• 생명과학회지
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• 제33권1호
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• pp.102-113
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• 2023

본 총설에서는 지난 수년 동안 자궁내막 염증 관련 새롭게 밝혀진 에스트로겐과 프로게스테론 수용체의 기능 중 지엽적 에스트로겐의 합성, 특이적 에스트로겐 수용체의 조절, 프로게스테론 저항성 그리고 스테로이드 호르몬의 작용에 의한 자궁내막 조직세포의 염증반응, 분화 및 생존에 대한 세포 및 분자적 조절기전들을 고찰한다. 자궁내막 조직 기질세포의 비정상적인 후성유전체적 변화는 자궁내막증의 발병과 진행에 중요한 요인으로 작용한다. 특히, 에스트로겐 수용체 유전자들의 차별적 메틸화는 기질세포내 ERα로부터 ERβ로의 발현 우세도 전환을 유도하여, ERβ-매개 염증반응, 프로게스테론 저항성 및 레티노이드 합성장애 등의 비정상적인 에스트로겐 반응을 초래한다. 이 기질세포는 또한 PGE2 및 SF-1 매개에 의한 스테로이드 합성효소의 발현유도를 통하여 지엽적 에스트로겐의 생성을 촉진하며, 증가된 에스트라디올은 다시 ERβ에 피드백으로 작용하여 COX-2 촉진을 통한 염증반응의 악순환을 야기한다. 높은 ERβ의 발현은 중간엽 줄기세포의 염색질 구조변화릉 야기하여 프로게스테론 저항성을 획득하고, 이는 반복적 생리에 따른 지속적 노출로 자궁내막 조직의 염증을 형성하며, 이후에는 ERβ-매개 에스트로겐과 TNF-α 및 TGF-β1을 포함한 염증 유발 인자들이 작용하여 염증 조직세포의 부착, 혈관생성 및 생존과 기질세포의 분화조절장애를 유도한다. 따라서, 생리주기의 역동적인 호르몬 변화와 이에 따르는 자궁내막 조직의 핵수용체 신호전달 조절기전에 대한 구체적인 이해는 정상적인 생식기능을 유지하면서 자궁내막증과 같은 비정상적 염증질환을 치료하기 위한 새로운 안목을 제공할 수 있을 것으로 기대된다.

• Clinical and Experimental Reproductive Medicine
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• 제30권4호
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• pp.317-324
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• 2003

목 적: 본 실험의 목적은 자궁내막세포를 분리 및 배양법 확립과 함께 불멸화 시키는 것이다. 방 법: 자궁내막에서 상피세포(epithelial cells)와 기질세포(stromal cells)의 분리는 Satyawaroop 등(1979)의 방법에 기초를 두었다. 자궁내막에서 상피세포와 기질세포의 순수 분리도를 확인하고, 불멸화된 기질세포에서 SV40 large T antigen을 확인하기 위하여 면역형광 염색(immunocytochemistry)과 Western blot 기법을 이용하였다. 정상 기질세포의 경우 subconfluence (60%) 상태에서 transfection을 진행하였다. 순수 분리된 plasmid DNA와 Qiagen 사의 superfect를 이용하여 transfection을 실시하였다. 결 과: 본 연구에서 우리는 두 가지 형태의 자궁내막 세포의 분리 및 배양에 성공하였다. 상피세포는 다면체의 형태를 띠며, 선(grandular)조직의 조각으로부터 나선형으로 자란다.기질 세포는 길쭉한 형태를 띠며, 상피세포에 비해 오래 살고, 빠르게 증식하여 나란한 형태로 배열된 세포 다발(cell bundle)을 형성한다. 이렇게 분리된 세포들은 95%의 균질성을 보였으며, 면역형광염색과 western blot을 통해 확인 하였다. 한편 SV40(Simian Virus 40) large T 항원을 암호화 하고 있는 염기 서열을 포함한 플라스미드 벡터로 안정적인 트랜스펙션을 시킴으로써 불멸화 된 자궁내막의 기질 세포주를 확립하였다. 불멸화 된 세포는 그 세포가 유래한 정상의 세포와 동일한 표현형을 가지고 있었다. 결 론: 본 연구에서, 우리는 자궁내막에서 상피세포(epithelial cells)과 기질세포(stromal cells)를 분리하여 배양법을 확립하였다. 동시에 SV40 large T antigen을 이용하여 불멸화된 세포주를 확립하였다. 이렇게 확립된 세포주는 자궁의 생리작용 연구 및 자궁내막증(Endometriosis)과 자궁암(Endometrial cancer) 등과 같은 여러 자궁관련 질병 연구에 많은 도움이 될 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제23권3호
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• pp.263-275
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• 2019

Based on our preliminary results, we examined the possible role of low-dose and chronic-exposing of the chemicals those are known as endocrine disrupting chemical (EDC), on the proliferation of uterine endometrium and the localization of steroid receptors. Immunohistochemical or immunofluorochemical methodology were employed to evaluate the localization of antigen identified by monoclonal antibody Ki 67 protein (MKI67), estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), and progesterone receptor (PGR). In $133{\mu}g/L$ and $1,330{\mu}g/L$ di(2-ethylhexyl) phthalate (DEHP) and $50{\mu}g/L$ nonylphenol (NP) groups, the ratio of MKI67 positive stromal cells was significantly increased but not in $500{\mu}g/L$ NP group. The ratios of MKI67 positive glandular and luminal epithelial cells were also changed by the chronic administration of NP and DEHP in tissue with dose specific manner. ESR1 signals were localized in nucleus in glandular and luminal epithelia of control group but its localization was mainly in cytoplasm in DEHP and NP administered groups. On the other hand, it was decreased at nucleus of stromal cells in $1,330{\mu}g/L$ DEHP group. The colocalization patterns of these nuclear receptors were also modified by the administration of these chemicals. Such a tissue specific and dose specific localization of ESR2 and PGR were detected as ESR1 in all the uterine endometrial tissues. These results show that the chronic lows-dose exposing of NP or DEHP modify the localization and colocalization of ESRs and PGR, and of the proliferation patterns of the endometrial tissues.

• Clinical and Experimental Reproductive Medicine
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• 제49권4호
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• pp.259-269
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• 2022

Objective: Animal-free scaffolds have emerged as a potential foundation for consistent, chemically defined, and low-cost materials. Because of its good potential for high biocompatibility with reproductive tissues and well-characterized scaffold design, we investigated whether polyglycolic acid (PGA) could be used as an animal-free scaffold instead of natural fibrin-agarose, which has been used successfully for three-dimensional human endometrial cell culture. Methods: Isolated primary endometrial cells was cultured on fibrin-agarose and PGA polymers and evaluated various design parameters, such as scaffold porosity and mean fiber diameter. Cytotoxicity, scanning electron microscopy (SEM), and immunostaining experiments were conducted to examine cell activity on fabricated scaffolds. Results: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and SEM results showed that endometrial cells grew and proliferated on both scaffolds. Immunostaining showed cytokeratin and vimentin expression in seeded cells after 7 days of culture. On both scaffolds, an epithelial arrangement of cultured cells was found on the top layer and stromal arrangement matrix on the bottom layer of the scaffolds. Therefore, fibrin-agarose and PGA scaffolds successfully mimicked the human endometrium in a way suitable for in vitro analysis. Conclusion: Both fibrin-agarose and PGA scaffolds could be used to simulate endometrial structures. However, because of environmental and ethical concerns and the low cost of synthetic polymers, we recommend using PGA as a synthetic polymer for scaffolding in research instead of natural biomaterials.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.147-152
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• 2009

Lysophosphatidic acid (LPA), a simple phospholipid-derived mediator implicated in diverse biological actions, acts through the specific G-protein coupled receptors, LPA receptor (LPAR) $1{\sim}5$. Our previous study showed that LPAR3 is expressed in the uterine endometrium in a cell type- and stage-specific manner and LPA via LPAR3 increases PTGS2 expression in the uterine endometrium during the period of implantation. Although LPAR3 is considered to be predominant LPA receptor in the uterine endometrium, other LPA receptors might playa role to mediate LPA functions in the uterine endometrium during pregnancy. Among LPARs, we investigated expression of LPAR1 during the estrous cycle and pregnancy in this study. Uterine endometrial tissue samples were collected from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Northern blot analysis determined that LPAR1 mRNA was constitutively expressed in the uterine endometrial tissues during the estrous cycle and pregnancy of all stages. Analysis by immunoblotting revealed that LPAR1 proteins were present in the porcine uterine endometrium during the estrous cycle and pregnancy. Immunohistochemical experiments demonstrated that LP AR1 protein was localized to endometrial epithelium and stromal cell, specifically to nuclei of these cell types. Results in this study show that LPAR1 is constitutively expressed in the uterine endometrium during the estrous cycle and pregnancy. These results suggest that LPA via LPAR1 may playa role in the uterine endometrial function throughout pregnancy in pigs.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.307-311
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• 2006

The purpose of this study was to determine effects of oxytocin and $interleukin-1{\alpha}$ on in vitro development of bovine embryo cultured with endometrial epithelial and stromal cells isolated from bovine uterus. The expressions of COX-2 mRNA in bovine endometrium were also studied. When embryos were cultured with epithelial cells, the rate of blastocysts was significantly (p<0.05) higher in embryos treated with oxytocin than that of control group. The rate of hatched blastocysts was also significantly (p<0.05) higher in embryos treated with oxytocin than those of two control groups. On the other hand, when the embryos were cultured with stromal cells, the rate of blastocysts were significantly (p<0.05) higher than those of groups treated with $IL-1{\alpha}$, oxytocin and control with stromal cells than that of control group without stromal cells. The rate of blastocysts hatched were also significantly (p<0.05) higher in group treated with $IL-1{\alpha}$ than those of control group without stromal cells and oxytocin group. In another experiment, COX-2 gene was expressed in embryo group treated with oxytocin during the co-culture of embryos with epithelial cells. In contrast, COX-2 mRNA was expressed in group treated with $IL-1{\alpha}$ when the embryos were cultured with stromal cell. This result shows that oxytocin and $IL-1{\alpha}$ were stimulate embryo development in vitro when embryos were cultured with epithelial and stromal cells, and can affect the development of bovine embryos in the uterus.

• 한국수정란이식학회지
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• 제27권4호
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• pp.245-252
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• 2012

The purpose of the present study was to investigate the effect of IFN-${\tau}$ 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-${\tau}$ (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin $E_2$ and $F_{2{\alpha}}$ 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-${\tau}$ (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN-${\tau}$ group (p<0.05). Although IFN-${\tau}$ did not affect $PGE_2$ and $PGF_{2{\alpha}}$ production in epithelial cells, it decreased $PGE_2$ and $PGF_{2{\alpha}}$ production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN-${\tau}$ (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-${\tau}$ could improve the implantation environment of uterine by maintenance of high P4 concentration.

• 한국수정란이식학회지
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• 제12권3호
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• pp.229-246
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• 1997

Implantation is a most important biological process during pregnancy whereby conceptus establishes its survival as well as maintenance of pregnancy. During the periimplantation period, both uterine endometriurn and conceptus synthesize and secrete a host of growth factors and cytokines which mediate the actions of estrogen and /or progesterone and also exert their steroid-independent actions. Growth factors expressed by the materno-conceptal unit en masse have important roles in cell migration, stimulation or inhibition of cell proliferation, cellular differentiation, maintenance of pregnancy and materno-conceptal communications in an autorcrine /paracrine manner. The present review focuses on the role of the intrauterine IGF system during periimplantation conceptus development. The IGF system comprises of IGF- I and IGF- II ligands, types I and II IGF receptors and six or more IGF-binding proteins(IGFBPs). IGFs and IGFBPs are expressed and secreted by uterine endometrium with tissue, pregnancy stage and species specificities under the influence of estrogen, progesterone and other growth factor(s). Conceptus also synthesizes components of the IGF system beginning from a period between 2-cell and blastocyst stages. Maternal IGFs are utilized by both maternal and conceptal tissues; conceptus-derived growth factors are believed to be taken up primarily by conceptus. IGFs enhance the development of both maternal and conceptal compartments in a wide range of biological processes. They stimulate proliferation and differentiation of endometrial cells and placental precursor cells including decidual transformation from stromal cells, placental formation and the synthesis of some steroid and protein hormones by differentiated endometrial cells or placenta. It is also well-documented in a number of experimental settings that both IGFs stimulate preimplantation embryo development. In slight contrast to these, prenatal mice carrying a null mutation of IGF and /or IGF receptor gene do not exhibit any apparent growth retardation until after implantation. Reason (s) for this discrepancy between the knock-out result and the in vitro ones, however, is not known. IGFBPs, in general, are believed to inhibit IGF action within the materno-conceptal unit, thereby allowing endometrial stromal cell differentiation as well as dampening ex cessive placental invasion into maternal tissue. There is evidence, however, indicating that IGFBP can enhance IGF action depending on environrnental conditions perhaps by directioning IGF ligand to the target cell. There is also a third possibility that certain IGFBPs and their proteolytic fragments may have their own biological activities independent of the IGF. In addition to IGFBPs, IGFBP proteases including those found within the uterine tissue or lumen are thought to enhance IGF bioavailability by degrading their substrates without affecting their bound ligand. In this regard, preliminary results in early pregnant pigs suggest that a partially characterized IGFBP protease activity in uterine luminal fluid enhances intrauterine IGF bioavailability during conceptus morphological development. In summary, a number of in vitro results indicate that IGFs stimulates the development of the rnaterno-conceptal unit during the periimplantation period. IGFBPs appear to inhibit IGF action by sequestering their ligands, whereas IGFBP proteases are thought to enhance intrauterine bioavailability of IGFs. Much is remaining to be clarified, however, regarding the roles of the individual IGF system components. These include in vivo evidence for the role of IGFs in early conceptus development, identification of IGF-regulated genes and their functions, specific roles for individual IGFBPs, identification and characterization of IGFBP proteases. The intrauterine IGF club house thus will be paying a lot of attention to forthcoming results in above and other areas, with its door wide-open!

• Clinical and Experimental Reproductive Medicine
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• 제35권1호
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• pp.49-60
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• 2008

목 적: 자궁내막조직에서 분리한 상피세포와 기질세포를 삼차원 공배양을 통한 탈락막화 유도에서 성호르몬과 TGF-${\beta}1$의 역할을 알아보고 2-세포기 생쥐배아와 탈락막화가 유도된 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 포배기배아의 내세포괴와 영양막세포수 및 부착율을 알아보기 위해 시행되었다. 연구방법: 인간 자궁내막조직에서 분리된 기질세포와 상피세포의 표지인자인 cytokeratin과 vimentin에 대한 면역조직 화학염색을 실시하여 분리를 확인하였으며, 성호르몬 우세환경 (progesterone, estrogen)에서 분리된 세포를 단일배양 혹은 3차원 공배양을 통하여 RT-PCR법으로 TGF-${\beta}1$, 수용체-1, -2, integrin-${\beta}3$, prolactin의 발현을 조사하였다. 배양액군을 대조군으로 하여 2-세포기 생쥐배아와 탈락막화 유도와 유도하지 않은 인간 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 부착율과 부화된 포배의 영양막세포와 내세포괴수를 비교하였다. 결 과: 상피세포 표지인자인 cytokeratin과 기질세포 표지인자인 vimentin을 이용하여 면역조직화학염색을 한 결과 각각 95% 이상에서 양성반응을 나타내어 자궁내막조직으로부터 상피세포와 기질세포가 성공적으로 분리되었음을 확인하였다. 분리된 상피세포와 기질세포를 단일배양에서는 성호르몬의 조건에 관계없이 TGF-${\beta}1$과 수용체 type-1, type-2, integrin-${\beta}3$, prolactin mRNA가 발현되지 않았다. 공배양에서는 progesterone 우세환경일 경우 TGF-${\beta}1$ 수용체 type-2를 제외한 모든 mRNA가 발현하였으나 estrogen 우세환경에서는 TGF-${\beta}1$ 수용체 type-2와 prolactin이 발현되지 않았다. 2-세포기 생쥐배아를 배양액군, 비탈락막군 및 탈락막군으로 나누어 공배양하였을 때 포배기 발달율은 차이가 없었으나 부화율 (92%)과 부착율 (82%)은 탈락막군이 유의하게 높았으며 (p<0.05), 비탈락막군의 공배양에서 다수의 영양막세포가 투명대를 완전히 빠져나오지 않은 상태로 부착한 비정상형태를 보였다. 부화된 생쥐 포배기배아의 내세포괴수는 탈락막화에 관계없이 공배양한 포배의 내세포괴수가 유의하게 많았으며 (p<0.05), 영양막세포수는 탈락막군에서 배양액군과 비탈락막군보다 유의하게 많았다 (p<0.05). 결 론: 자궁내막조직에서 상피세포와 기질세포를 분리하여 다시 삼차원적 공배양을 통하여 progesterone (100 nM), estrogen (1 nM)과 TGF-${\beta}1$ (10 ng/ml)을 첨가하면 체외에서 탈락막화를 유도할 수 있으며, 탈락막화를 유도한 자궁내막 세포와 2-세포기 생쥐배아를 공배양하였을 때 탈락막화가 부화율, 부착율 및 영양막세포수에 유효한 영향을 미치는 것을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.83-87
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• 1999

Since the blastocyst is broken and spreads out on a flat plastic culture dish (two dimensional culture) during in vitro development, it has been difficult to study the implantation process. It also has been difficult to analyse the interactions between endometrial epithelial and stromal cells because of the lack of a long-term in vitro model which can stimulate in vivo characteristics, as these cells eventually fail to proliferate or cease to express differentiated functions. Recently nontransformed cell lines, CUE-P and CUS-V2, derived from rat endometrial epithelium and stroma were reported. In this study, morphology of CUE-P and CUS-V2 was examined and oxytocin gene expression by CUE-P cells was demonstrated by RT-PCR. The CUE-P cells have a cuboidal morphology and CUS-V2 cells resemble fibroblast and exhibit a spindle-like morphology. In RT-PCR, same size of PCR products of oxytocin gene at hypothalamus, uterus and CUE-P cells were demonstrated. These results showed three dimensional culture system could be made by using the new cell lines.