This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5 in 2% in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2 water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100M -mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

소 체외수정란의 체외 발육에 와 의 농도가 미치는 영향에 대하여 두가지 배양액과 서로 다른 Co-culture 체계를 이용하여 검토하였다. 체외 수정후 40~44시간에 회수간 2~8 세포기 수정란을 여러가지 gas조건하의 다른 배양조건에서 무작위로 옮겨 실험을 수행하였다. 5% 와 ,10% 및 20% 조건에서 배양을 실시한 결과, 상실배가 이상 발육된 체외 발육성적은 각각 22.1% 15.0% 및 21.1%였다.(p<0.05). 한편 배양액에 따른 헤외

가축의 일란성 쌍태를 생산하기 위한 기술 개발을 확립하고자 상실배 및 포배기에 있는 BALB/c 계통의 생쥐 수정란을 micromanipulator로 분할 수정란을 작출하고 이를 체외배양을 실시하여 발달성적을 조사하였으며, 외과적 및 비외과적 이식을 실시하여 착상율 및 산자생산 성적을 조사한 결과는 다음과 같다. 1. 상실배 및 포기배에 있는 총 811개의 정상적인 수정란을 분할하여 이중에서 666(82.1%)개가 분할시의 물리적인 손상이 없이 분할되었

Spatiotemporal expressions of microRNAs (miRNAs) are altered by the physiological states of cells which could be influenced by microenvironment. Function of miRNAs has been focused as a new regulator of gene expressions and cell differentiation in human health and diseases. We found and identified the several miRNAs, which were related to developmental competence of preimplantation and implantation process of mouse blastocysts and outgrowth embryos by microarray-based bioinformatical studies. In this study, we evaluated three miRNAs expressions related to third cleavage event in conditioned media (CM) and blastocysts. Mouse 2-cell stage embryos were collected and monitored for 9 hours. The embryos were divided two groups as early third cleavage before 9 hours of collection and late third cleavage after 9 hours of collection. They were cultured to blastocyst stage up to day-5 after hCG injection. The total number of cells and the number of cells with fragmented DNA were assessed in blastocysts by terminal dUTP nick-end labelling (TUNEL) staining and DAPI staining. Mean cell number of early third cleavage group was significantly higher than that of late third cleavage group (105.3±8.0 vs 81.8±7.0, p<0.05), but apoptotic index was not different. The miRNAs of CM and blastocysts from early and late group were prepared, and quantified by qRT-PCR with TaqMan probes. The expression levels of three miRNAs (mmu-let-7b, mmu-miR-183, and mmu-miR-429) in CM and blastocysts were slightly upregulated in late third cleavage group. Our study suggested that the expression level of miRNAs could be altered with embryo quality, and miRNAs in CM may be used to predict miRNAs expression of embryos and developmental competence.

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

The anandamide signaling plays various roles in directing reproductive processes. Mouse embryos are shown to express high levels of CB1 receptor (CB1R). It has recently been shown that an analog of anandamide induces autophagy-mediated cell death through stimulation of ER stress response in glioma cells. Since adverse effects of high levels of anandamide agonists on embryo development and implantation are well known, we hypothesized that anandamide mediates an autophagic response in embryonic cells as in cancer cells via highly abundant CB1R on embryos. We tested this hypothesis by using a stable anandamide agonist, Methanandamide (MET) in three embryonic cell systems, i.e., mouse embryonic fibroblasts (MEF), trophoblast stem (TS) cells, and preimplantation embryos from mice. RT-PCR, immunofluorescence staining, and Western blot analysis were used to examine the effects of anandamide on autophagy in these systems. In MEF cells, the conversion of LCI to LCII was heightened by methanandamide (MET), and AM251, a selective CB1 antagonist partially reversed the effects of MET. Treating MEF cells with a high level of MET induces clustering of GFP-LC3, seen as large puncta throughout the cytoplasm. At 28 nM concentration, MET also weakly increased LC3II in TS cells. When MET was injected to day 4 pregnant mice, autophagy was increased in blastocysts in utero as demonstrated by the increased number of LC3 puncta. Formation of numerous autophagic vacuoles was also confirmed by electron microscopic observation. In conclusion, this work suggests that the anandamide-CB1 signaling pathway may be one inducer of autophagy in embryonic cells.

The aim of this study was to investigate the effect of glucose on embryonic development of mouse embryos. Two cell embryos were recovered from ICR female mice(3-4weeks) at 46~50 hrs after hCG 5 IU injection (mated just after hCG injection) and cultured in 50 DMEM droplets supplemented with nothing (control: n=46), glucose 0.5mM (Group A; n=46) or glucose 3.15 mM(Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Total blastocyst formation rates was lower (NS) in glucose groups (group A: 52.2% : B. 47.8%) than control group (60.9%). ZiB rates was the highest (P<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates were the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell number compared with the others (control: 39.2 ; group A: (45.6). The ICM proportion (% ICM of total cells) in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2 ; group B: 13.9%). This study shows that a low dose of glucose added to culture medium increases the ICM proportion of blastocysts in mice.

소 배반포로부터 배아주 (embryonic stem, ES) 유사세포를 분리하기 위해서는 영양외배엽 (trophectoderm, TE) 세포를 제거하는 것이 효과적이다. 따라서 본 실험은 효과적으로 TE를 제거하기 위한 calcium ionophore A23187 (CIPA) 처리조건을 확립하고, 분리해낸 ES 유사세포의 in vitro 다능성 (pluripotency)을 검증하고자 수행하였다. CIPA 농도 및 처리시간을 달리 하였을 때 50 M에서