This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.

Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In this report, we examined the cytotoxic effects of chitosan nanoparticles on mouse embryos at the blastocyst stage and in vivo implantation by embryo transfer. Blastocysts treated with 250 nm chitosan nanoparticles exhibited significantly increased apoptosis and a corresponding decrease in total cell number, which was concentration‐dependent. Moreover, the TUNEL positive signal in the embryos exposed to chitosan nanoparticles showed an increased of the ICM and the implantation success rate was lower than that of their control counterparts. Our results collectively indicate that in vitro exposure to chitosan nanoparticles induces apoptosis and retards implantation development after transfer to host mice. The results collectively show that chitosan nanoparticles have the potential to induce embryo cytotoxicity. Further studies are required to establish effective protection strategies against the cytotoxic effects of these nanoparticles.

The organotypic raft culture model has been shown to be a useful method for examining the effects of biochemical manipulation on various epithelial cell types, using in vitro conditions that simulate the in vivo environment of the tissue of origin. To investigate this method as a model for photodynamic therapy (PDT), we cultures the oral squamous carcinoma cell line YD-10B on fibroblast-containing collagen gels at the air-liquid interface and assessed the efficacy of PDT using a photosensitiser 5-ALA-hexenyl ester. PDT effect on YD-10B organotypic raft cultures after twenty- four hours was determined. The number of residual cells was significantly reduced in comparison to control at all four different conditions. PCNA immunostaining and TUNEL assay revealed that PDT inhibited cell proliferation and increased apoptosis. These findings suggest that the organotypic raft culture model provides an effective and rapid laboratory method of investigating strategies for PDT on oral precancerous lesions and squamous cell carcinomas

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes (156.0+-68.2 vs 134.7+-54.8)). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total (160.0+-69.0 vs 156.7+-68.4) and apoptotic cells (14.0+-10.4 vs 11.8+-6.4)) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

본 연구에서는 hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 rtTA2SM2 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이된 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다.

본 연구는 안과에서 주로 사용되는 생체 염색시약으로 Fluorescein, Rose Bengal, Erythrosin B, Lissanúne Green B 올 인간의 결박세포인 Clone1-5c-4 cell line올 배 양하여 cytotoxicity릎 조사하기 위하여 1%, 0.25% 의 농도보 5분， 15분 동안 처리하 여 MTT assay, SRB assay를 실시하였다. 각 염색사약의 세포독성올 바교 검정한 결과， Rose Bengal과 Lissamine Green B에서 서I 포 증식 저해용이 30% 이상 나타났 고， 회복효과를 보기 위하여 시약 제거 후 1 일 배양하여 Ml‘T. SRB assay를 실시한 결과는 Rose Bengal에서 거의 비슷한 수준이었으나 Lissamine Green B 에서는 더 증 가하였다. 또한 5, 15분 동안 시약처리 직 후 subculture활 실시한 후 세포의 부착과 중식정도를 관찰하였고， 회복효과를 보기 위해 3 잊 후에 세포의 형태를 관찰한 결과， Rose Bengal은 거의 차이 가 없었으나 Fluorescein 에서는 회복효과를 보였다.0.25% trypsin을 처리하여 세포에 영향을 주어 세포의 인위적인 손상올 형태학적으로 비교 한 염색상 세포 저해 정도는 Rose Bengal 이 가장 높-았고， Fluorescein 이 제일 낮았 다. 이러한 결과로 볼 때 일부 생체 염색시약이 인간의 결막세포인 Clone1•5c -4 세 포증식을 저해하는 것으로 사료된다.

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.