PP2A-B55α, a regulatory subunit of PP2A plays an important roles in regulating cell proliferation and survival. However, the functions for PP2A-B55α in mouse early embryo development is not clear. The objective of present were to investigate the expression patterns and to explore its biological function during mouse early development. Thetranscripts of PP2A-B55α were detected at all developmental stages in mouse embryo and decreased during embryo development. Immunostaining revealed that PP2A-B55α was present in both the nucleus and cytoplasm in early cleavage stage embryos. In the late embryonic development, PP2A-B55α was predominantly located in the cytoplasm. Knockdown (KD) of PP2A-B55α using double strand RNA not affect the proportion of cleaved embryos, but resulted in significantly decreased development to blastocyst stage and reduced total cell number in blastocyst. KD PP2A-B55α is able to induce sustained DNA damage and reduced the transcripts of non-homologous end joining (NHEJ) or homologous recombination (HR) pathways relative genes in mouse early embryo. KD PP2A-B55αcaused apoptosis and increase the transcripts of pro-apoptotic genes in blastocyst. Furthermore, The KDPP2A-B55α showed significantly lower cell proliferating rates (from 5-Bromo-deoxyuridineassayresults) in blastocysts and to talareas of out growth potential was decreased. These observation provide novel in sight into PP2A-B55α expression patterns in mouse early embryos and down-regulation of PP2A-B55α negatively impacted blastocyst development, total cell number, DNA damage, apoptosis, and proliferation and post-hatchingevents.

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst (99.7 ± 12.4) compared to the post-thaw blastocyst (94.8 ± 15.1). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups (74.7 ± 14.6, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different (0.0 ± 0.0 vs. 1.9 ± 3.1, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group (5.4 ± 4.4) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles, in addition to recycling protein and ATP synthesis. Although programmed cell death (PCD) is very important during embryogenesis, the mechanism underlying the dynamic development during this process remains largely unknown. In order to obtain insights into autophagy and it's relation with apoptosis in early embryo development, we first evaluated LC3 gene expression levels in mouse embryos developing in vitro. qRT-PCR revealed high expression levels from 1- to 4 cell stage embryo, and then expression decreased during morula and blastocyst formation. Indirect immunocytochemistry showed protein synthesis of LC3 in these stage embryos. Introducing of autophagy inhibitor, 3-MA (2mM) significantly decreased both developmental rate (54.85±11.0%) and total cell number (n=71±8), but increased apoptosis rate (5.68± 1.9%) at the blastocyst. Real time RT-PCR confirmed reduced expression of selected autophagy related genes, including ULK1, Atg4A, B, C, D, Atg5, Atg8, Gabarap, Atg9A, B and Atg16L. Treatment of autophagy inducer, rapamycin (50 ng/㎖) increased both mRNA expression and protein synthesis of LC3 and apoptosis rate (16.11±3.42%), but decreased developmental rates (50.16±9.78) and total cell numbers (n=60±7) as compared to control developmental rate (70.74±12.9%), Total cell number (89.8±9) and apoptotic cell death (1.11±0.7%). These results suggest that autophagy is related with apoptosis in mouse embryo, which possibly give a role for early development.

세포의 대사과정에서 생성되는 활성산소종(reactive oxygen species : ROS)은 세포의 성숙과 발생 과정을 저해하며, 인간의 생식 수관에서 불임의 원인이 된다. 많은 세포생물학적 연구를 통해 ROS에 대한 세포 내의 보호 기작이 밝혀지고 있다. Activating transcription factor 4(ATF4)는 세포 내에서 산화적 스트레스를 비롯한 여러 스트레스 요인으로부터 세포를 보호하는 기작에 관여하는 중요한 인자로서, 스트레스

생쥐 초기 배아의 형태형성에 영향을 주는 세포질내 인자의 기원과 작용기작을 연구하기 위해 단백질 합성과 단백질 활성화 효소 (protein kinase)의 억제제를 처리한 배아의 세포질로 재조합된 배아에서 발생과 RNA합성, 단백질 인산화를 조사하였다. 단백질 합성 억제제인 cycloheximide (CHX)가 함유된 배양액에서 24시간 배양한 1-세포 배아의 제핵된 세포질을 두 개의 전핵을 모두 가진 절반의 1-세포 배아와 재조합한 P+P-CHX군의

인슐린 유사 성장 호르몬 1과 2 (IGF-1 & IGF-2)는 착상 전 초기배아 발생을 조절하는 중요한 요소이다. 생쥐 착상 전 초기배아에서 IGF-1의 역할에 관한 연구를 위해, IGF-1과 IGF-1 수용체의 전사물의 존재 여부를 난자와 착상 전 초기배아에서 조사하였다. 새로이 고안된 IGF-1 primer를 이용하여 난자에서 전사물을 검출하였다. 그리고, PCR 산물을 제한효소인 Msp I으로 절단하여 확인하였다. 이 실험에서 IGF-1과 IGF

이온 비의존적으로 작동하는 포도당 수송체 (glucose transporter 1, Glut1)는 생쥐 배아의 세포막을 경계로 포도당을 수송하는 주요통로이다. 성장인자 가운데 insulin-like growth factor-I (IGF-I)은 생쥐배아에서 포도당의 유입을 증가시키는 것으로 알려져있으나 이러한 효과가 IGF-I 의한 Glut1의 전사조절 효과에 기인한 것인지는 알려져 있지 않다. 본 연구는 포도당과 IGF-I 생쥐의 착상전 배아 발생과 G

Phospholipase C (PLC)는 다양한 세포주에서 세포내 신호전달에 중요한 역할을 한다고 알려져 있으나, 생쥐 난자성숙 과정과 착성전 배아발생 과정에서 PLC의 역할과 발현은 아직 연구된 바 없다. 본 연구에서는 난자성숙과 착상전 배아발생 과정에서 생쥐의 PLC β1과 γ1의 유전자 발현을 조사하기 위하여 한 개의 난자 혹은 배아에서 추출된 total RNA를 사용하여 경쟁적 RT-PCR 방법으로 mRNA를 정량하였다. PL