOC-II medium. The results obtained are as follows. 1. In the absence of glucose and phosphate in the culture medium, 15.2% of porcine embryos developed 4-cell stage or further but morula formation was completely blocked. 2. The addition to 5mM glucose in the culture medium decreased rate of embryo reached 4-cell stage or further but increased morula stage as compared with addition to 0mM and 2.5mM glucose, regardless of the presence of phosphate. The addition to glucose in the culture medium without phosphate increased rate of embryo reached 4-cell stage or further and morula stage as compared with both addition to glucose and phosphate, regardless of added glucose concentration.

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.219-223
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• 2004

Embryogenic calli were induced from petioles of Aralia elata on MS solid medium supplemented with 1.0 mg/L 2,4-D. When embryogenic calli were transferred to MS liquid medium supplemented with 1.0 mg/L 2,4-D, embryogenic cells and embryogenic cell clusters were developed after 2 weeks of culture. Embryogenic cells were filtered through a 250 ${\mu}{\textrm}{m}$ sieve and the passed cells were proliferated and maintained in MS liquid medium supplemented with 1.0 mg/L 2,4-D. Embryogenic cell clusters entrapped on the sieve were transferred to 1/2 MS liquid medium without plant growth regulators, globular-shaped embryos were developed from embryogenic cell clusters after 2 weeks of culture. Numerous early stage somatic embryos could be developed to heart-shaped, torpedo-shaped, cotyledonary embryos and plantlets in 5 L bioreactor. Above results suggest that effective somatic embryo proliferation can be achieved via bioreactor culture systems in Aralia elata.