• Clinical and Experimental Reproductive Medicine
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• 제22권2호
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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.

• 한국수정란이식학회지
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• 제23권1호
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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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• 제16권5호
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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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• 제17권2호
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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.

• 한국가축번식학회지
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• 제16권4호
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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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• 제24권2호
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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).

• 대한수의학회지
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• 제39권1호
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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.

• 한국가축번식학회지
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• 제21권1호
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• pp.61-69
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• 1997

본 연구는 EGF와 anti-EGF가 초기 생쥐배아의 발생 및 부화에 미치는 영향을 알아보고자 실행되었다. 초기 2세포기부터 상실배까지의 배아를 EGF와 anti-EGF를 각각 처리한 Ham's F10 배양액에서 배양하여 그 발생률과 부화율을 대조군과 비교하였다. EGF 처리시 배양시간에 따른 발생률은 증진되었으나 통계학적 유의성은 없었다. EGF 처리군에서의 부화율(57.5, 62.5, 65.0, 62.5%)은 대조군(35%)에 비하여 유의하게 (p<0.01) 높았다. Anti-EGF 처리시 각 발생시기별 1:1000 실험군의 발생률은 대조군과 차이가 없었다. 그러나, 1:100 실험군의 경우, 2∼4세포기의 배아는 모두 4∼8세포기에서 정지되었고, 8세포기와 상실배의 포배형성은 48시간 이상 지연되었으며 부화 역시 대조군에 비해 억제되었다(8세포기; 2%, 44%, 상실배; 6.2%, 58.3%). 이 실험에서, EGF는 생쥐 배아의 포배형성과 부화를 증진시키는 반면, anti-EGF는 이를 억제하였다. Anti-EGF 처리시 나타나는 발생정지 현상은 anti-EGF가 배아에서 만들어지는 EGF와 반응하여 EGF의 작용을 억제시키기 때문으로 사료된다. 그러므로 EGF는 paracrine mode로서 뿐만 아니라 antocrine mode로서 생쥐 초기배아의 발생에 중요한 인자로 작용함을 알 수 있었다.

• 농업과학연구
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• 제22권1호
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• pp.96-102
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• 1995

본 연구는 돼지 체외수정란의 발육에 있어서 적절한 에너지 급원을 구명하기 위하여 수행하였는 바, BMOC-II 배양액을 기본배양액으로 하여 여기에 해당과정을 통하여 에너지를 생산하는 glucose와 이 과정에 중요하게 작용하는 phosphate를 실험설계에 따라 첨가한 배 양액에 체외에서 발생된 돼지의 수정란을 배양하여 수정란의 발육에 미치는 영향을 조사하였다. 1. 배양액에 Glucose와 phosphate가 모두 첨가되지 않은 배양액에서 배양된 돼지의 수정란은 15.2%가 4-세포란 이상으로 발육하였으나, 상실배로는 전혀 발육하지 못하였다. 2. 배양액에 첨가된 glucose의 농도는 phosphate의 첨가유무에 관계없이 농도가 높은 처리에서 4-세포기 이상으로 발달한 비율은 감소하였으나, 상실배에 도달한 비율은 증가하는 경향을 나타냈고, phosphate와 함께 첨가되었을 때보다 단독으로 첨가하였을 때가 첨가농도에 관계없이 돼지 체외수정란의 발육능을 증가시켰다.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.219-223
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• 2004

두릅나무의 엽병을 1.0 mg/L 2,4-D가 포함된 MS 고체배지에서 배발생캘러스를 유도하였다. 배발생세포와 배발생세포괴는 1.0 mg/L 2.4-D가 포함된 MS 액체배지에서 배발생캘러스를 2주간 현탁배양하여 대량으로 얻었다. 그물망을 통과한 배발생세포는 1.0 mg/L 2.4-D가 포함된 MS 액체배지에서 배양하면 배발생능이 소실되지 않고 지속적으로 유지 및 증식시킬 수 있었다. 그물망을 통과하지 못한 배발생세포괴를 식물생장조절물질이 첨가되지 않은 1/2 MS 액체배지에 옮겨 2주간 배양하면 구상형의 체세포배로 발달하였다. 구상형의 체세포배는 5 L의 bioreactor를 이용하여 배양하면 심장형, 어뢰형, 자엽형의 배와 유식물체로 발달하였다. Bioreactor 배양을 통해 두릅나무의 체세포배를 효과적으로 대량증식 시킬 수 있었다.