Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

This study was performed to evaluate whether vitrification method using ethyle glycol and eletron microscopic (EM) grid could be used far the cryopreservation of human oocytes in ART program. Surplus oocytes were obtained from consented IVF patients. These surplus human oocytes were frozen with our vitrification method, Oocytes were exposed to 1.5M ethylene glycol (EG) in DPBS far 2,5 minutes, followed by 5.5M EG plus 1.0M Sucrose in DPBS for 20 seconds. Then oocytes were transferred onto the EM grid and the grid was plunged into LN2 for storage. For thawing, oocytes containing EM grid were sequentially transferred in 1.0M, 0.5M, 0.25M, 0.125M and 0 M sucrose in DPBS solution at the intervals of 2.5 minutes. Thawed and survived oocytes were provided for ICSI. Embryos from vitrified oocytes were transferred to uterus of the patient on 4 to 5 days after ovulation in natural cycles of on 15 to 17 day of hormone replacement cycles. A total of 370 oocytes from 26 patients were thawed and 159 (43.0%) of them survived. One hundred thirty four oocytes (84.3%) were fertilized normally and 126 pre-embryos were transferred to 26 patients, resulting in 5 clinical pregnancies. The pregnancy rate per transfer was 19.2% and implantation rate was 4.0%. Among the five pregnant, 4 patients delivered 4 healthy babies and the one patient was 32-week ongoing pregnancy. From this results, vitrification using ethylene glycol as cryoprotectant and EM grid is a rapid and simple method that can be effectively applied for the cryopreservation of human oocytes in ART program.

A total of 92 unfertilized human oocytes were treated with ethanol (EtOH), calcium ionophore A23187 (CI) or electric pulse (EP) for activating pronuclear formation and subsequent development. In Experiment 1, there was a significant (P=0.0001) treatment effect on the activation of unfertilized oocytes. No spontaneous activation was occurred in the control, but activation treatments induced PN formation with various efficacy. More unfertilized oocytes (UFOs) were activated after EtOH or EP treatment than after CI treatment. EP was as effective (63.6 %) as EtOH, but fragmentation was observed in 43% of UFOs activated by EP. Proportion of UFOs that formed presumptive haploid PN (2 PNs+1 PB or 1 PN +2 PBs) was 33.3, 0 and 28.6% after EtOH, CI and EP treatments, respectively. In Experiment 2, a significant (P=0.0362) effect of immature oocytes (IOs) status on activation was fecund. IOs at the GVBD-MI oocytes had higher potential to form PN than those at the GV stage or with abnormal morphology (25 vs. 77.8%). The results of this study clearly demonstrated that the treatment of 10% ethanol for 5 min effectively induced the activation of UFOs. IOs could form pronucleus with high efficacy by ethanol treatment, as long as they grew beyond the GVBD stage.

미성숙의 Germinal Vesicle(GV 단계에서 성숙한 Metaphase II(MII) 단계가 되는 난자성숙 과정은 핵과 세포질의 성숙을 통해 이루어지며, 이를 통해 수정과 배 발달을 할 수 있는 능력을 갖게 된다. GV 난자는 prophase I 단계에 arrest 되어 있다가 meiosis 과정을 거쳐 성숙한 MII로 되는데 이를 조절하는 기작에 대해서는 거의 알려져 있지 않다. 따라서 본 연구는 미성숙 난자와 성숙 난자간의 유전자 발현의 차이

본 연구에서는 이러한 초기 난포 발달 과정 중 원시난포-1차 난포 변화과정 시기에 발현하는 유전자를 알아보고 자 수행하였다. 원시난포로만 이루어져 있는 생쥐의 생후 1일자 난소와 원시난포 및 1차 난포로만 이루어져 있는 5일자 난소의 RNA와 총 80개의 annealing control primer(ACP) primer를 사용하여 PCR을 수행하여 서로 다르게 발현하는 유전자 (differentially expressed genes; DEG) 41개를

성장을 멈추고 있는 원시난포(primordial follicle)에서 난포발달이 개시되어 1차난포(primary follicle)로 발달하는 조절기전은 잘 알려져 있지 않다. 이 초기 난포발달 과정에 관여하는 유전자를 알아내기 위해 suppression subtractive hybridization(SSH)을 사용하였다. 생후 1일과 5일째의 생쥐 난소로부터 얻은 cDNA로 forward와 reverse subtraction을 수행하여 각각 day1과