• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.67-74
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• 2000

Objective: This study was conducted to investigate the effect of vitrification on the implantation and the pregnancy of human blastocysts. Method: The transfer of the frozen-thawed blastocysts by the slow freezing or vitrification was performed between January 1998 and July 1999. The zygotes derives from IVF were cocultured with cumulus cells in YS medium containing 20% hFF for 5days. Two or three of the best balstocysts produced on day 5 were transferred into the uterus, and then supernumerary blastocysts were randomly divided into two groups. One was frozen by slow freezing and the other was frozen by vitrification method. The slow freezing procedure was performed in two steps (5% glycerol and 9% glycerol + 0.2 M sucrose for 10 min, respectively) using programmed freezer ($-2^{\circ}C$/min to $-7^{\circ}C$, manual seeding at $-7^{\circ}C$, $-0.3^{\circ}C$/min to $-38^{\circ}C$ and plunged into $LN_{2}$). The blastocysts frozen by slow freezing were thawed at $36^{\circ}C$ then removed glycerol in 7 steps. The vitrification procedure was performed in three steps (10% glycerol for 5 min, 10% glycerol + 20% ethylene glycol for 5 min, 25% glycerol + 25% ethylene glycol and directly $LN_{2}$ within 1 min). The blastocysts frozen by vitrification were thawed at $20^{\circ}C$ water then removed cryoprotectant in 3 steps. In each group, thawed blastocysts were cocultured with cumulus cells in YS medium containing 20% hFF for 18h and transferred into the uterus. The implantation rate was evaluated per transferred blastocysts and the pregnancy rate was evaluated per transfers. Results: The survival rate of vitrified group (74.5%) was higher than slow freezing group (68.0%), but not significant. When 98 thawed blastocysts of vitrification were transferred in 40 cycles, 19 pregnancies (clinical pregnancy rate; 47.5%) were established. One miscarriage occurred in the eighth week of pregnancy (ongoing pregnancy rate; 45.0%). 7 pregnancies were ongoing, 11 pregnancies went to term, and 16 healthy infants were born. The Implantation rate was 31.6%. These results were higher than those obtained by the slow freezing (clinical pregnancy rate; 40.3%, ongoing pregnancy rate; 32.5% and implantation rate; 25.3%), but not significant. Conclusion: Vitrification is a simple, quick and economical method when compared to slow freezing. It will be chosen as a good method of human embryo freezing in IVF-ET programs.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.47-52
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• 2016

The objective of this study was to investigate the result of in vivo embryo collection and pregnancy rate after embryo transfer using sex-sorted sperm of Korean brindle cattle. Donor Korean brindle cattle superovulation treated by decreasing dose of FSH injection. Embryos were recovered on 7 days after the third artificial insemination. Control group semen straw used artificial insemination contained 20 million sperm. Sex-sorted semen straws contained 4 million sperm or 10 million sperm. As for the result of the recovery of the in vivo embryos derived from sex-sorted sperm, the number of transferable embryos was significantly highly recovered to be $6.20{\pm}2.28/donor$ from the control group and was significantly lowly recovered to be $1.57{\pm}1.72/donor$ from the group treated at a sperm concentration of $10{\times}10^6$ (p<0.05). The number of unfertilized embryo was $0.8{\pm}1.30/donor$ in control group which was significantly lower than the group treated at a sperm concentration of $4{\times}10^6$ (p<0.05). However, there was no significant difference in the number of undeveloped ova between control and treatment groups. Pregnancy rate after embryo transfer was shown to be 35.00% in control group and 12.50% in treatment group. The karyotype analysis of the calf derived from sex-sorted sperm resulted in a similar chromosomal distribution pattern (2n=60, XX) compared to those of common Korean native cattle.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.1-7
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• 2005

Telomeres consisting of (TTAGGG)n tandem repeat DNA sequences and associated proteins are essential for chromosome stability and related with cell senescence, apoptosis and cancer. The telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. This study was carried out to identify the distribution of telomeres on mouse chromosomes and also to analyze the amount of telomeres and telomerase activity of mouse embryos at early embryonic stages. Germ cells and early embryos from 1 cell to blastocyst stage were analyzed. The amount of telomeres was analyzed by quantitative fluorescence in situ hybridization technique(Q-FISH) using a human telomeric DNA probe, and telomerase activity was measured by telomeric repeat amplification protocol assay(TRAP). In results, the telomeres on mouse chromosomes were distributed at the ends of all autosomes and sex chromosomes. Although the quantity of telomeres varied among chromosomes, most of chromosomes had higher amount in q-arm telomeres than in p-arm telomeres. The results of Q-FISH indicated that the relative amount of telomeres of mouse embryos in each embryonic stage was approximately the same except the higher amount in blastocysts. Using TRAP assay on mouse embryos, telomerase activity was detected in all preimplantation stages from mature oocytes to blastocysts. Especially the telomerase activity was significantly increased at the morula and blastocyst stage. In conclusion, there may be a close association between the amount of telomeres and telomerase activity in early embryonic stages, and analysis of telomere quantity and telomerase activity on early development will be helpful for the investigation of embryogenesis and embryonic cell differentiation in mice.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.191-200
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• 2005

Many studies have shown that the development of mouse early 2-cell embryos in vitro is related with the intracellular $Ca^{2+}$ changes. In ICR strain mouse, the development of embryos arrests at early 2-cell stage, but the arrested early 2-cell embryos can be rescued by the addition of $Ca^{2+}$-related materials. Acetylcholine (ACh) increases intracellular $Ca^{2+}$ concentration ([$Ca^{2+}$]i) via the mAChR-PLC-IP3 pathway in mouse oocytes. We examined whether ACh rescues 2-cell block in mouse. In early 2-cell embryos, ACh increased [$Ca^{2+}$]i in a dose-dependent manner (p<0.001), and had an effect on rescue of 2-cell block and embryonic development. To identify the signal pathway involved in ACh-induced rescue of 2-cell block, we first applied an agonist of ACh receptor (AChR). Like ACh, carbachol increased intracellular $Ca^{2+}$ concentration ([$Ca^{2+}$]i) and atropine, an antagonist of ACh receptor, blocked the ACh-induced $Ca^{2+}$ increase. In $Ca^{2+}$-free medium, ACh also increased [$Ca^{2+}$]i, indicating that $Ca^{2+}$ increased by ACh is mainly released from the intracellular $Ca^{2+}$ store. The ACh-induced $Ca^{2+}$ increase was blocked by PLC inhibitor (U73122), ryanodine receptor (RyR) antagonist (dantrolene), and CaM KII inhibitor (KN-93), but not by IP3R antagonists (xestospongin C). These results show that ACh increases intracellular $Ca^{2+}$ concentration via mAChR/PLC/RyR, and this contributes to the rescue of 2-cell block.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.35-43
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• 2006

Ions play important roles in various cellular processes including fertilization and differentiation. However, it is little known whether how ions are regulated during early embryonic development in mammalian animals. In this study, we examined changes in $Ca^{2+}\;and\;K^+$ concentrations in embryos and oviduct during mouse early embryonic development using patch clamp technique and confocal laser scanning microscopy. The intracellular calcium concentration in each stage embryos did not markedly change. At 56h afier hCG injection when 8-cell embryos could be Isolated from oviduct, $K^+$ concentration in oviduct increased by 26% compared with that at 14h after injection of hCG During early embryonic development, membrane potential was depolarized (from -38 mV to -16 mV), and $Ca^{2+}$ currents decreased, indicating that some $K^+$ channel might control membrane potential in oocytes. To record the changes in membrane potential induced by influx of $Ca^{2+}$ in mouse oocytes, we applied 5 mM $Ca^{2+}$ to the bath solution. The membrane potential transiently hyperpolarized and then recovered. In order to classify $K^+$ channels that cause hyperpolarization, we first applied TEA and apamin, general $K^+$ channel blockers, to the bath solution. Interestingly, the hyperpolarization of membrane potential still appeared in oocytes pretreated with TEA and apamin. This result suggest that the $K^+$ channel that induces hyperpolarization could belong to another $K^+$ channel such as two-pore domain $K^+(K_{2P})$channel that a.e insensitive to TEA and apamin. From these results, we suggest that the changes in $Ca^{2+}\;and\;K^+$ concentrations play a critical role in cell proliferation, differentiation and reproduction as well as early embryonic development, and $K_{2P}$ channels could be involved in regulation of membrane potential in ovulated oocytes.