• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.179-187
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• 1991

This experiment was carried out to develop a new technique of identifying XX of XY-bearing bisected embryos prior to implantation by immunological method. H-Y antiserum prepared in inbred Wastar female rats by repeated immunization with spleen cells from males of the same strain. The reactivity of H-Y antibody was confirmed by culturing mouse embryos in the medium containing H-Y antiserum and complement obtained from the guinea pig. The optimal condition for the activity of H-Y antibody was also investigated by culturing embryos under the concentraton or affected H-Y antibody was also investigated by culturing embryos under the concentration or affected H-Y antibody and culture rate. However, production of live young or sex rates of male and female from embryos transferred with psudopregnant. The biological test with the morula stage embryos showed that H-Y antibody was formed in all female rats immunized with spleen cell, but it was formed only in 80% female rats immunized with the antigen. When the bisected mouse embryos were cultured in vitro for 5~6 hours in morula stage, of 457 bisected embryos 81.4% of then were developed to the blastocyst stage. When the concentration rate of complement to H-Y antiserum varied from 1.0~5.0${mu}ell$, the lysis-rate of embryo was 19.5 to 67.3%. The concentration rate of complement did not influence the lysis-rate of embryos(P<0.05). The morphology embryos of bisected, zona-free and intact embryos showed the embryos lysis rate of 58.6, 42.7 and 48.5% respectively(P<0.05). Pregnancy rate were 50.0, 45.5 and 57.1% in psudopregnant recipient transferred with bisected, zona-free and intact blastocyst embryos. However, production of live youngs, sexual rate of male or female was 24(50.0:50.0), 22(45.5:55.5) and 36(58.3:41.7)mice, but affected and non affected half embryos with H-Y antiserum treatment was 23.1 and 26.7%. Also production of live youngs and sexual rate was 14(92.9:7.1) and 17(17.6:82.4)mice in affected and non affected half embryos in H-Y antiserum treatment(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.37-43
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• 1993

In 27 patients with the past history of poor response to the gonadotropin superovulation induction due to poor follicular growth or permature surge of endogenous luteinizing hormone, the effectiveness of pituitary supperssion with the gonadotropin releasing hormone agonist(GnRH-a) in in vitro fertilization(IVF) program was evaluated in 43 cycles using a combination regimen of D-Trp-6 LHRH(Decapeptyl, Ferring)and FSH/hMG from June, 1989 to August, 1990 at Korea University Hospital IVF Clinic. At midluteal phase of menstrual cycle, Decapeptyl-CR was administered by long-term protocol to minimize initial agonistic effect of endogenous gonadotropins. After the confirmation of pituitary suppression, about 2-3 weeks after GNRH-a administration, ovarian follicle growth was stimulated with FSH/hMG and followed by transvaginal ultrasonic measurement of follicle size and by monitoring of serm E2 and LH if necessary. When compared with the control group stimulated with gonadotropin regimen only, the cancellation rate and occurrence rate of premature LH surge during gonadotropin treatment were significantly lower in study group(11.6% and 2.4%, respectively). There is no significant differences in the mean number of aspirated oocytes, fertilization/cleavage rate, embryo transfer(ET) rate, and mean number of embryos transferred between the two groups. The pregnancy rate per treatment cycle, 16.3%, and per ET cycle, 23.3%, were significantly higher in the study group compared with those of control group. These data suggest that GnRH-a therapy is effective for previous poor responder In gonadotropin superovulation induction for IVF.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.1-12
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• 1992

본 연구는 소의 난포란의 체외성숙과 체외수정에 영향을 미치는 요인을 구명하기 위하여 미숙 난포란을 채취하여 형태적 분류를 통해 우수한 란을 공시한 후 난포의 크기, 정액의 형태, 수정능득법, 혈청, 호르몬, 난포액, 난구세포등을 첨가한 TCM-199 배양액에서 배양하면서 체외성숙 및 수정율을 조사하였는 바 그 결과는 다음과 같다. 1. 소 난포란을 채취하여 배양을 통해 형태적 분류를 했을때 A형란은 61.4%, B형란은 12.1%, C형란은 19.2%, D형란은 4.2%였으며 발생중지 또는 퇴화란은 3.0%였다. 또한 A, B, C형란을 배양액에 배양했을때 난포란의 체외성숙율은 각각 89.1%, 78.0%, 52.6%였으며, 수정율은 각각 78.1%, 66.1%, 33.3%였다. 2. 소 난포의 크기를 1-2mm, 3-5mm 및 5mm이상으로 분류하여 채취한 난포란의 수는 각각 67개, 98개, 63개 였으며, 이를 TCM-199 배양액에서 배양했을때의 체외성숙 및 수정율은 각각 56.7%와 44.8%, 82.5%, 72.4%와 46.0%와 28.6%였다. 3. 소 난포란의 체외수정에 있어서 정소상체 미부정자, 희택정액 및 동결정액을 이용하여 매정하였을때 체외수정율과 분할율은 각각 63.3%, 73.3%, 70.0%와 32.7%, 37.8%, 38.3%였다. 4. 소 난포란의 체외수정에 있어서 m-KRB 처리법, HIS처리법, Ca-IA처리법, BFF처리법 및 heparin처리법으로 각각 수정능획득을 유기하였을때 체외성숙 및 분할율은 각각 53.1%, 28.1%, 33.9%와 17.7%, 50.8%와 26.2%, 48.1%와 22.8% 및 58.8%와 32.8%로서 heparin 처리법이 가장 높았다. 5. 소 난포란의 체외성숙과 수정에 있어서 각 농도의 우태아혈청과 FSH, HCG, $\beta$-estradiol을 첨가한 TCM-199배양액에서 배양했을때의 체외성숙 및 수정율은 각각 76.0-82.3%와 26.2-70.0%로서 무첨가에 비해 첨가가 높았다. 6. 발정우혈청 및 우태아혈청 5-20%를 첨가한 배양액에서 배양했을때의 체외성숙율은 각각 71.7-76.9%, 74.0-80.6%였으며 체외수정율은 51.9-58%와 26.2-30.0%로서, 체외수정율의 경우 발정우혈청의 첨가가 우태아혈청의 첨가에 비해 높았다. 7. 난포액20-30%를 첨가한 배양액에서 배양했을때의 난포란의 체외성숙율은 각각 68.0%와 64.6%, 수정율은 각각59.6%와 60.4%로서 난포액10%, 50%를 첨가한 배양액에서 배양시의 체외성숙율과 수정율에 비해 높았다. 8. 1$\times$10 6 /ml의 난구세포를 첨가한 배양액에서 배양했을때의 난포란의 체외성숙율과 수정율은 각각 76.5%와 61.7%로서 FCS 10%와 1$\times$10 4 -10 5/ml 와 1 $\times$10 8/ml난구세포를 첨가한 배양액에서 배양시의 체외성숙율과 수정율에 비해 높았다.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.147-156
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• 2005

본 연구는 NCSU-23과 PZM-3 배양액에 EGF, $\beta-ME$와 glucose의 첨가가 돼지 난자의 체외성숙에 미치는 영향과 배양조건을 다르게 하여 계대배양한 섬유아세포를 이용한 핵이식배를 다른 배양액과 산소조건에서 배양하였을 때 체외발생율에 미치는 영향을 조사하였다. 핵이식 배를 20ng/ml EGF를 첨가 또는 첨가하지 않은 NCSU-23 및 PZM-3 배양액에서 배양하였을 때 배반포로의 체외 발생율은 각각 $12.0\pm1.3\%,\;9.6\pm1.9\%,\;10.9\pm2.1\%,\;9.1\pm2.3\%$였다. 핵이식 배를 $25{\mu}M\;\beta-ME$를 첨가 또는 첨가하지 않은 NCSU-23 및 PZM-3 배양액에서 144시간 배양하였을 때 배반포로의 체외 발생율은 각각 $9.6\pm1.7\%,\;7.3\pm2.3\%,\;11.9\pm1.8\%$와 $7.4\pm2.1\%$였다. $\beta-ME$를 첨가한 PZM-3 배양액에서 배양하였을 때 배반포로의 체외 발생율은 $\beta-ME$를 첨가하지 않은 배양액에서 배양한 배보다 높은 체외발생율을 나타냈다. (p<0.05). 핵이식 배를 1.5mM glucose를 첨가 또는 첨가하지 않은 NCSU-23 및 PZM-3 배양액에서 배양하였을 때 배반포로의 체외 발생율은 각각 $9.4\pm2.2\%,\;6.8\pm2.7\%,\;10.9\pm2.4\%$와$8.9\pm2.6\%$였다. Glucose를 첨가한 NCSU-23과 PZM-3 배양액에서 배양하였을 때 배반포로의 체외 발생율은 glucose를 첨가하지 않은 배양액에서 배양한 배보다 높은 체외 발생율을 나타냈다. 핵이식 배를 NCSU-23 및 PZM-3 배양액과 $5\%$ 및 $20\%$ 산소 조건에서 배양하였을 때 배반포로의 체외 발생율은 각각 $11.1\pm1.8\%,\;9.8\pm1.4\%,\;12.5\pm1.6\%$와$10.9\pml.5\%$였다. NUSU-23과 PZM-3 배양액에서 $5\%$ 산소 조건에서 배양하였을 때 $20\%$산소 조건에서 배양한 난자보다 높은 체외 발생율을 나타났다. 섬유아세포를 NCSU-23 배양액에서 배양하여 공여자세포로 이용하여 10 및 $11\~15\;passage$ 이내로 계대배양하였을 때의 융합율은 $60.0\~73.3\%,\;52.5\%$였다. 섬유아세포를 PZM-3 배양액에서 배양하여 공여자세포로 이용하여 10 및 $11\~15passage$ 이내의 계대배양시의 융합율은 $60.4\~75.1\%$ 및 $58.7\%$였다.

• Development and Reproduction
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• v.3 no.1
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• pp.39-44
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• 1999

In the present study, we investigated the effect of maturity and motility of spermatozoa on the formation of pronuc-leus and subsequent developmental capacity of the human embryo in vitro. The fertilization was performed by means of intracytoplasmic sperm injection (ICSI) in HEPES-buffered m-TCM-199 medium. In the first part of the experiment, motile or im-motile human spermatozoa ejaculated were injected into cumulus-enclosed human oocytes matured in vivo. Significantly (p<0.002) higher proportion of oocytes that was injected with motile spermatozoa formed 2 pronuclei than the oocytes injected with immotile spermatozoa (79.8% vs 51.7%). In the second part of the experiment, cumulus-enclosed human oocytes matured in vivo were injected with motile or immotile spermatozoa collected from testes. There was no difference between motile and immotile spermatozoa. In the third part of the experiment, using modified Tyrode's medium containing 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin and 10% human follicular fluid, we found that the development of oocytes that formed 2 pronuclei were able to develop to 9-16 cells regardless of maturity and motility of spermatozoa.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.135-142
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• 2018

Objective: To prospectively evaluate the efficacy and safety of a fixed early gonadotropin-releasing hormone (GnRH) antagonist protocol compared to a conventional midfollicular GnRH antagonist protocol and a long GnRH agonist protocol for in vitro fertilization (IVF) in patients with polycystic ovary syndrome (PCOS). Methods: Randomized patients in all three groups (early antagonist, n = 14; conventional antagonist, n = 11; long agonist, n = 11) received 21 days of oral contraceptive pill treatment prior to stimulation. The GnRH antagonist was initiated on the 1st day of stimulation in the early antagonist group and on the 6th day in the conventional antagonist group. The GnRH agonist was initiated on the 18th day of the preceding cycle. The primary endpoint was the number of oocytes retrieved, and the secondary endpoints included the rate of moderate-to-severe ovarian hyperstimulation syndrome (OHSS) and the clinical pregnancy rate. Results: The median total number of oocytes was similar among the three groups (early, 16; conventional, 12; agonist, 19; p= 0.111). The early GnRH antagonist protocol showed statistically non-significant associations with a higher clinical pregnancy rate (early, 50.0%; conventional, 11.1%; agonist, 22.2%; p= 0.180) and lower incidence of moderate-to-severe OHSS (early, 7.7%; conventional, 18.2%; agonist, 27.3%; p= 0.463), especially among subjects at high risk for OHSS (early, 12.5%; conventional, 40.0%; agonist, 50.0%; p= 0.324). Conclusion: In PCOS patients undergoing IVF, early administration of a GnRH antagonist may possibly lead to benefits due to a reduced incidence of moderate-to-severe OHSS in high-risk subjects with a better clinical pregnancy rate per embryo transfer. Further studies with more subjects are required.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.5-14
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• 2003

Objective: Present study was aimed to verify the effect of granulocyte macrophage-colony stimulating factor (GM-CSF) in the preimplantation development of mouse embryos and the involvement of the mitogen activated protein kiase (MAPK) in the GM-CSF signaling. Methods: Two-cell embryos were cultured for 96 h in the presence or absence of GM-CSF (0, 0.4, 2, 10 ng/ml) and PD98059, a MEK inhibitor (10 ${\mu}M$). Morphological development, cell number per blastocyst, and apoptotic nuclei, were eamined. MAPK activity of embryonic immunoprecipitate by MAPK (ERK1/2) antibody was measured by in vitro phosphorylation of myelin basic protein. Results: At post hCG 122 h the embryonic development among the experimental groups was significantly different (p=0.018). The rate of blastocyst development and cell number per embryo were the highest in 2 ng/ml GM-CSF treatment group. The percent of apoptotic cells of the GM-CSF-treated embryos was the lowest among the group. In blastocysts, GM-CSF treatment transiently increased MAPK activity. PD098059 attenuated the effect of GM-CSF on the morphological development, increase in cell number per blastocyst, down regulation of apoptosis, and upregulation of MAPK activity, suggesting that activation of MAPK cascade possibly mediated the embryotropic effect of GM-CSF. Conclusion: This result suggested that GM-CSF potentiated the development of preimplantation mouse embryos by activation of MAPK.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.7
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• pp.855-862
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• 2010

Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful pathways. The Mx protein has direct antiviral activity and inhibits a wide range of viruses by blocking an early stage of the viral replication cycle. Cloning, characterization, and expression of Mx in vivo and in vitro have been conducted. The chicken Mx gene spans 21 kb and is made up of 14 exons and 13 introns, of which the promoter region was analyzed. The real-time PCR results showed that Mx expression was increased in chicken embryo fibroblasts (CEF) after 12- and 24-h induction with polyI: C. Induction of Mx expression by poly I: C in vivo revealed tissue-specific patterns among the chicken tissues tested. A trace expression of Mx was detected in healthy chicken liver tissues from adult chickens without inducement; the expression levels in the liver, heart, and gizzard were higher than in the muscle and kidney. This is the first report to demonstrate the expression of a glutathione-S-transferase-tagged-Mx fusion protein of 75 KDa, as well as the biological activity tested by SDS-PAGE and western blotting.