The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5106 cells/ml; 71.6%, 5.0 106/ml; 71.9%, l.0 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5 106 cells/mi; 43.6%, 5.0 106/ml; 46.8%. l.0 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.

거창지역의 쥬라기 화강암에서 발달하는 결과 평행한 압열인장강도(σt)의 특성을 분석하였다. 여섯 방 향의 결에 대한 평가는 미세균열의 길이와 위의 강도에 대한 파라미터의 값을 이용하여 수행하였다. 각 방향에 속하는 다섯 시험편의 강도값은 다섯 그룹으로 분류하였다. 이들 다섯 그룹의 강도값은 그룹 A < B < C < D < E의 순으로 증가한다. 위의 미세균열과 강도 사이의 밀접한 상관성을 도출하였다. 이 연구의 분석 결과는 다음과 같다. 첫째, 3개 결 사이의 강도의 변화 및 특성을 보여 주는 도면을 작성하였다. 위의 도면에 서는, 각 그룹에 속하는 여섯 방향의 강도값을 1번 결(리프트 1 및 리프트 2), 2번 결(그레인 1 및 그레인 2) 그리고 3번 결(하드웨이 1 및 하드웨이 2)의 순으로 배열하였다. 다섯 그룹의 강도분포선은 R1의 방향에 집중한다. 그래서 강도차(Δσt)를 지시하는 위의 다섯 선 사이의 폭은 R1 방향에서 가장 협소하다. 관련 도면으로부터, 각 결을 형성하는 두 방향 사이의 변화 특성을 도출하였다. 그레인 2(2)-시험편은 그레인 1(1)-시험편 에 비하여 보다 높은 강도값 그리고 보다 낮은 강도차값을 보여 준다. 이러한 현상은 시험편 H2(2)와 H1(1) 사이의 사례와 부합한다. 위의 시험편 (2)의 강도 특성은 시험편 (1)에 비하여 보다 낮은 미세균열의 밀도 그리고 하중 방향에 평행 배열하는 미세균열의 분포에 있어서의 보다 높은 균일도를 시사한다. 위의 도면에서는 각 그룹에 속하는 여섯 강도값이 증가하는 순으로 배열하였다. 그룹 D 및 E 양쪽에 속하는 시험편의 강도값은 R1 < R2 < G1 < H1 < G2 < H2의 순으로 나타난다. 따라서, 그룹 D 및 E의 강도값은 여섯 방향의 결의 평가를 위한 지시자값이 될 수 있다. 둘째, 경사각(θ)과 강도차 사이의 상관도를 작성하였다. 위의 두 파라미터의 값은 두 방향 사이를 연결하는 다섯 강도분포선을 통하여 획득하였다. 일번 면(그레인 1-하드웨이 1, R'), 2번 면(리프트 1-하드웨이 2, G') 및 3번 면(리프트 2-그레인 2, H')과 관련된 도면으로부터, 일차함수의 기울기값은 R'(0.391) < G'(0.470) < H'(0.485)의 순으로 증가한다. 3개 면 중에서, 3번 면과 관련된 도면은 다섯 그룹 사이의 가장 높은 분포밀도를 보여 준다. 1번 결(리프트 1-리프트 2, R), 2번 결(그레인 1-그레 인 2, G) 및 3번 결(하드웨이 1-하드웨이 2, H)과 관련된 도면으로부터, 일차함수의 기울기값은 R(0.407) < H(0.453) < G(0.460)의 순으로 증가한다. 3개 결 중에서, 일번 결과 관련된 도면은 하부 구간에 속하는 그룹의 가장 높은 빈도수를 보여 준다. 종합하면, 3개 면 · 3개 결 사이의 경사각의 분포 폭은 H' < G < R' < R < G' < H의 순으로 증가한다. 섯째, 미세균열의 길이와 관련된 파라미터 그리고 인장강도 사이의 상관성 분 석을 수행하였다. 이들 파라미터는 빈도수(N), 총 길이(Lt), 평균 길이(Lm), 중앙 길이(Lmed) 및 밀도(ρ)를 포 함할 수 있다. 위의 미세균열에 대한 개개 파라미터(X) 그리고 다섯 그룹에 해당하는 다섯 수준의 인장강도 (Y) 사이의 상관도를 작성하였다. 다섯 종류의 상관도로부터, 상관계수의 값(R2)은 다섯 수준의 강도와 함께 증가한다. 각 도면으로부터 도출한 다섯 상관계수의 평균값은 0.22(N) < 0.34(Lt) < 0.38(ρ) < 0.57(Lmed) < 0.58(Lm)의 순으로 증가한다. 넷째, 그룹 E에 해당하는 최대강도(X) 그리고 위의 다섯 파라미터(Y) 사이의 상 관도를 작성하였다. 관련 도면으로부터, 상관계수의 값은 0.61(N) < 0.81(Lt) < 0.87(ρ) < 0.93(Lm) < 0.96 (Lmed)의 순으로 증가한다. 가장 높은 상관성을 갖는 두 파라미터는 최대 강도와 함께 중앙 길이이다. 미세 균열과 강도 사이의 상기 상관성 분석을 통하여, 이 연구에서 도출한 결과물에 대한 신뢰성을 제고할 수 있다.

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.

In animal development, the mechanisms by which localized factors and organelles in egg cytoplasm were exactly distributed into each daughter cell are essential for formation of various cell types. During ascidian Halocynthia roretzi embryogenesis, ooplasmic mitochondria were mainly segregated into muscle and neural precursor cells. At the 32-cell stage, localized mitochondria in the B6.2 blastomeres were preferentially distributed into the B7.4 muscle precursors compared with the B7.3 mesenchyme/ notochord precursors. When the B6.2 blastomeres were isolated from the early 32-cell stage embryos and then allowed to divide 2 times of cell division, the resultant partial embryos showed symmetric distribution of mitochondria, and the partial embryos were composed of equal size cells. In normal development, cell fates of the B7.3 blastomere were correlated with the unequal cleavage of B6.2 lineage cells that normally occurs in the next two-cell division stages to produce a large B8.5 mesenchyme and a small B8.6 notochord cell. Mitochondria are distributed asymmetrically in both cells. When embryos were treated with FGF receptor inhibitor SU5402 and MEK inhibitor U0126 between the 32-cell and the early 64-cell stages, the resultant embryos showed equal cleavage pattern and symmetric distribution of mitochondria in daughter cells of the B6.2 blastomeres. However, blocking of Nodal and Notch signaling did not affect the cell division pattern and mitochondrial distribution in the B6.2 lineage blastomeres between the 32-cell and 110-cell stages. Therefore, it is likely that FGF/MEK signaling is involved in asymmetric distribution of mitochondria and unequal cleavage of the B6.2 lineage blastomeres in ascidian embryo.

Iris nertschinsk has been used generally as a decorative plant. However, it has been almost used as a medicine for therapy on various human diseases. In this study, we demonstrate the anti-tumor effect of Iris nertschinsk on human breast cancer cells. Firstly, we found that Iris nertschinsk dose-dependently induced cell death in human breast cancer cell lines, MCF7 and MDA-MB231. Moreover, phosphorylation of p53 was induced after Iris nertschinsk treatment in MCF7 cells, which has a functional p53, but not in MDA-MB231 cells, which has a dysfunctional p53. We next examined whether Iris nertschinsk induces caspase-dependent cell death. Caspase-7 was cleaved after Iris nertschinsk treatment in MCF7 cells. Interestingly, either caspase-3 or caspase-7 was cleaved in MDA-MB231 cells that p53 had been phosphorylated by Iris nertschinsk treatment, indicating that Iris nertschinsk induces apoptosis through the cleavage of caspase-3, -7 in human breast cancer cell lines, MCF7 and MDA-MB231, but related to the status of p53. Therefore, these results suggest that Iris nertschinsk could be used as a treatment for human breast cancer. This research is supported by National Institute of Agricultural Biotechnology research grant.

This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.