Importance of the in vitro model of tissues or organs is now evident in tissue engineering and cell biology research. Till now, two-dimensional culture systems have been using for in vitro cell culture, and have contributed to cell function studies despite their limitations. Three-dimensional (3D) culture has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, unlike conventional monolayer cell culture. In our laboratory, we are developing 3D culture systems of bovine endometrial cells as a tool for the analysis of uterine endometrial functions. Among them, this lecture introduces spheroid culture and Matrigel culture. 1. Spheroid culture; Spheroids are a spherical mass composed of cells and extracellular matrices (ECMs). We have regenerated multicellular spheroids composed of bovine endometrial stromal and epithelial cells using ascorbate (1). Expression of MMPs, which are key enzymes for the tissue remodeling of the endometrium, were analyzed using the spheroid. E2, P4 and type-I IFN did not affect the gene expression of MMPs in the spheroid. However, treatment of type-I IFN increased the clearance of MMPs in the supernatant. These results suggest that IFN indirectly regulates endometrial tissue remodeling through clearance of MMPs. 2. Matrigel culture; It is reported that cells form lumens automatically by culturing cells in Matrigel (2). Matrigel is a solubilized basement membrane extracted derived from EHS mouse sarcoma cells. The bovine endometrial epithelial cells cultured in 15% Matrigel formed a circular or elliptical gland-like structure. Gene expressions of glandular epithelial specific factors (FOXA2, SERPINA14 and GRP) were significantly high in the Matrigel, compared to the monolayer cultured cells, except FOXA2. Further, SERPINA14 expression was affected by neither P4 nor IFN. However, when epithelial cells in Matrigel were co-culture with stromal cells, SERPINA14 expression increased significantly in the treatment of both P4 and IFN. These results suggest that bovine endometrial epithelial cells cultured in Matrigel show properties similar to the glandular epithelial cells in vivo, and regulated by the factors produced by the stromal cells. Finally, by using these 3D culture systems, it becomes possible to clarify not only factors regulating embryo elongation and implantation but also regulation of their expression. It will be able to reveal the mechanism of the embryo elongation and implantation to contribute to the improvement of the embryo transplantation technique. (1) Yamauchi N, Yamada O, Takahashi T, Imai K, Sato T, Ito A, Hashizume K. A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate. Placenta. 2003; 24(2-3):258-69. (2) Eritja N, Llobet D, Domingo M, Santacana M, Yeramian A, Matias-Guiu X, Dolcet X. A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis. Am J Pathol 2010; 176:2722-2731.

The aim of this study was to investigate change of plasminogen activators (PAs) and their inhibitors (PAIs) mRNA and protein expression level by heat stress in porcine endometrial cells. The endometrial epithelial cells were isolated from endometrial epithelium in porcine uterus and cultured in different temperature conditions (38.5 and 41.5℃) for 24 h. Expression of urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor-1 (PAI-1) and -2 (PAI-2) mRNA in epithelial cells were analyzed using reverse transcription-PCR and protein levels were measured by immunofluorescence. In result, mRNA expression of uPA, tPA, PAI-1 and PAI-2 were decreased in 41.5℃ than 38.5℃ culture condition, however, significant differences were no detected. uPA, tPA and PAI-2 protein were mainly expressed in nucleus, whereas PAI-1 was distributed in cytoplasm and nucleus. uPA and tPA protein levels were increased by heat stress treatment and significant difference was only detected in tPA level (p<0.05). In contrast, two types of PAIs protein level were decreased in 41.5℃ cultured group compared with 38.5℃ group. In present study, tPA protein expression was upregulated by heat stress in porcine endometrial cells. This result suggest that change of tPA by heat stress may be related to blood flow into uterus and intrauterine microenvironments, and could directly and indirectly influence to reproductive performance in pigs.

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.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

The aim of this study was to establish a three dimensional (3D) culture system of endometrial cells and to examine the plasminogen activators (PAs) activity in porcine uterine. The 3D culture system in porcine endometrial cells was composed to mixture 3D gel, stromal cells and epithelial cells. The 3D culture system was used to identify normal structure search as uterine tissue and PAs expression in this study. In results, porcine endometrium epithelial cells forming a top monolayer and endometrium stromal cells developed as fibroblast-like within 3D matrix scaffold. Expression of urokinase-type PA (uPA) and tissue-type PA (tPA) were observed during the 3D culture using immunofluorescence. PA activity in 3D-cultured endometrial cells was no significant difference between the tissue type, but 2D culture system were significantly lower than in 3D-cultured endometrial cells (P<0.05). Therefore, basic system and functional aspect of 3D culture could be established with similar system of endometrium tissue. We suggest that this study was assumed applicable as baseline data to investigate mechanism between porcine uterus cells in vitro.

The purpose of the present study was to investigate the effect of IFN- on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and concentration of progesterone (P4) in endometrial cells. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses of IFN- (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin and levels in the culture media were analyzed by enzyme immunoassays and total RNA was extracted from the cells for RT-PCR. P4 concentrations of blood samples were assayed by chemiluminescent immuno assays system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN- (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN- group (p<0.05). Although IFN- did not affect and production in epithelial cells, it decreased and production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN- (1 ug/ml). The results indicate that PG production was mediated by COX-2 expression in stromal cells but it was not affected in epithelial cells and this suggest that treatment of IFN- could improve the implantation environment of uterine by maintenance of high P4 concentration.

임신의 성립 및 유지에 중요한 역할을 하는 자궁내막세포에 통로의 존재를 확인하기 위하여 본 연구를 수행하였다. 통로는 일반적으로 중추신경계에 풍부하게 존재하면서 세포의 안정막 전압을 유지시킨다. 역전사 중합 효소 중합 반응과 면역 세포 화학 염색 방법을 이용하여 자궁내막세포에 존재하는 통로를 조사한 결과, TASK-1, TASK-3, TREK-1, TREK-2 및 TRAAK의 발현이 확인되었다. TASK-3와 TREK-1은 핵을 포함한 세포 전역에 발현

This study aimed to investigate changes in the activity and mRNA expression of plasminogen activators (PAs) induced by 17β-estradiol (E₂), human chorionic gonadotropin (hCG), and interleukin-1β (IL-1β) in porcine endometrial cells. Endometrial cells were isolated from the epithelium and cultured to 80% confluence. They were then treated for 24 h with E₂ (0.2, 2, 20, and 200 ng/mL), IL-1β (0.1, 1, 10, and 100 ng/mL), and hCG (0.5, 1, 1.5 and 2 IU/mL). mRNA expressions of urokinase-type (uPA) and tissue-type (tPA) PAs were analyzed using reverse transcription PCR, and activities were measured using a PA activity assay. mRNA expressions of uPA and tPA increased with E₂ treatment; however, this was not significant. Similarly, treatment with hCG did not influence the mRNA expressions of PAs. Interestingly, treatment with 0.1 ng/mL IL-1β significantly reduced the mRNA expression of uPA, but did not affect that of tPA. Treatment with 2, 20, and 200 ng/mL E₂ increased PA activity compared with the control group; treatment with 0.1 and 1 ng/mL IL-1β significantly increased PA activity compared with the other IL-1β treatment groups, whereas treatment with 10 and 100 ng/mL IL-1β decreased. Treatment with 2 IU/mL hCG increased PA activity compared with the other treatment groups, although there were no significant differences between the hCG and control groups. In conclusion, the activity and mRNA expression of PAs were differently regulat-ed by the hormone/cytokine and its concentration in porcine endometrial cells. Therefore, understanding PA regulatory mechanisms may help to improve the reproductive potential of domestic animals.

포배의 착상은 구조 및 기능적으로 준비된 자궁내막의 체계적 반응에 의하여 진행되는데, 자궁 내막에서 엄격히 통제된 급격한 세포의 증식과 분화가 진행된다. 구강 상피세포암의 억제자로 알려진 Doc-1이 자궁에서 스테로이드 호르몬에 의하여 발현되며 세포 증식을 조절할 것으로 추정되어 왔으나, 그 정확한 발현 변화에 대한 이해가 불충분하였다. 따라서 본 연구에서는 체외에서 탈락막 반응 모델 확립을 통하여 세포의 증식과 분화 진행과정에서 Doc-1의 발현 조절