To investigate the effect of co-transfer of trophoblastic vesicle (TV) with frozen-thawed in vitro Produced (IVP) bovine embryo on pregnancy rate, IVP blastocysts were transferred to synchronized recipients. Elongated blastocysts were recovered at Day 13 to 15, and dissected more than 4 pieces to removed the embryonic disc. Throphoblastic fragments were cultured for 48 hours to make throphoblastic vesicles (TVs). TVs were cryopreserved in ethylene glycol or vitrification solution and frozen-thawed TVs were co-transferred to recipients with frozen-thawed IVP embryos. 1 The recovery rate of elongated blastocyst on Day 13 to 15 was 22.5% (18/80) and the size of recovered elongated blastocysts was 0.2∼5.0mm. 2. Eighteen elongated blastocysts were dissected into 88 pieces and 61.4% of those pieces were formed to TV (54/88) 3. The viability of frozen-thawed TV in ethylene glycol was higher than in vitrified solution (92.8% vs. 68.8%) 4. The pregnancy rate in co-transfer with frozen-thawed TV and IVP blastocyst was better than transfer only IVP blastocysts (50.0% vs. 23.1%).

본 논문은 인공 진피와 조직공학용 scaffold로 이용하기 위해 다공성 membrane로서 gelatin-based sponge의 효율성을 연구하였다. 불용성의 다공성 membrane은 1-ethyl-(3-3dimethylaminopropyl)carbodiimide(EDC)로 가교하여 제조하였다. Fourier-transformed infrared (FT-IR) spectroscopy, scanning electron microscopy(SEM) 그리고 Instron analysis로 다공성 membrane의 특성을 조사하였다. 다공성 membrane은 용적당 큰 표면적을 제공하는 micro porous한 구조를 가지고 있다. Gelatin/hyaluronic acid (HA) membrane의 공경크기는 40~200μm이다. HA의 첨가는 다공성 membrane의 기계적 강도와 세포부착능력에 영향을 미쳤다. Gelatin/HA 다공성 membrane의 압축강도는 collagen과 비슷하며, 세포배양과 인공진피 transplantation에 있어서의 충분한 기계적 강도를 가지고 있다. Fibroblasts를 함유한 진피기질을 제조하기 위해 직경 8mm의 다공성 membran에 4×10(sup)5cells/membrane의 세포밀도로 fibroblast를 배양하였다. GH91 porous membrane에서의 fibroblast 부착성은 GH55 porous membrane에서보다 우수하였다. 삼차원 구조의 gelatin/HA membrane matrix에서의 fibroblast의 배양은 생체내 조건과 유사한 생리적 환경을 제공하였다.

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 /mL), and higher rate of encleation was obtained at 7.5 and 15 /mL than at /mL. In Experiment 2, oocytes enucleated in 7.5 /mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 ) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 or 90 , but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

The objective of this study was to identify a follicular fluid ingredient inhibiting the cumulus oocyte complex (COC) expansion. Thus, follicular fluid or liquid chromatographic fractions of follicular fluid was supplemented in COC culture medium. And COCs were incubated for 48 hours to investigate about cumulus expansion and also the first polar body extrusion. The results obtained were as follows; 1. The fluid of medium follicle significantly inhibited the COC expansion. 2. The fluid of large follicle inhibited the COC expansion. 3. Follicular fluid showed six major fractions at retention volumes (RVs) 1.83, 1.91, 2.15, 2.34, 2.53 and 2.74 ml after separation with Superose 12 column. Of the major fractions, fractions RV2.15, RV2.34, RV2.53 and RV2.74 inhibited both COC expansion and polar body extrusion. Especially, fractions of RV2.15 and RV2.53 significantly inhibited COC expansion, oocyte denudation and polar body extrusion. In conclusion, porcine follicular fluid contained a COC expansion inhibiting ingredient (CEI) that may be contained largely in fractions RV2.15 and RV2.53. And CEI may inhibit oocyte maturation by inhibition of oocyte denudation and extrusion of the first polar body.