The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37/with hFF, Group II: 32/withGroup III: 37/without, Group IV:32 /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.91.9%. After 24 hours culture, sperm motility was 23.52.1% (Group I), 8.11.1% (Group II), 10.4 1.4% (Group III) and 4.00.8% (Group IV), respectively. After 48 hours, the motility had been changed as 322.3% (Group I), 14.31.7% (Group II), 5.3 1.4% (Group III) and 4.30.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37 was higher than those of group II at 32. But, sperm viability of group I cultured at 37 was lower than those of group II at 32 (54.44.1% vs. 59.43.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

본 연구에서는 한방의학에서 가장 중요한 약재중의 하나인 녹용을 정자의 처리에 있어 녹용약침액 상태로 배양액에 첨가 후 정자에 미치는 영향을 운동성 측면에서 비교해 보았다. 정상정액군에서는 Ham's F-10 배양액에 0.3% BSA가 첨가된 배양액과 Ham's F-10 배양액에 0.3% BSA와 녹용약 침액이 같이 첨가된 배양액에서 24시간 배양한 정자의 운동성이 높았으며, 48시간동안 배양 후의 운동성은 BSh와 녹용약침액이 같이 들어간 경우에서 다른

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

This experiment was undertaken to investigate the effect of administration of Agyoju or Mokhyang on the maintenance of pregnancy, delivery and sex ratio in the mice in different gestation periods. Adult female mice were administered orally at three different periods, from ovulation to implantation (Exp.1), from post-implantation to organogenesis (Exp.2), and from fetal growth to parturition (Exp.3). In Experiment 1, number of fetus implanted and mean body weight were not significantly different. However, the delivery of male offspring was significantly increased in Agyoju and Mokhyang administrated groups than control group. In Experiment 2, the number of fetuses implanted, live offsprins and their body weight at delivery were significantly increased in the Agyoju administered group than Mokhyang and control groups. In Experiment 3, the number of live offspring and sex ratio were not different in both treatments and control group. However, mean body weight at delivery was significantly increased in both treatment groups than that of control group. These results suggest that 1) Agyoju and Mokhyang have beneficial effects in maintenance of pregnacy, and that 2) The action of unknown component(s) in Agyoju may be related to selection of male spermatozoa for fertilization in vivo, and that 3) the administration of Agyju of Mojhyang during mid-and late-pregnance periods were shown the increment of body weight of live offspring without decrease of litter size.

Txnip는 Thioredoxin (Trx)과 결합하여 그 기능을 억제함으로써 세포 내의 산화환원 환경을 조절하고, Txnip를 과발현시키면 Trx와는 독립적으로 glucose uptake와 lactate output을 억제한다고 알려져 있지만, 난자 내에서의 역할은 아직 밝혀진 바가 없다. 따라서 본 연구는 RNA interference (RNAi)를 이용하여 난자 내의 Txnip의 발현을 효과적으로 억제하고, Txnip가 생쥐 난자 성숙에 미치는 영향을 알아보고자 수행하였다. ICR 생쥐에 PMSG를 주사하고 44시간 후에 cumulus-oocyte complex(COC)형태의 GV 난자를 채취하였다. Cumulus cell을 물리적으로 제거한 후 Txnip dsRNA를 GV난자의 세포질 내에 미세 주입하여 Txnip RNAi를 수행하였다. 0.2mM IBMX가 첨가된 M16 배양액에서 24시간 배양 후 plain M16 배양액에서 16시간 배양하면서 난자의 성숙율과 표현형을 관찰하고, 염색체와 spindle의 모양은 immunofluorescence staining으로 동시에 관찰하였다. Time-Lapse imaging으로 cytoplasmic streaming, polar body emission 등의 표현형 및 시간차 등을 비교하였다. Txnip RNAi 결과, Txnip RNAi 된 난자는 MI 단계에서 정지하는 표현형을 보였고, 세포질이 응축되고, 난자 중심부에 spindle과 염색체가 분리되지 않은 상태로 뭉쳐서 존재함을 관찰하였다. IBMX 배양액 안에서 Txnip knockdown이 시작될 때 이미 난자의 세포질 안에 과립형성이 증가하고 cytoplasmic streaming이 어려워지는 것을 관찰하였으며, 세포질의 rigidity가 증가하는 것을 관찰하였다. Txnip은 lactate의 생산을 억제하기 때문에 Txnip 발현을 억제시키면 반대로 세포 내 lactate 생산이 증가된다고 알려져 있어, 본 연구진은 lactate의 농도를 높인 배양액에서 난자를 배양하였다. 이 때 시간이 지날수록 난자의 중심부에 과립이 형성되는 현상 즉, Txnip RNAi를 수행한 난자에서 나타나는 현상과 유사한 표현형을 확인하였다. 따라서 Txnip가 난자 성숙과정에서 lactate의 생성과 관련하여 작용할 것이라는 가설을 세우고, 따라서 Txnip의 감소로 증가된 lactate로 인해 glycogen granule이 과형성되고, 그 결과 cytoplasmic streaming이 현저히 줄어들면서 spindle과 염색체의 이동이 방해되는 것으로 추측된다. 이번 연구를 통해서 생쥐 난자의 포도당 대사와 Txnip의 연관성을 알 수 있었고, 이는 생쥐 난자의 대사적 조절기전과 난자 성숙과의 연관 관계를 규명하는데 중요한 역할을 할 것이라고 사료된다.

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

본 연구는 생쥐의 난소 및 정소 조직에서 발달 단계에 따라 나타나는 일주기성 clock유전자의 발현과 단백질의 발현 양상을 알아보고자 하였다. 생쥐의 난소 및 정소에서 일주기성 변화와 연관된 유전자(Period1(Per1), Period2(Per2), Period3(Per3), Cryptochromel(Cry1), Cryptochrome2 (Cry2), Clock, Bmall)와 시교차 상핵에서 분비되어 표적 조직 또는 기관으로 전달되는 물질로 알려진