人子宫内膜上皮细胞紧密连接及其蛋白在胚胎着床中的作用及调节机制研究
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摘要
研究背景：人类的受精卵仅约一半能发育到妊娠20周后,而其中70-80%的失败归因于着床失败。胚胎着床失败是早期妊娠丢失的主要原因。尽管IVF-ET及其相关技术取得了很大进展,胚胎着床仍是决定妊娠率的主要环节。然而人类对胚胎着床分子机制的认识远远落后于对其它生殖过程的认识。IVF及其衍生技术中,促卵泡发育理论和药物几经更新,然而对促进胚胎着床、改善子宫内膜容受性方面几无良策。
人类分泌中期的子宫内膜在雌孕激素以及各种生长因子、细胞因子、粘附分子组成的网络系统精细调控下表现为对胚泡最大的容受性,这一时期也称为胚泡着床窗口期。在这一时期子宫内膜上皮细胞在形态、结构上都经历了重大的改变以适应胚泡的侵入。子宫内膜上皮包括腔上皮和腺上皮,两者都是由极性的单层立方上皮细胞构成,但是两者在胚胎着床窗口期形态和功能出现了不同的变化。容受期宫腔上皮出现细胞极性的下降：细胞高度下降,微绒毛消失,并由胞饮突取代,细胞表面电荷减少,细胞表面粘附分子分布由基侧膜转向顶侧膜。容受期腺上皮细胞则细胞高度增加,表现为最大的分泌活性和细胞极性。两种细胞在胚胎着床中有不同的作用,宫腔上皮与胚泡发生接触、粘附,子宫腺体的分泌为围着床期以及早期妊娠的胚胎的发育提供“蛋白乳”的作用。
紧密连接是上皮细胞最富特征性的结构,也是上皮细胞屏障功能和维持细胞极性、调节细胞旁小分子物质通透性的重要结构,由多种穿膜蛋白组成。其中Occludin是第一个被发现的TJ组成蛋白,其生物学作用表现为多样性,除了调节紧密连接的屏障功能外还可能参与细胞增生与分化。
紧密连接及其蛋白组成在人容受期与非容受期子宫内膜宫腔上皮细胞和腺上皮细胞是否发生了变化?紧密连接蛋白Occludin是否受到性激素的调节,是否参与了子宫内膜上皮细胞的生物学功能?这一系列问题至今尚未见报道,本课题针对上述问题进行了研究和探索。
第一部分月经周期子宫内膜上皮细胞紧密连接形态学及其蛋白组成研究
目的：检测月经周期人子宫内膜上皮细胞紧密连接超微结构及其组成蛋白Occludin、Zo-1、claudin-1,-2,-3,-4,-5的定位和定量表达
方法：透射电镜检查人正常子宫内膜宫腔上皮细胞和腺上皮细胞紧密连接超微结构在月经周期中的变化；免疫荧光和免疫印迹法检测TJ组成蛋白Occludin、Zo-1、claudin-1、-2、-3、-4、-5在人子宫内膜上皮细胞中的定位与分布、表达量的周期性变化。
结果：透射电镜研究结果显示人子宫内膜宫腔上皮细胞紧密连接在分泌中期比增生中晚期浅、宽；而子宫内膜腺上皮细胞中分泌中期紧密连接比增生中晚期深、复杂、更加多的分支。共聚焦免疫荧光显微镜检查提示子宫内膜宫腔上皮细胞紧密连接蛋白Occludin,Zo-1表达在增生中晚期信号明显强于分泌中期,荧光定量统计分析两者有显著差异。子宫内膜腺上皮细胞Occludin,Zo-1的表达趋势与腔上皮细胞刚好相反,在分泌中期信号明显强于增生中晚期,荧光定量统计分析两者有显著差异。所有检测的紧密连接蛋白Occludin、Zo-1、claudin-1、-2、-3、-4、-5均可在分泌中期的腺上皮细胞膜顶端表达；在增生中晚期腺上皮细胞claudin-2、5表达阴性,claudin-1、-3在侧基底膜表达；Occludin、Zo-1除了在细胞顶端外还在胞浆、侧基底膜不连续表达。子宫内膜组织western blot分析在分泌中期Occludin、claudin-1,-3,-4蛋白表达为最高峰。
结论：人子宫内膜上皮细胞紧密连接及其组成蛋白在月经周期呈周期性变化。其中宫腔上皮细胞分泌中期紧密连接及其组成蛋白下调；腺上皮细胞紧密连接及其蛋白表达上调
第二部分子宫内膜上皮细胞紧密连接及其蛋白Occludin在胚泡粘附中的作用
目的：明确子宫内膜上皮细胞紧密连接在子宫内膜与滋养细胞粘附中的调节作用
方法：用透射电镜、蛋白免疫印迹法、免疫荧光化学法、跨膜上皮细胞电阻(TER)检测非容受性子宫内膜上皮细胞HEC1-A细胞和容受性子宫内膜上皮细胞RL95-2细胞在TJ形态、功能、TJ蛋白组成和分布的差异；利用子宫内膜上皮细胞株-滋养细胞球(JAR spheroids)粘附的胚胎着床模型检测两种子宫内膜上皮细胞胚泡粘附率的差异；对滋养细胞球进行活细胞荧光染料标记,利用双色免疫荧光共聚焦显微镜检测在HEC-1A细胞与滋养细胞球发生粘附处TJ蛋白Occludin表达的变化;观察HEC1-A细胞Occludin siRNA干扰后检测TJ功能、胚泡粘附率的变化；检测着床促进因子白细胞介素p-1对HEC-1A细胞TJ蛋白Occludin和胚泡粘附率的调节。
结果：透射电镜显示HEC-1A细胞具有极性,紧密连接位于细胞顶端,免疫荧光染色显示TJ蛋白Occludin、Zo-1表达于细胞膜。RL95-2细胞无TJ结构,Occludin、Zo-1表达于细胞浆。western blot结果显示HEC-1A细胞Zo-1, Occludin蛋白表达显著高于RL95-2细胞。而Claudin-4在HEC-1A细胞阳性表达,RL95-2细胞中阴性表达。HEC-1A细胞TER为(163.0±23.0)Q×cm2, RL95-2细胞TER为(92.3±11.0)Ω×cm2,两者相比有显著差异(P<0.001)。HEC-1A细胞与JAR spheroids粘附率显著低于RL95-2细胞。在JAR细胞球粘附处,HEC-1A细胞Occludin呈现为不连续、点状分布。对HEC-1A细胞的Occludin经siRNA干扰后,细胞在TJ重装过程中TER峰值下降。白细胞介素1-p下调HEC1-A细胞Occludin表达,并上调HEC1-A细胞胚泡粘附率。
结论：非容受性子宫内膜上皮细胞HEC-1A存在功能性的紧密连接。HEC-1A细胞TJ蛋白Occludin下调后能够促进子宫内膜上皮细胞与胚泡的粘附；在JAR细胞球粘附处,HEC-1A细胞的紧密连接蛋白Occludin为不连续的点状分布。本研究提示紧密连接蛋白Occludin下调参与子宫内膜容受性的建立。
第三部分Occludin表达的性激素调控及其在子宫内膜腺上皮细胞增生和分泌中的作用
目的：明确子宫内膜上皮细胞Occludin表达的性激素调控；阐明Occludin在子宫内膜上皮细胞增生和分泌中的作用。
方法：雌激素、雌孕激素联合培养增生期子宫内膜腺上皮细胞。Western blot检测上皮细胞Occludin、Claudin-4的表达。以IK细胞为腺上皮细胞模型,对其Occludin进行siRNA基因干扰,采用流式细胞技术检测细胞对凋亡诱导剂H202的敏感性,通过细胞周期检测细胞的增殖率；并采用蒽酮法检测细胞糖原含量的变化。
结果：孕激素上调65kD Occludin、Claudin-4表达;对IK细胞Occludin基因干扰后,细胞凋亡敏感性下降；细胞增殖率增加；糖原分泌下降。
结论：孕激素上调TJ蛋白65kD Occludin、Claudin-4表达,提示孕激素能增强TJ功能。Occludin能够抑制子宫内膜上皮细胞的增生,并促进其分泌。提示Occludin介导孕激素对子宫内膜上皮细胞的增生抑制和促分化作用。
It has been shown that only 50 to 60 percent of conceptions in women could develop and survive past 20 weeks of gestation. Around 40-50% of conception and pregnancy failed at different time of gestation. Because approximately 75 percent of these failures are due to implantation failure, implantation failure is a main factor resulting in early pregnancy loss. So embryo implantation rate determines the success of the in vitro fertilization (IVF) and the birth rate. However, the mechanisms involved in embryo implantation remain largely unknown. The knowledge about the process of embryo implantation we have is well less than the knowledge about the other process of human reproduction. Although the ART protocol and pharmaceuticals for ovarian stimulation are being renovated and reproved constantly, on the contrary, we nearly have no measure about improving the uterine receptivity.
The phenotypes of endometrial luminal and glandular epithelium change dramatically from the proliferative to the mid-secretory phase of the menstrual cycle, when the endometrium becomes receptive for embryo implantation under regulation of ovarian hormones, cytokines, cell growth factors, adhesion molecules. Endometrial luminal and glandular epithelium are composed of the polarity epithelial cells. In embryo implantation window, two types of epithelial cells undergo different changes in morphology. A loss of apico-basal polarity could be observed in luminal epithelial cells, while gland epithelial cells showed the most polarity and secretory activity. The different morphology between two cell populations highlights the functional difference, with the luminal epithelial cells initial contact and attachment with the embryo and the glandular epithelial cells expressing secretory proteins for conceptus recognition, survival, and development.
However, little is known about the association of TJ functions with the establish of the endometrial receptivity. The purpose of the present study was 1) to investigate the alteration of TJ morphology and TJ protein in human uterine epithelium during the menstrual cycle; 2) to determine whether TJ function associated with the endometrial receptivity; 3) to determine whether expression of TJ proteins in uterine gland epithelial cells had a correlation with serum 17β-estradiol (E2) and/or progesterone; and 4) to determine whether Occludin regulates the uterine epithelial cell proliferation and differentiation.
Part1 Alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycle
Objective:To investigate alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycle
Methods:We examined the dynamically changes of morphology of TJs between adjacent uterine epithelial cells including luminal epithelial cells and gland epithelial cells during the menstrual cycle with transmission electroscope. Then we investigated the location and expression of TJ proteins including Occludin、Zo-1、Claudin-1,-2,-3,-4,-5 in the proliferative and secretary endometrium with immunochemistry techniques, respectively.
Results:the morphological analysis of uterine epithelial cells with transmission electroscope showed that the depth of TJ between gland epithelial cells in mid-secretary phase increased about 2-fold along the lateral membrane and present more complex and curled when compared with mid-proliferative phase. However, in the luminal epithelial cells, the TJ depth in mid-secretary phase was reduced significantly compared with the mid-proliferative phase.
All examined TJ proteins including Occludin. Zo-1、Claudin-1,-2,-3,-4,-5 with confocal immunofluorescence were located at TJ region in mid-secretary phase GE cells. On the other hand, Claudin-2 and -5 were not expressed in mid/late proliferative phase GE cells. During mid/late proliferative phase, Claudin-1, Claudin-3 and Claudin-4 were faintly stained in the basallaterial membrane, and Zo-1, Occludin localized in punctuate manner at the uppermost portion of the lateral membrane and cytoplasm. The endometrial expression levels of TJ proteins in mid-secretary phase were higher than those in other phases with Western blot analysis. Conclusion:Dynamically changes of structural and molecular components of TJs in uterine epithelial cells were detected during proliferative and secretive phases of the menstrual cycle. In LE cells the TJ depth and expression of TJ proteins were downregulated during mid-secretary phase, while GE cells showed an oppositive result.
Part 2 The roles of tight junction between uterine endometrial epithelial cells in the embryo implantation
Objective:To investigate the roles of tight junction in regulation of adhesion between uterine endometrial epithelial cells and embryo cells.
Methods:Endometrial cell lines RL95-2 and HEC-1A were used as models of receptive and nonreceptive cells, respectively. Attachment assay of JAR spheroids to the endometrial cell was used as embryo implantation model to evaluate the hEEC receptivity to embryo under different cultured conditions. We compared the HEC1-A and RL95-2 cell lines in TJ ultrastructure and TJ protein composition with transmission electron microscope, Western blot and immunofluorescent confocal microscope, respectively. We examined the TJ function by measurement of cell transcellular electrical resistance (TER) with Millicell-ERS. The embryo adhesion rate in HEC1-A and RL95-2 cell lines were examined. The expression of Occludin at the adhesion parts was examined with three-color laser confocal inspection. Then, we investigated the embryo adhesion rate in the HEC1-A cells pretreated with siRNA to interfere Occludin gene and TER during TJ assembled under the calcium switch assay with EGTA. Additionally, we treated HEC1-A cells with interleukin-1βfor 48 hours, and examined the expression level and location of Occludin with Western blot and immunofluorescence, and the adhesion rate of HEC-1A cells to JAR spheroids.
Results:The transmission electron microscopy showed that HEC1-A cells, not RL95-2 cells, were polarized and had typical tight junctions at the apex of the lateral membrane cells. In HEC1-A the tight junction proteins Zo-1 and Occludin were expressed in the cell membrane, while they were expressed in the cytoplasm of RL95-2 cells. Western blot results showed that the expression of Zo-1 and Occludin in the HEC1-A was significantly higher than that in RL95-2 (P<0.01). Claudin-4, a component of tight junction skeleton, was strongly expressed in HEC1-A cells, but no expression was detected in RL95-2 cells. The TER of monolayer HEC1-A cells was higher than that of RL95-2 cells (163.0±23.0)Ω×cm2 vs (92.3±11.0)Ω×cm2(P＜0.01). In hEEC and JAR spheroids attachment experiments, the adhesion rate of HEC1-A cells to JAR spheroids was significantly higher than that of RL95-2 cells (P＜0.01). Confocal laser microscopic analysis showed that Occludin might move from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells, remaining disrupted and point-like distribution in the cell membrane. After Occludin siRNA interfering, the expression of Occludin in the membrane was decreased in HEC1-A cells, and meanwhile the embryo adhesion rate increased. However, the peak TER was attained more slowly and its value was lower in cultured HEC-1A cells pretreated with Occludin siRNA during TJ reassembled in calcium switch experiments.
Conclusion:HEC-1A cell presents TJ and TJ protein in TJ region, while RL95-2 cell did not express TJ and TJ proteind in cell border. Downregulated Occludin protein expression by siRNA interference in HEC1-A cells results in an increase in the embryo adhesion rate, the translocation of Occludin from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells. These results suggest that down-regulation of the uterine endometrial epithelial cell tight junctions may be related to embryonic adhesion.
Part 3 The roles of Occludin in regulation of epithelial cell differentiation and proliferation and the alteration of TJ proteins in cultured human endometrial epithelial cells in the presence of ovarian steroid hormones
Objective:To investigate the effects of E2 and progesterone (P4) on the expression of TJ proteins Occludin and Claudin-4 in the cultured human endometrial epithelial cells and the roles of Occludin in the regulation of epithelial differentiation and proliferation.
Method:The endometrial gland epithelial cells in proliferative phase was collected from 6 women who underwent hysterectomy for leomyoma. Endometrial GE cells were isolated and cultured in vitro in the presence of 17β-estradiol (E2,10-8mol/L) or E2 (10-8mol/L)+P4(10-6mol/L) for 48h. The control group received no treatment. The levels of Occludin and Claudin-4 proteins in the cultured human endometrial epithelial cells were analyzed with Western blot. Ishikawa (IK) cells were used in these experiments. IK cells have glandular characteristics and are able to secrete some cellular factors similar to normal endometrial cells. IK cells were widely considered as suitable cell lines for studying functions of normal endometrial glandular epithelial cells. We used IK cells as a GE cell model and treated these cells with Occludin siRNA to downregulate Occludin expression. IK cells apoptosis was examined using H2O2 as apoptogen and analyzed with flow cytometry. The cell proliferation was examined with cell cycle flow cytometry analysis. Glycogen level in IK cells was also analyzed.
Results:E2 (lOnM) had little effect on Claudin-4 protein expression and P4 significantly increased the 65 kD/50kD ratio of Occludin band and Claudin-4 protein. Pretreatment of IK cells with Occludin siRNA significantly decreased the cell glycogen level, increased cell proliferative rate and reduced apoptosis sensitivity to H2O2.
Conclusion:Progesterone upregulates the protein expression of 65kD Occludin and Claudin-4 in primary cultured endometrial gland epithelial cells, suggesting that progesterone may promote the TJ assembled in uterine GE cells. siRNA targeting human Occludin in IK cells led to downregulate apoptosis and increase cell proliferation, reduce cell glycogen production, suggesting that Occludin may play a role in mediating the progesterone effects on GE cell apoptosis and on secretory activity.
人类分泌中期的子宫内膜在雌孕激素以及各种生长因子、细胞因子、粘附分子组成的网络系统精细调控下表现为对胚泡最大的容受性,这一时期也称为胚泡着床窗口期。在这一时期子宫内膜上皮细胞在形态、结构上都经历了重大的改变以适应胚泡的侵入。子宫内膜上皮包括腔上皮和腺上皮,两者都是由极性的单层立方上皮细胞构成,但是两者在胚胎着床窗口期形态和功能出现了不同的变化。容受期宫腔上皮出现细胞极性的下降：细胞高度下降,微绒毛消失,并由胞饮突取代,细胞表面电荷减少,细胞表面粘附分子分布由基侧膜转向顶侧膜。容受期腺上皮细胞则细胞高度增加,表现为最大的分泌活性和细胞极性。两种细胞在胚胎着床中有不同的作用,宫腔上皮与胚泡发生接触、粘附,子宫腺体的分泌为围着床期以及早期妊娠的胚胎的发育提供“蛋白乳”的作用。
紧密连接是上皮细胞最富特征性的结构,也是上皮细胞屏障功能和维持细胞极性、调节细胞旁小分子物质通透性的重要结构,由多种穿膜蛋白组成。其中Occludin是第一个被发现的TJ组成蛋白,其生物学作用表现为多样性,除了调节紧密连接的屏障功能外还可能参与细胞增生与分化。
紧密连接及其蛋白组成在人容受期与非容受期子宫内膜宫腔上皮细胞和腺上皮细胞是否发生了变化?紧密连接蛋白Occludin是否受到性激素的调节,是否参与了子宫内膜上皮细胞的生物学功能?这一系列问题至今尚未见报道,本课题针对上述问题进行了研究和探索。
第一部分月经周期子宫内膜上皮细胞紧密连接形态学及其蛋白组成研究
目的：检测月经周期人子宫内膜上皮细胞紧密连接超微结构及其组成蛋白Occludin、Zo-1、claudin-1,-2,-3,-4,-5的定位和定量表达
方法：透射电镜检查人正常子宫内膜宫腔上皮细胞和腺上皮细胞紧密连接超微结构在月经周期中的变化；免疫荧光和免疫印迹法检测TJ组成蛋白Occludin、Zo-1、claudin-1、-2、-3、-4、-5在人子宫内膜上皮细胞中的定位与分布、表达量的周期性变化。
结果：透射电镜研究结果显示人子宫内膜宫腔上皮细胞紧密连接在分泌中期比增生中晚期浅、宽；而子宫内膜腺上皮细胞中分泌中期紧密连接比增生中晚期深、复杂、更加多的分支。共聚焦免疫荧光显微镜检查提示子宫内膜宫腔上皮细胞紧密连接蛋白Occludin,Zo-1表达在增生中晚期信号明显强于分泌中期,荧光定量统计分析两者有显著差异。子宫内膜腺上皮细胞Occludin,Zo-1的表达趋势与腔上皮细胞刚好相反,在分泌中期信号明显强于增生中晚期,荧光定量统计分析两者有显著差异。所有检测的紧密连接蛋白Occludin、Zo-1、claudin-1、-2、-3、-4、-5均可在分泌中期的腺上皮细胞膜顶端表达；在增生中晚期腺上皮细胞claudin-2、5表达阴性,claudin-1、-3在侧基底膜表达；Occludin、Zo-1除了在细胞顶端外还在胞浆、侧基底膜不连续表达。子宫内膜组织western blot分析在分泌中期Occludin、claudin-1,-3,-4蛋白表达为最高峰。
结论：人子宫内膜上皮细胞紧密连接及其组成蛋白在月经周期呈周期性变化。其中宫腔上皮细胞分泌中期紧密连接及其组成蛋白下调；腺上皮细胞紧密连接及其蛋白表达上调
第二部分子宫内膜上皮细胞紧密连接及其蛋白Occludin在胚泡粘附中的作用
目的：明确子宫内膜上皮细胞紧密连接在子宫内膜与滋养细胞粘附中的调节作用
方法：用透射电镜、蛋白免疫印迹法、免疫荧光化学法、跨膜上皮细胞电阻(TER)检测非容受性子宫内膜上皮细胞HEC1-A细胞和容受性子宫内膜上皮细胞RL95-2细胞在TJ形态、功能、TJ蛋白组成和分布的差异；利用子宫内膜上皮细胞株-滋养细胞球(JAR spheroids)粘附的胚胎着床模型检测两种子宫内膜上皮细胞胚泡粘附率的差异；对滋养细胞球进行活细胞荧光染料标记,利用双色免疫荧光共聚焦显微镜检测在HEC-1A细胞与滋养细胞球发生粘附处TJ蛋白Occludin表达的变化;观察HEC1-A细胞Occludin siRNA干扰后检测TJ功能、胚泡粘附率的变化；检测着床促进因子白细胞介素p-1对HEC-1A细胞TJ蛋白Occludin和胚泡粘附率的调节。
结果：透射电镜显示HEC-1A细胞具有极性,紧密连接位于细胞顶端,免疫荧光染色显示TJ蛋白Occludin、Zo-1表达于细胞膜。RL95-2细胞无TJ结构,Occludin、Zo-1表达于细胞浆。western blot结果显示HEC-1A细胞Zo-1, Occludin蛋白表达显著高于RL95-2细胞。而Claudin-4在HEC-1A细胞阳性表达,RL95-2细胞中阴性表达。HEC-1A细胞TER为(163.0±23.0)Q×cm2, RL95-2细胞TER为(92.3±11.0)Ω×cm2,两者相比有显著差异(P<0.001)。HEC-1A细胞与JAR spheroids粘附率显著低于RL95-2细胞。在JAR细胞球粘附处,HEC-1A细胞Occludin呈现为不连续、点状分布。对HEC-1A细胞的Occludin经siRNA干扰后,细胞在TJ重装过程中TER峰值下降。白细胞介素1-p下调HEC1-A细胞Occludin表达,并上调HEC1-A细胞胚泡粘附率。
结论：非容受性子宫内膜上皮细胞HEC-1A存在功能性的紧密连接。HEC-1A细胞TJ蛋白Occludin下调后能够促进子宫内膜上皮细胞与胚泡的粘附；在JAR细胞球粘附处,HEC-1A细胞的紧密连接蛋白Occludin为不连续的点状分布。本研究提示紧密连接蛋白Occludin下调参与子宫内膜容受性的建立。
第三部分Occludin表达的性激素调控及其在子宫内膜腺上皮细胞增生和分泌中的作用
目的：明确子宫内膜上皮细胞Occludin表达的性激素调控；阐明Occludin在子宫内膜上皮细胞增生和分泌中的作用。
方法：雌激素、雌孕激素联合培养增生期子宫内膜腺上皮细胞。Western blot检测上皮细胞Occludin、Claudin-4的表达。以IK细胞为腺上皮细胞模型,对其Occludin进行siRNA基因干扰,采用流式细胞技术检测细胞对凋亡诱导剂H202的敏感性,通过细胞周期检测细胞的增殖率；并采用蒽酮法检测细胞糖原含量的变化。
结果：孕激素上调65kD Occludin、Claudin-4表达;对IK细胞Occludin基因干扰后,细胞凋亡敏感性下降；细胞增殖率增加；糖原分泌下降。
结论：孕激素上调TJ蛋白65kD Occludin、Claudin-4表达,提示孕激素能增强TJ功能。Occludin能够抑制子宫内膜上皮细胞的增生,并促进其分泌。提示Occludin介导孕激素对子宫内膜上皮细胞的增生抑制和促分化作用。
It has been shown that only 50 to 60 percent of conceptions in women could develop and survive past 20 weeks of gestation. Around 40-50% of conception and pregnancy failed at different time of gestation. Because approximately 75 percent of these failures are due to implantation failure, implantation failure is a main factor resulting in early pregnancy loss. So embryo implantation rate determines the success of the in vitro fertilization (IVF) and the birth rate. However, the mechanisms involved in embryo implantation remain largely unknown. The knowledge about the process of embryo implantation we have is well less than the knowledge about the other process of human reproduction. Although the ART protocol and pharmaceuticals for ovarian stimulation are being renovated and reproved constantly, on the contrary, we nearly have no measure about improving the uterine receptivity.
The phenotypes of endometrial luminal and glandular epithelium change dramatically from the proliferative to the mid-secretory phase of the menstrual cycle, when the endometrium becomes receptive for embryo implantation under regulation of ovarian hormones, cytokines, cell growth factors, adhesion molecules. Endometrial luminal and glandular epithelium are composed of the polarity epithelial cells. In embryo implantation window, two types of epithelial cells undergo different changes in morphology. A loss of apico-basal polarity could be observed in luminal epithelial cells, while gland epithelial cells showed the most polarity and secretory activity. The different morphology between two cell populations highlights the functional difference, with the luminal epithelial cells initial contact and attachment with the embryo and the glandular epithelial cells expressing secretory proteins for conceptus recognition, survival, and development.
However, little is known about the association of TJ functions with the establish of the endometrial receptivity. The purpose of the present study was 1) to investigate the alteration of TJ morphology and TJ protein in human uterine epithelium during the menstrual cycle; 2) to determine whether TJ function associated with the endometrial receptivity; 3) to determine whether expression of TJ proteins in uterine gland epithelial cells had a correlation with serum 17β-estradiol (E2) and/or progesterone; and 4) to determine whether Occludin regulates the uterine epithelial cell proliferation and differentiation.
Part1 Alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycle
Objective:To investigate alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycle
Methods:We examined the dynamically changes of morphology of TJs between adjacent uterine epithelial cells including luminal epithelial cells and gland epithelial cells during the menstrual cycle with transmission electroscope. Then we investigated the location and expression of TJ proteins including Occludin、Zo-1、Claudin-1,-2,-3,-4,-5 in the proliferative and secretary endometrium with immunochemistry techniques, respectively.
Results:the morphological analysis of uterine epithelial cells with transmission electroscope showed that the depth of TJ between gland epithelial cells in mid-secretary phase increased about 2-fold along the lateral membrane and present more complex and curled when compared with mid-proliferative phase. However, in the luminal epithelial cells, the TJ depth in mid-secretary phase was reduced significantly compared with the mid-proliferative phase.
All examined TJ proteins including Occludin. Zo-1、Claudin-1,-2,-3,-4,-5 with confocal immunofluorescence were located at TJ region in mid-secretary phase GE cells. On the other hand, Claudin-2 and -5 were not expressed in mid/late proliferative phase GE cells. During mid/late proliferative phase, Claudin-1, Claudin-3 and Claudin-4 were faintly stained in the basallaterial membrane, and Zo-1, Occludin localized in punctuate manner at the uppermost portion of the lateral membrane and cytoplasm. The endometrial expression levels of TJ proteins in mid-secretary phase were higher than those in other phases with Western blot analysis. Conclusion:Dynamically changes of structural and molecular components of TJs in uterine epithelial cells were detected during proliferative and secretive phases of the menstrual cycle. In LE cells the TJ depth and expression of TJ proteins were downregulated during mid-secretary phase, while GE cells showed an oppositive result.
Part 2 The roles of tight junction between uterine endometrial epithelial cells in the embryo implantation
Objective:To investigate the roles of tight junction in regulation of adhesion between uterine endometrial epithelial cells and embryo cells.
Methods:Endometrial cell lines RL95-2 and HEC-1A were used as models of receptive and nonreceptive cells, respectively. Attachment assay of JAR spheroids to the endometrial cell was used as embryo implantation model to evaluate the hEEC receptivity to embryo under different cultured conditions. We compared the HEC1-A and RL95-2 cell lines in TJ ultrastructure and TJ protein composition with transmission electron microscope, Western blot and immunofluorescent confocal microscope, respectively. We examined the TJ function by measurement of cell transcellular electrical resistance (TER) with Millicell-ERS. The embryo adhesion rate in HEC1-A and RL95-2 cell lines were examined. The expression of Occludin at the adhesion parts was examined with three-color laser confocal inspection. Then, we investigated the embryo adhesion rate in the HEC1-A cells pretreated with siRNA to interfere Occludin gene and TER during TJ assembled under the calcium switch assay with EGTA. Additionally, we treated HEC1-A cells with interleukin-1βfor 48 hours, and examined the expression level and location of Occludin with Western blot and immunofluorescence, and the adhesion rate of HEC-1A cells to JAR spheroids.
Results:The transmission electron microscopy showed that HEC1-A cells, not RL95-2 cells, were polarized and had typical tight junctions at the apex of the lateral membrane cells. In HEC1-A the tight junction proteins Zo-1 and Occludin were expressed in the cell membrane, while they were expressed in the cytoplasm of RL95-2 cells. Western blot results showed that the expression of Zo-1 and Occludin in the HEC1-A was significantly higher than that in RL95-2 (P<0.01). Claudin-4, a component of tight junction skeleton, was strongly expressed in HEC1-A cells, but no expression was detected in RL95-2 cells. The TER of monolayer HEC1-A cells was higher than that of RL95-2 cells (163.0±23.0)Ω×cm2 vs (92.3±11.0)Ω×cm2(P＜0.01). In hEEC and JAR spheroids attachment experiments, the adhesion rate of HEC1-A cells to JAR spheroids was significantly higher than that of RL95-2 cells (P＜0.01). Confocal laser microscopic analysis showed that Occludin might move from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells, remaining disrupted and point-like distribution in the cell membrane. After Occludin siRNA interfering, the expression of Occludin in the membrane was decreased in HEC1-A cells, and meanwhile the embryo adhesion rate increased. However, the peak TER was attained more slowly and its value was lower in cultured HEC-1A cells pretreated with Occludin siRNA during TJ reassembled in calcium switch experiments.
Conclusion:HEC-1A cell presents TJ and TJ protein in TJ region, while RL95-2 cell did not express TJ and TJ proteind in cell border. Downregulated Occludin protein expression by siRNA interference in HEC1-A cells results in an increase in the embryo adhesion rate, the translocation of Occludin from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells. These results suggest that down-regulation of the uterine endometrial epithelial cell tight junctions may be related to embryonic adhesion.
Part 3 The roles of Occludin in regulation of epithelial cell differentiation and proliferation and the alteration of TJ proteins in cultured human endometrial epithelial cells in the presence of ovarian steroid hormones
Objective:To investigate the effects of E2 and progesterone (P4) on the expression of TJ proteins Occludin and Claudin-4 in the cultured human endometrial epithelial cells and the roles of Occludin in the regulation of epithelial differentiation and proliferation.
Method:The endometrial gland epithelial cells in proliferative phase was collected from 6 women who underwent hysterectomy for leomyoma. Endometrial GE cells were isolated and cultured in vitro in the presence of 17β-estradiol (E2,10-8mol/L) or E2 (10-8mol/L)+P4(10-6mol/L) for 48h. The control group received no treatment. The levels of Occludin and Claudin-4 proteins in the cultured human endometrial epithelial cells were analyzed with Western blot. Ishikawa (IK) cells were used in these experiments. IK cells have glandular characteristics and are able to secrete some cellular factors similar to normal endometrial cells. IK cells were widely considered as suitable cell lines for studying functions of normal endometrial glandular epithelial cells. We used IK cells as a GE cell model and treated these cells with Occludin siRNA to downregulate Occludin expression. IK cells apoptosis was examined using H2O2 as apoptogen and analyzed with flow cytometry. The cell proliferation was examined with cell cycle flow cytometry analysis. Glycogen level in IK cells was also analyzed.
Results:E2 (lOnM) had little effect on Claudin-4 protein expression and P4 significantly increased the 65 kD/50kD ratio of Occludin band and Claudin-4 protein. Pretreatment of IK cells with Occludin siRNA significantly decreased the cell glycogen level, increased cell proliferative rate and reduced apoptosis sensitivity to H2O2.
Conclusion:Progesterone upregulates the protein expression of 65kD Occludin and Claudin-4 in primary cultured endometrial gland epithelial cells, suggesting that progesterone may promote the TJ assembled in uterine GE cells. siRNA targeting human Occludin in IK cells led to downregulate apoptosis and increase cell proliferation, reduce cell glycogen production, suggesting that Occludin may play a role in mediating the progesterone effects on GE cell apoptosis and on secretory activity.
引文
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