• Plant Resources
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• v.5 no.1
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• pp.29-44
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• 2002

In this study, plant regeneration through in vitro culture from plantlet stems of Yooja (C. junos Sieb.) and trifoliate orange (P. trifoliata Rafin.) was attempted to make mass-production system of virus-free plants having the same genotype with mother plant. In order to investigate physiological change depending on the developmental stage of plant regeneration, the changes of total protein, peroxidase and esterase activity and their isozyme patterns as well were examined in 1/2 MS medium. The results are as follows : 1. The MS medium for the optimal callus induction and shoot formation was utilized. The medium was supplemented either with 2,4-D and Kinetin or with BA and NAA. The optimal concentrations were the combination of 1.0mg/ 2,4-D +0.3mg/ Kinetin and 1.0mg BA +0.3mg NAA in callus induction and shoot formation, respectively. 2. For the plant regeneration from somatic embryos, 1/2 MS medium was used with supplements of growth regulators (free, 1.0mg/ IBA +1.0mg/ BA ,0.5mg/ IBA +0.5mg/ BA). Shooting and rooting were the best in the treatment of 0.5mg/ IBA and 0.5mg/ BA combination. 3. The total protein content has a tendency of increase with the developmental stage of embryo, but it was decreased at the plantlet. Also it was the highest at 8 and 6 weeks stage in C. junos Sieb. and P. trioliata Rafin, respectively. In the SDS-PAGE pattern of protein, C. junos Sieb. showed bands of 29.0 and 40kDa at 10 weeks. The 45,66 and 97.4 kDa bands at 10 weeks of culture were shown in P. trifoliata Rafin. 4. The highest esterase activity was shown at the 6 and 8 weeks of culture in C.junos Sieb. and P. trifoliata Rafin.., respectively. 5. Esterase isozyme patterns were shown difference according to the developmental stage. In C. junos Sieb. a new band was observed at pl 7.7 following 4 weeks culture. On the other hand, new bands in P. trifoliata Rafin. were observed at pl 7.5~6.5 following 4 and 6 weeks culture, respectively.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.80-80
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• 2003

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell and morula stages were vitrified in EFS40 by a 1-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Day -1 to -2 of synchrony, i.e., at a point in pseudopregnancy that was 1-2 days earlier than the embryos. However, although about half the vitrified embryos transferred into oviducts on Day -1 developed to term, only a minority of embryos transferred at later times did so, whether vitrified (10-34%) or fresh (24-33%), suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos (-63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day-1). A majority of vitrified morulae also developed to term (52-68%) in a wider range of recipients (Day 0 to -1), the greatest success occurring with recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos and morulae developed to term when there was appropriate synchronization between embryo and recipient. Thus vitrification of preimplantation stage rat embryos does not appear to impair their developmental potential in vivo.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.73-73
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• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.2
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• pp.157-164
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• 1999

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP $(400\;{\mu}M)$) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.148-152
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• 2011

Objective: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics. Methods: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LAICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics. Results: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients. Conclusion: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.45-56
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• 1990

Maternal serum ${\beta}$-specific human chorionic gonadotropin(${\beta}$-hCG) and pregnancy-specific ${\beta}_1$-glycoprotein($SP_1$) levels were determined more than one per week during 11-41 days post embryo transfer(ET) in 21 consecutive pregnancies after in vitro fertilization(IVF), which included 8 normal singleton pregnancies, 3 twin pregnancies, 4 clinical abortions, 1 ectopic pregnancy, and 5 preclinical abortions. The sensitivity of serum ${\beta}$-hCG and $SP_1$ radioimmunoassays was 3mIU/ml and 0.7ng/ml relatively. At the 7th to 8th week of gestation, ultrasonographic confirmation of fetal pole and fetal heartbeat was performed. Both serm ${\beta}$-hCG and $SP_1$ levels showed logarithmic increase, but log[$SP_1$] had more steep rising curve and had wider variation than log[${\beta}$-hCG] in normal singleton pregnancies. In 3 twin pregnancies and one ectopic pregnancy, both serum ${\beta}$-hCG and $SP_1$ levels located within the 95% confidence interval of the mean levels of 8 normal singleton pregnancies(normal range). In 2 clinical abortions which had a fetal pole without heartbeat, serum ${\beta}$-hCG level showed lower limit of the normal range or just below, but all $SP_1$ levels showed within the normal range. In other 2 clinical abortions which were diagnosed as blighted ovum, both serum ${\beta}$-hCG levels from 11 days post-ET and serum $SP_1$ levels from later days compared with ${\beta}$-hCG were below the normal range. In 5 preclinical abortions, serum $SP_1$ levels were within the normal range but serum ${\beta}$-hCG levels were far below the normal range. In conclusion, both serum ${\beta}$-hCG and $SP_1$ levels increased exponentially with similar pattern in normally conceived pregancy after IVF-ET. Both serum ${\beta}$-hCG and $SP_1$ levels could predict outcome of early pregnancy to a certain degree, but log[${\beta}$-hCG] levels had more significant correlation with outcome of pregnancy compared with log[$SP_1$] levels. In addition, ultrasonographic examination of fetal poles and fetal heartbeats gives very important clinical information and prognosis.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.447-456
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• 1999

Objective: The purpose of this study was to evaluate outcome of intracytoplasmic sperm injection (ICSI) using epididymal and testicular sperm in patients with azoospermia. Methods: From March, 1993 to May, 1999, a retrospective clinical analysis was done of a total of 140 cycles in 112 patients who underwent ICSI. Subjects were divided into three groups: ejaculated-ICSI group included 42 cycles in 34 patients with ejaculated sperm who underwent ICSI due to severe oligospermia and past history of failed or poor fertilization in the previous in vitro fertilization and embryo tranfer (IVF-ET) cycles, microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection (MESA-ICSI) group included 50 cycles in 42 patients with congenital absence of the vas deferens (CAVD) or unreconstructable obstructive azoospermia and testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) group included 48 cycles in 36 patients with no spermatozoa which can be retrieved from epididymis or non-obstructive azoospermia. Results: Normal two-pronuclear fertilization rates were similar in three groups: 64.4% for ejaculated-ICSI group, 59.4% for MESA-ICSI group and 60.4% for TESE-ICSI group. The pregnancy rates were 26.2%, 26.0% and 25.0% respectively. There were no significant differences in the fertilization, cleavage, and clinical pregnancy rates among ICSI cycles using ejaculated, epididymal and testicular sperm. Conclusion: Epididymal and testicular sperm obtained in azoospermic patients can fertilize oocyte successfully and may lead to be similar fertilization rates and clinical pregnancy rates to ejaculated sperm.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.109-109
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• 2003

Even though success in birth of live offspring from nuclear transfer(NT) using somatic cells in many species, detailed information on processes or mechanisms of development are not well known. Cytoplasm of bovine oocyte has been known to support the development of nuclear transferred embryos using nuclear donor cells from different species. Therefore, interspecies NT might be used to find answers of some questions in basic aspect of nuclear transfer In this study, we examined the developmental potential of reconstructed embryos when bovine oocyte as a cytoplasm recipient and mouse embryonic fibroblast as a nuclear donor were used. The nuclear transfer units were aliocated in Group 1 (murine block media and normal media) and Group 2. (bovine block media and normal media). NT units were not blocked at 2-cell stage regardless of types of medium. On mouse media, poor development of interspecies NT units was observed compared to bovine media. However, as NT units cultured in bovine normal medium, embryos developed over 8-cell stage. Further studies performed to increase the developmental rate in condition of antioxidant treatment. Despite low development, bovine-murine interspecies nuclear transferred embryos could develop to blastocysts and they showed that blastocyts rate of antioxidant group was superior to those of non-antioxidant group. Next, we investigated gene expression pattern which is carried out for zygotic activation. The Xist gene is expressed in female mouse embryo after zygotic activation of 4-cell stage. But interspecies nuclear transferred embryos do not express Xist gene at 4-cell stage. As a result, it is suggested that the bovine cytoplasm controls the early preimplantation development in interspecies NT However, the development of later stages might require genomic control from transferred donor nucleus. Therefore, even though the involvement of several other factors such as mitochondrial incompatibility, effective development of embryos produced by interspecies NT requires proper genomic activation of donor nucleus after overcoming the cytoplasmic control of recipient oocytes.