In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

The purpose of this experiment was to determine the effects of thiol compounds, -mercaptoethanol(-ME) and cystearrone with buffalo rat liver cell(BRLC) co-culture on the development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro inaturation(IVM) and in vitro fertilization(IVF). Bovine IVM /IVF embryos developed to 2~8 cell stage were co-cultured with BRLC in GRlaa with or without thiol compounds. The developmental rate beyond morulae stage in CRlaa containing 0, 10,25 and 50M -ME with BRLG were 63.0, 74.0, 72.3 and 77.1%, respectively. And the developmental rate with 0, 25, 50 and 75M cystearnine with BRLC were 69.6, 77.6, 81.0 and 76.8%, respectively. The developmental rate beyond morulae stage of GRlaa containing thiol compound with BRLG group was higher than that of control group. The intracellular GSH concentrations of blastocysts cultured for 5 days in GRlaa containing 0 and 50M -ME or cysteamine with BRLG were 81.2 and 86.4, 83.2 and 84.2pM, respectively. The intracellular GSH concentrations of blastocysts in GRlaa containing thiol compounds with BRLG was slightly higher than that of control group The cell numbers of blastocysts were not difference in all experimental groups. These results indicate that thiol compounds with BRLG co-culture was increased the percentage of developed into morulae and blastocysts, and intracellular GSII concentrations of blastocysts embryos.

The objective of this study was to investigate the effects of thiol compounds with bovine oviduct epithlial crlls(BOEC) co culture on development and intracellular glutathione(GSH) concentrations of bovine embryos derived from IVM /IVF oocytes. In experiment 1 and 2, embryos developed to 2~8 cell stage after in vitro fertilization were co-cultured with BOEC in CRaa with or without -mercaptoethanol(-ME) and cysteamine. The percentage of embryos that developed to morulae and blastocysts in 0,10, 25 and 5OM -ME with BOEC was 48.1, 64.0, 72.9 and 75.9%, respectively. Twenty-five and 5OM -ME groups were significantly higher than in 0 and 1OM - -ME groups(PM cysteamine with BOEC was 50.0, 53.2, 72.0 and 66.7%, respectively. Fifty M cysteamine group was significantly higher than any other groups (Paa with 0 and 5OM -ME or cysteamine were 68.5, 77.8, 78.7 and 80.0pM, respectively. Fifty M -ME group was significantly higher than that of control(P<0.05), but cysteamine group was not. Cell numbers of blastocysts were not difference in all experimental groups. These experiments indicate that -ME and cysteamine with BOEC co-culture can affect the development and intracellular GSH concentrations of bovine embryos produced by IVM /IVF docytes.

This study was conducted to investigate the effects of preincubation time, concentration and exposure time of sperm on in vitro fertilization of porcine follicular oocytes rnatured in in vitro. The results obtained are as follows ; 1. Effect of preincuhation time for porcine sperm capacitation on in vitro fertilization in medium with heparin was investigated. Normal fertilization rate was highest in 15 min(26.4%). However, there were no significant differences among preincuhation times of 5~90 min, 2. Normal fertilization rates of sperm concentrations were 17.0~26.5%, and normal fertilization rate from l l05cell /ml concentration was also higher than those of other sperm concentration. 3. Normal fertilization rates of sperm exposure time of 4, 8, 12, 16 and 20 hours were 6.1, 20.8, 27.8, 25.0 and 26.7%, respectively. Normal fertilization rate from sperm exposure time of 12 hours was also higher than that of other sperm exposure times.

1. 본 연구에서는 공동배양 배지에 Agrobacterium 성장 억제물질인 silver nitrate를 첨가하고 변온과 여과지처리를 추가하여 공동배양 기간을 7일로 늘였으며, 또한 항산화 물질 3종을 공동배양 배지에 첨가하여 세포의 oxidative burst를 최소화함으로써 벼 형질전환효율을 높일 수 있었다. 또한 이 방법을 적용하여 형질전환이 어려운 품종을 대상으로도 형질전환 식물체를 작성할 수 있었다. 2. 벼 형질전환체의 70%에서 도입유전자