• Development and Reproduction
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• v.7 no.2
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• pp.61-68
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• 2003

Zygote genome should entail a complex process of epigenetic reprogramming including a global DNA demethylation to reach a totipotency or pluripotency during early mammalian development. In this study, we have analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant DNA methylation patterns were observed in various genomic regions of cloned embryos except single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in viかo or in vivo. Abnormal methylation profiles were also specifically represented in trophectoderm cells of cloned embryos, which probably result in widespread gene dysregulation in extraembryonic region or placental dysfunction familiar to cloned animals. Our findings suggest that developmental failures of cloned embryos are due to incomplete epigenetic reprogramming of donor genomic DNA. Understanding the epigenetic reprogramming processes of donor genome will clearly define the faulty development of cloned embryos.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.263-268
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• 2000

The development of single blastomeres isolated from 2-, 4- and 8-cell mouse embryos and the ability of such blastomeres to survive slow freezing were studied. Of 223, 60 and 188 single blastomeres isolated from 2-, 4- and 8-cell mouse embryos, respectively, 111 blastomeres (49.8%) from 2-cell embryos, 12 blastomeres (20.0%) from 4-cell embryos and blastomeres (16.5) from 8-cell embryos developed into blastocysts after culture for 96 hrs. The recovery rate was 54.2% (65/120), 46.4% (13/28) and 24.3% (17/70) of blastomeres derived from 2-, 4- and 8-cell embryos following freezing and thawing and the survival of frozen-thawed blastomeres was 27.1% (16/59), 36.4% (4/11) and 17.6% (3/17), and respectively. The apparently six normal fetuses were obtained from frozen-thawed blastomere from 2-cell embryos after transferring into the recipients. These results indicate that mouse btastomeres isolated from preimplatation stage embryos can survive storage in liquid nitrogen following slow freezing.

• Animal Bioscience
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• v.36 no.9
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• pp.1327-1335
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• 2023

Objective: When evaluating individuals with the same parent and no phenotype by pedigree best linear unbiased prediction (BLUP), it is difficult to explain carcass grade difference and select individuals because they have the same value in pedigree BLUP (PBLUP). However, single step GBLUP (ssGBLUP), which can estimate the breeding value suitable for the individual by adding genotype, is more accurate than the existing method. Methods: The breeding value and accuracy were estimated with pedigree BLUP and ssGBLUP using pedigree and genotype of 408 Hanwoo cattle from 16 families with the same parent among siblings produced by fertilized egg transplantation. A total of 14,225 Hanwoo cattle with pedigree, genotype and phenotype were used as the reference population. PBLUP obtained estimated breeding value (EBV) using the pedigree of the test and reference populations, and ssGBLUP obtained genomic EBV (GEBV) after constructing and H-matrix by integrating the pedigree and genotype of the test and reference populations. Results: For all traits, the accuracy of GEBV using ssGBLUP is 0.18 to 0.20 higher than the accuracy of EBV obtained with PBLUP. Comparison of EBV and GEBV of individuals without phenotype, since the value of EBV is estimated based on expected values of alleles passed down from common ancestors. It does not take Mendelian sampling into consideration, so the EBV of all individuals within the same family is estimated to be the same value. However, GEBV makes estimating true kinship coefficient based on different genotypes of individuals possible, so GEBV that corresponds to each individual is estimated rather than a uniform GEBV for each individual. Conclusion: Since Hanwoo cows bred through embryo transfer have a high possibility of having the same parent, if ssGBLUP after adding genotype is used, estimating true kinship coefficient corresponding to each individual becomes possible, allowing for more accurate estimation of breeding value.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.101-112
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• 2004

Chloride($Cl^-$) channels play critical roles in cell homeostasis and its specific functions such as volume regulation, differentiation, secretion, and membrane stabilization. The presence of these channels have been reported in all kinds of cells and even in frog oocytes. These essential role of $Cl^-$­ channels in cell homeostasis possibly play any role in egg homeostasis and in the early stage of development, however, there has been no report about the presence of $Cl^-$­ channel in the mammalian oocyte. This study was performed to elucidate the presence of $Cl^-$­ channels in hamster eggs. When allowing only $Cl^-$­ to pass through the channel of the egg membrane by using impermeant cation such as N-methyl-D-glucamine(NMDG), single channel currents were recorded. These channel currents showed typical long-lasted openings interrupted by rapid flickering. Mean open $time({\tau}o)$ was 43${\pm}$10.14 ms(n=9, at 50 mV). The open probability(Po) was decrease with depolarization. The current-voltage relation showed outward rectification. Outward slop conductance(32${\pm}$5.4 pS, n=22) was steeper than the inward slop conductance(10${\pm}$1.3 pS). Under the condition of symmetrical 140 mM NaCl, single channel currents were reversed at 0 mV(n=4). This reversal potential(Erev) was shifted from 0 mV at 140 mM concentration of internal NaCl(140 mM [Na+]i) to ­9.8${\pm}$0.5 mV(n=4) at 70 mM [Na+]i and 11.5${\pm}$1.9 mV at 280 mM [Na+]i(n=4) respectively, strongly suggesting that these are single $Cl^-$­ channel currents. To examine further whether this channel has pharmacological property of the $Cl^-$­ channel, specific Cl­ channel blockers, IAA-94(Indanyloxyacetic acid-94) and DIDS(4, 4'-diisothiocyan ostillben- 2-2'disulfonic acid) were applied. IAA-94 inhibited the channel current in a dose-dependent manner and revealed a rapid and flickering block. From these electrophysiological and pharmacological resluts, we found the novel $Cl^-$­ channel present in the hamster oocyte membrane. The first identification of $Cl^-$­ channel in the hamster oocyte may give a clue for the further study on the function of $Cl^-$­ channel in the fertilization and cell differentiation.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.191-199
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• 2004

This study was conducted to examine in vitro development of porcine embryos constructed by the microinjection of cultured fetal fibroblast cells into porcine oocytes matured in vitro. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5％ FBS, at 38.5$^{\circ}C$ for 6 to 8 days in 5％ $CO_2$ and air. In experiment 1, fusion rates of nuclear transfer embryos did not differ for fetal fibroblast cells incubated in 5％ FBS + NCSU-23 or 5％ FBS + TL Heaps medium, nor did fusion rates of donor cells differ between 1-8 hr incubation durations. Fusion rates for the four treatment subclasses ranged from 72.1％ to 78.0％. In experiment 2, Pre-synchronization in medium containing 0.1 $\mu\textrm{g}$/m Hoechst 33342 an increase from 0 and 8 versus 15 h culture an increased percentage of porcine fibroblast cells in G2/M at the end of the synchronization period (12.4％, 17.5％ and 47.6％). Neither an increase in the concentration of H 33342 (0.2-1.6 $\mu\textrm{g}$/$m\ell$) nor a longer exposure time (12h, 18h and 24h) increased the proportion of porcine G2/M fibroblasts. In experiment 3, fusion rates did not differ significantly far nuclear transfer embryos constructed using donor cells cultured in 5％ FBS + NCSU-23 medium for 1-2, 6-8 or 12-14 days (60.0％, 73.3％ and 62.5％), respectively. The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1-2 days was 44.0％, significantly less than 56.7％ and 50.0％. for 6-8 or 12-14 days duration of culture, respectively. In experiment 4, the proportions of nuclear transfer embryos that developed to the $\geq$2 cell and to the blastocyst stage were not affected significantly by culture medium (5％ FBS + NCSU-23 or 5％ FBS + TL-Heaps) or by $O_2$ concentration of the culture (5％ vs 10％). Rates of development to the $\geq$2 cell stage ranged from 65.9％ to 70.1％, and development rates to the blastocyst stage ranged from 9.8％ to 12.5％ for the four treatment subclasses. Developmental rate was highest for embryos cultured in 5％ FBS + NCSU-23 under a gas atmosphere of 5％ $O_2$ in air.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.137-146
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• 2006

The objective of this study was to examine the effect of donor cell types, the source of recipient oocytes and estrous synchronization on pregnancy and delivery rates of somatic cell nuclear transfer (SCNT) embryos in Korean native goats. Recipient oocytes were surgically collected after superovulation. Ear cells and fetal fibroblasts were collected and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. The zonae pellucidae of in vivo- and in vitro-matured oocytes were partially drilled using a laser system. Single somatic cell was transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3 M mannitol. After the fusion, embryos were activated by Ionomycin+6-DMAP. NT embryos were cultured in mSOF medium supplemented with 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 12 to 20 hr. One hundred and two SCNT embryos were transferred into 20 recipients and pregnancy rate at days 30 was 20.0%. Of them, one developed to term and delivered 1 kid. Ear cells showed significantly higher fusion (63.8 vs. 26.5%) and pregnancy rates (20.0 vs. 0.0%) than those of fetal fibroblast (p<0.05). The recipients synchronized by CIDR showed significantly lower pregnancy rates compared to that of recipient in natural estrus ($0.0{\sim}25.0%$ vs. 100%) (p<0.05). Cloned kid was born from the recipient in natural estrus. For the synchronization of estrus between recipient and donor, there was no difference between treatments (${\pm}0$ vs. +12 hr) in pregnancy rate. The first healthy cloned kid (Jinsoonny) was produced by transfer of SCNT embryos derived from in vivo oocytes and ear cells into a recipient goat whose estrus was synchronized with the donor. These results imply that donor cells for nuclear transfer may affect the success rate, and the estrus synchronization between donor and recipient animals can also be important.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.459-465
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• 1997

The present study was undertaken to determine the effect of NT time on the rate of fusion a and suhseguent development In vitro and determine the optimal strength and duration of DC pulse for electrofusion of IVF donor embryo nuclei and IVM recipient oocytes. The recipient oocytes were enucleated 25 ~ 2Sh after IVM and further cultured for 18 ~ 20h prior to fusion for oocyte aging. IVF embryos as donor nuclei were C co cultured with BOEC for 16- to 32-cell stage development. The transfer time of donor bIas tomeres was 1~3h post-enucleation in early NT group and 1 ~ 18h post-enucleation in late NT group, respectively and fusion was performed 43~4Sh post-IVM. The fusion rate did not differ between the early NT and late NT group, but the rate of cleavage and 8- to 16-cell stage embryos in the late NT group was more higher than that in the early NT group. The fusion, cleavage and M+B development was high from O.7SkV /cm DC than from 1.0kV /cm DC voltage, resulting in 17.6% M+B from 0.75kV /cm DC voltage. No difference in fusion rate was among pulse durations, but 50 and 70 usec pulse duration showed slight high cleavage and M + B d development. The results indicate that the best NT time of IVF donor blastomeres into the enucleated oocytes was 42~44 post-IVM and the most suitable condition for electrofusion was a single 0.7SkV /cm DC voltage for SO~70$\mu$sec.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.1
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• pp.28-32
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• 2012

Objective: This study was performed to assess the prognostic value of serum hCG, progesterone, and inhibin A levels measured at 11 days post-ET for predicting pregnancy outcome in women participating in IVF. Methods: Between May 2005 and April 2008, sera were obtained from 70 infertile women who underwent IVF-ET at 11 days post-ET and stored. HCG, progesterone, and inhibin A levels were measured by commercial enzyme-linked immunosorbent assay kits. The predictive accuracy of hCG, progesterone, and inhibin A levels for establishment of intrauterine pregnancy and ongoing pregnancy was calculated by receiver-operating characteristic curve analysis. Results: For the prediction of intrauterine and ongoing pregnancy, serum hCG was better than progesterone and inhibin A. The predictive performance of progesterone and inhibin A was similar. The serum progesterone and inhibin A levels were significantly correlated each other (r=0.915, p=0.010). Conclusion: A single measurement of the serum hCG level is sufficient to predict pregnancy outcome in IVF-ET patients.

• Journal of Yeungnam Medical Science
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• v.38 no.1
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• pp.53-59
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• 2021

Background: There are no guidelines for the optimal incubation time or temperature to improve pregnancy outcomes in testicular sperm extraction-intracytoplasmic sperm injection (TESE-ICSI) cycles. We aimed to evaluate whether a 24-hour in vitro culture of testicular spermatozoa affects pregnancy outcomes in TESE-ICSI cycles. Methods: This was a retrospective study of 83 TESE-ICSI cycles using testicular spermatozoa in 46 couples with male partners suffering from nonobstructive or obstructive azoospermia. Sperm retrieval was performed either on the oocyte retrieval (OR) day (65 cycles in 33 couples; group A) or on the day before OR (18 cycles in 13 couples; group B) followed by in vitro culture for 24 hours. The clinical characteristics and pregnancy outcomes, including the number of retrieved oocytes, fertilization rates, embryo transfer rates, implantation and clinical pregnancy rates, were compared between the two groups. Results: There were no differences in terms of clinical characteristics except for the levels of luteinizing hormone (LH) in males. Group B had higher LH levels than group A (4.56±1.24 IU/L vs. 3.67±1.07 IU/L, p= 0.017). Group B showed higher fertilization rate (72.4%±32.1% vs. 59.2%±21.7%, p=0.045), implantation rate (35.0%±34.1% vs. 14.0%±21.5%, p=0.010), pregnancy rate per cycle (80% vs. 39%, p=0.033), and clinical pregnancy rate per cycle (80% vs. 37.5%, p=0.024) than those of group A. Conclusion: Testicular sperm retrieval performed on the day before OR followed by in vitro culture can potentially improve pregnancy outcomes.