RhoA在小鼠胚胎围着床期子宫内膜的表达及其意义

目的探讨RhoA在小鼠胚胎植入过程中的表达及其生物学作用。方法取胚胎围种植期（D0至D6天）昆明小鼠子宫作切片，用免疫组织化学方法和图像分析方法对RhoA在子宫内膜中的表达进行定位和半定量分析。结果RhoA主要分布于小鼠子宫内膜腺上皮、腔上皮和基质细胞。受精后，RhoA在子宫内膜的含量高于未孕组（P＜0．05）；受精后第4d（植入期），小鼠子宫内膜腔上皮中RhoA的含量明显增多，明显高于未孕及植入前各时间组（P＜0．05），并逐渐向基质细胞延伸，第5d广泛分布在蜕膜细胞和基质细胞，到第6天大部分子宫内膜基质区域及腺上皮的细胞中有大量的RhoA分布。RhoA在小鼠子宫内膜组织中的表达强度在受精后呈逐渐上升趋势。结论①RhoA可能参与了胚胎的种植和子宫内膜容受性的建立；②还可能间接参与植入后胚胎的滋养层扩展、分化及子宫内膜蜕膜化反应。     

ATF4基因在小鼠动情周期中子宫内膜的表达

目的探讨转录激活因子4(ATF4)在小鼠动情周期中子宫内膜的表达规律。方法采用RT-PCR、免疫组织化学和Western blot法分别检测小鼠子宫内膜中ATF4 mRNA和蛋白在动情前期、动情期、动情间期和动情后期的表达规律。结果 ATF4 mRNA在动情期表达显著高于其他3期(P0.05);ATF4蛋白主要在腺上皮细胞和腔上皮细胞胞质表达,其表达规律与RT-PCR的结果基本一致。结论 ATF4在小鼠动情周期子宫内膜中呈动态表达,提示ATF4可能参与了动情周期子宫内膜变化的调控。     

溶血磷脂酸受体3在小鼠胚胎围着床期子宫内膜的表达及意义

目的 探讨溶血磷脂酸受体3(LPA-R3)nRNA和蛋白在小鼠胚胎着床过程中表达的动态变化. 方法 将78只雌性昆明小鼠随机分为真孕组(36只)、假孕组(36只)和对照组(6只),应用反转录.聚合酶链反应(RT-PCR)和Western blotting以及免疫组织化学SP法,检测胚胎围着床期(0～6d)LPA-R3 mRNA和蛋白在小鼠子宫内膜的表达和定位. 结果 LPA-R3主要分布于小鼠子宫内膜腔上皮、腺上皮和部分基质细胞的胞质.LPA-R3 mRNA和蛋白在小鼠胚胎着床过程中,3d时开始上升,4d时达峰值.5d、6d骤然下降至基础水平.假孕组子宫内膜LPA-R3mRNA和蛋白表达水平及变化规律与相应同期真孕组一致. 绪论 LPA-R3可能参与了胚胎的着床和子宫内膜容受性的建立过程.     

组织型纤溶酶原激活因子及其抑制因子在孕鼠子宫中的表达

本实验用原位杂交和免疫组织化学定位方法从ｍＲＮＡ和蛋白质水平研究组织型纤维溶酶原激活因子（ｔＰＡ）及其Ｉ型抑制因子（ＰＡＩ－１）在受孕第七天大鼠子宫中的表达，结果表明，在非着床点子宫腔上皮细胞，腔上皮附近的基质细胞和腺上皮细胞中ｔＰＡ和ＰＡＩ－ｍＲＮＡ都有表达，但ＰＡＩ－１ｍＲＮＡ的量很低，ｔＰＡ抗原分布于了宫腔上皮和腺上皮，ＰＡＩ－１抗原只能在腺上皮和表层基质中微弱地检测到，在着床点，ｔＰＡ和Ｐ     

小鼠胚胎与子宫内膜中整合素α5的表达

目的：观察整合素α5在小鼠早期胚胎和子宫内膜中的表达分布状况，探讨整合素在胚胎着床中的可能作用。方法：用免疫组织化学ABC法染色，对发育不同时期的小鼠胚卵和妊娠2d-6d、8d、13d以及非妊娠期的子宫内膜中整合素内的表达分布进行形态学观察与半定量分析。结果：整合素α5的阳性反应物质在小鼠早期胚卵胞浆内；非妊娠期子宫内膜和妊娠期的脱膜上皮与基质细胞内均有整合素α5表达，植入前表达相对较弱，植入后随妊娠的进展逐渐增强。结论：提示整合素α5在小鼠胚卵着床中起重要作用：推测它不参与胚卵对母体子宫内膜的识别和粘附，而是参与了粘附后的侵入及胚胎发育过程。     

ICAM-1在着床期小鼠PBLC及EC/DC中表达的研究

目的：研究细胞间粘附因子－１（ＩＣＡＭ－１）在着床期外周血淋巴细胞（ＰＢＬＣ）、子宫内膜及蜕膜细胞（ＥＣ／ＤＣ）中的不同表达特点及动态变化规律。方法：以着床期小鼠为动物模型,细胞分析采用单抗免疫荧光标主多参数流式细胞术分析技术。结果：发现ＩＣＡＭ－１在ＰＢＬＣ、ＥＣ／ＤＣ中的均存在明显的动态变化;其中ＩＣＡＭ－１的表达在ＰＢＬＣ中于妊娠第２天（Ｄ２）达最低小平,而在ＥＣ／ＤＣ中于妊娠第４天（Ｄ４）     

雌、孕激素对LPA3在小鼠子宫内膜围着床期表达的影响

目的 研究小鼠围着床期子宫内膜溶血磷脂酸受体3（LPA3）的表达以及雌、孕激素和孕激素拮抗剂RU486对其表达的影响。方法 用免疫组化法,检测妊娠第1~6天（d1~6）小鼠子宫内膜LPA3蛋白的表达规律; 用RT-PCR、Western blot检测子宫内膜LPA3的表达。结果 妊娠第1~6天小鼠子宫内膜组织均有LPA3表达,d3表达开始增强,d4最强,d5和d6 表达骤然下降。去势小鼠子宫内膜表达低水平LPA3 ;孕激素提高子宫内膜LPA3 的表达,且呈时间依赖性。雌激素对LPA3 表达无明显影响。雌、孕激素联合应用,雌激素降低孕激素对子宫内膜LPA3 表达的上调作用。RU486减弱孕激素的上调作用。结论　LPA3可能参与了胚胎的种植。雌、孕激素可能通过影响小鼠子宫内膜LPA3 的表达,共同参与小鼠子宫内膜容受性的建立。     

外源性LIF和IL—1对围着床期小鼠子宫内膜整合素β3亚素表达的调节

目的：研究外源性LIF及IL－1对围着床期子中内膜整合素β3表达的调节,探讨LIF参与着床的具体机制,方法：给孕2－4d的小鼠注射不同浓度的白血病抑制因子,白介素－1或白介素－1受体拮抗剂,应用免疫印迹法和RT－RCR法测定子宫内膜整合素β3的蛋白及mRNA表达水平。结果注射ＬＩＦ,ＩＬ－１子宫内膜整合素β３的表达水平升高（Ｐ＜０．０５）,且高剂量时升高更明显（Ｐ＜０．０１）,注射ＩＬ－１ra整合素β3水平下降（Ｐ＜０．０５）,结论：细胞因子ＩＬ－１及ＬＩＦ对围着床期子宫内膜表达整合素β３有促调节作用,二者参与着床与调节细胞粘附分子表达有关。     

白血病抑制因子在小鼠子宫内膜不同部位的表达研究

目的探讨白血病抑制因子(LIF)在着床窗口期小鼠子宫内膜不同部位的表达。方法运用半定量逆转录聚合酶链反应(RT-PCR)和免疫组化技术,分别从mRNA水平和蛋白质水平检测LIF在孕4d小鼠子宫内膜着床位点及着床旁组织表达量及位置的分布。结果LIFmRNA及蛋白在胚泡着床位点较着床旁组织表达明显增高(P<0.05)。免疫组化检测结果显示LIF蛋白表达于子宫内膜间质细胞及腺上皮细胞胞桨。结论在着床窗口期,LIF作用的发挥主要是通过在着床位点高表达而促进胚泡着床、胚胎发育。     

CXCR2在妊娠早期小鼠子宫内膜的表达

目的研究CXCR2在妊娠早期小鼠子宫内膜的表达情况.方法应用免疫组织化学和图像分析技术,观察CXCR2在妊娠1d、4d、5d、6d小鼠子宫内膜的表达部位及蛋白水平的变化规律;HE染色后光镜下观察子宫内膜的形态学变化.结果免疫组化染色结果显示:在子宫内膜腔上皮,CXCR2于妊娠4d表达最强,妊娠5d开始逐渐下降,妊娠6d降至妊娠1d水平;在子宫内膜腺上皮和基质,CXCR2的表达水平随妊娠天数的增加而逐渐升高.HE染色可见妊娠1d子宫内膜腔上皮和基质中有大量中性粒细胞浸润;妊娠4d腔上皮中未见中性粒细胞,基质中则有大量中性粒细胞浸润;妊娠5d和妊娠6d腔上皮中未见中性粒细胞,基质中中性粒细胞数量甚少.结论CXCR2可能通过与其配体白细胞介素-8结合而参与小鼠胚泡着床过程.     

白细胞介素-8在妊娠早期小鼠子宫内膜的表达及对胚泡着床的影响

目的：研究白细胞介素-8（IL-8）在妊娠早期小鼠子宫内膜的表达及其对小鼠胚泡着床的影响。方法：用免疫组化法及图像分析技术对IL-8在妊娠1～6d小鼠子宫内膜的表达进行定位和测定;子宫角注入IL-8抗体,探讨IL-8对胚泡着床的影响。结果：在子宫内膜腔上皮,IL-8主要定位于细胞的游离面,妊娠1d阳性反应最弱,4d表达最强,5d有所下降,6d又开始增强;在子宫内膜腺上皮,妊娠4d表达最弱,5d和6d最强;在子宫内膜的基质细胞,随妊娠天数增加,IL-8的表达逐渐增强。IL-8Ab明显抑制小鼠胚泡着床。结论：IL-8在妊娠早期小鼠子宫内膜持续表达,并参与胚泡的着床调控过程。     

Lefty is expressed in mouse endometrium in estrous cycle and peri-implantation period

BACKGROUND: Reproductive tissues are unique structures that exhibit cyclic stromal remodelling during menstrual cycles in humans. Ebaf/lefty participates in tissue remodelling of human endometrium by induction of matrix metalloproteases (MMP). METHODS: We describe the temporal expression and spatial distribution of lefty and tissue remodelling events in mouse endometrium. RT-PCR and real-time PCR were used to identify mRNA expression and western blots to analyse Lefty protein. Immunolocalization was performed with specific antibodies and horseradish peroxidase staining. RESULTS: Lefty was expressed in endometrium throughout the estrous cycle. Expression of MMP (MMP-2, -3, -7 and -14) was higher at estrus, metestrus and/or diestrus while collagen content of endometrium decreased in these phases. During pregnancy, lefty levels were higher on days 3-5 and were minimal by day 9. Similarly, expression of endometrial MMP was higher on days 3 and 5 of pregnancy and was low on day 9. During pregnancy, loss of collagen was initiated on day 3, persisted to day 5, and led to a significantly reduced collagen on day 9. Immunoreactive lefty decorated basal laminae, and was associated with extracellular matrix in stroma. CONCLUSIONS: Regulated expression and spatial distribution of lefty in mouse endometrium confines its biological impact on tissues that undergo remodelling during estrous cycle and pregnancy.     

Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

小鼠胚胎与子宫内膜中整合素α_5的表达

目的:观察整合素α5在小鼠早期胚胎和子宫内膜中的表达分布状况,探讨整合素在胚胎着床中的可能作用。方法:用免疫组织化学ABC法染色,对发育不同时期的小鼠胚卵和妊娠2 d～6 d、8 d、13 d以及非妊娠期的子宫内膜中整合素α5的表达分布进行形态学观察与半定量分析。结果:整合素α5的阳性反应物质在小鼠早期胚卵胞浆内;非妊娠期子宫内膜和妊娠期的脱膜上皮与基质细胞内均有整合素α5表达,植入前表达相对较弱,植入后随妊娠的进展逐渐增强。结论:提示整合素α5在小鼠胚卵着床中起重要作用:推测它不参与胚卵对母体子宫内膜的识别和粘附,而是参与了粘附后的侵入及胚胎发育过程。     

Cyclic and characteristic expression of phosphorylated Akt in human endometrium and decidual cells in vivo and in vitro

BACKGROUND: Akt is activated by phosphorylation and plays an important role in cell survival and maintenance of structure. METHODS: We investigated whether phosphorylated Akt was characteristically expressed in human endometrium in vivo and whether insulin-like growth factor-I (IGF-I) can activate Akt using cultured decidualized human stromal cells in vitro, using immunohistochemistry and Western blotting analysis. RESULTS: The levels of phosphorylated Akt protein increased markedly in the decidual tissues from ectopic pregnancy. The expression of phosphorylated Akt protein in stromal cells increased with the decidualization. The decidual cells showed strong cytoplasmic staining for phosphorylated Akt. However, cultured decidualized human stromal cells diminished phosphorylated Akt expression compared to control cells. IGF-I administration to decidualized human stromal cells significantly recovered pAkt expression. The effect of IGF-I on decidualized human stromal cells was blocked by an inhibitor of phosphatidylinositol-3 kinase (PI3K) (LY294,002). These results suggest that IGF-I may activate Akt via PI3K in human endometrium and decidua. The expression of phosphorylated Akt in stromal cells was only detected in the functional layer, where tissue remodelling occurs during menstruation or implantation. CONCLUSIONS: Akt activation may be involved in cell survival and extracellular matrix remodelling in human endometrium and decidua.     

Facilitation of decidualization by locally produced ghrelin in the human endometrium

Ghrelin acting via the growth hormone secretagogue receptor (GHS-R) stimulates GH secretion from pituitary glands. Both ligand and receptor are present in the pituitary, hypothalamus and many peripheral tissues including the uterus. This study demonstrates the cyclical expression of GHS-R and ghrelin in human endometrium. mRNA and protein for ghrelin and GHS-R were examined using RT-PCR and immunohistochemistry. Both ghrelin and GHS-R mRNA levels were highest in the secretory phase, with lower levels in the mid-proliferative phase and even lower expression in the menstrual phase. Immunoreactive ghrelin and GHS-R were confined predominantly to glandular epithelial and stromal cells with the greatest intensity of staining in secretory phase samples, consistent with the RT-PCR data. Additionally, we examined ghrelins effect on the decidualization of human endometrial stromal cells (HESCs) combined with sex steroid and cAMP treatments using prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) production as markers of decidualization. Ghrelin administered in combination with sex steroids to HESC, resulted in an increase in PRL and IGFBP-1 production above that obtained with cAMP, or sex steroids alone (P<0.001) whereas ghrelin in combination with cAMP inhibits the action of cAMP. These findings have potential clinical applications for the regulation of fertility.     

MUC4 gene polymorphism and expression in women with implantation failure

INTRODUCTION: The molecular mechanism of human embryo implantation is poorly understood. The role of MUC4 mucin, present in endometrial epithelium, has never been explored, and results obtained in animal studies strongly suggest a role in implantation. We investigated the role of MUC4 in human embryo implantation. METHODS AND RESULTS: We analysed the MUC4 variable number of tandem repeat (VNTR) polymorphism in three populations by Southern blot analysis: spontaneously fertile patients (C), infertile patients with repeated unexplained implantation failures after IVF (IF) and patients with a child after IVF (IVF-C). We found no differences in the size or allelic distribution of MUC4 VNTR between these three populations. We also examined, in IVF-C and IF groups, the endometrial expression of MUC4 mRNA as well as the expression of the MUC4 glycoprotein together with estrogen receptor (ER) and progesterone receptor (PR). No expression differences could be detected. However, we noticed a pattern of expression for MUC4 protein, which is limited to patches of cells in the luminal and glandular epithelium. CONCLUSIONS: We conclude that the different-sized MUC4 alleles do not interfere with implantation. The absence of coexpression of MUC4 and the steroid receptors suggests that MUC4 expression is not directly regulated by steroids.     

妊娠早期小鼠子宫内膜热休克蛋白70的免疫组化研究

目的 :研究妊娠早期小鼠子宫内膜热休克蛋白 70的表达。方法 :采用免疫组织化学方法。结果 :热休克蛋白 70主要存在于子宫内膜固有层的基质细胞及蜕膜细胞 ,内膜上皮、腺上皮中未见表达。与未孕小鼠相比 ,孕鼠热休克蛋白 70免疫反应阳性细胞显著增多 (P <0 .0 1 )且随妊娠日龄的增加而增加 (P <0 .0 1 )。结论 :热休克蛋白70可能参与了子宫内膜蜕膜反应中基质细胞的增殖 ,与蜕膜反应密切相关