环状RNA circCapzb在早孕小鼠围植入期子宫内膜中的表达

环状RNA(circular RNA,circRNA)是一类新型内源性非编码RNA,与多种疾病的发生、发展密切相关,但在胚胎着床的过程中罕见报道。该文旨在探讨环状RNA circCapzb在早孕小鼠围植入期子宫内膜中的表达。采用Real-time PCR检测正常妊娠小鼠孕第5天(d5)至第7天(d7)胚胎着床点及胚胎着床旁组织中circCapzb的表达水平;分别构建小鼠体内人工诱导蜕膜化模型和原代小鼠子宫内膜基质细胞体外人工诱导蜕膜化模型,采用Real-time PCR分别检测circCapzb在组织及细胞蜕膜化诱导模型中的表达;通过生物信息学预测circCapzb下游靶miRNA:miR-377-3p和miR-7005-5p,并采用Real-time PCR检测其在蜕膜化诱导模型中的表达。结果表明,circCapzb在小鼠孕第5天至第7天胚胎着床点的表达明显高于着床旁;circCapzb在组织及体内外细胞蜕膜化诱导模型中诱导组的表达明显高于未诱导组(对照组);circCapzb下游靶miR-377-3p和miR-7005-5p在组织及体内外细胞蜕膜化诱导模型中诱导组的表达明显低于未诱导组。该研究初步表明,circCapzb在小鼠早孕期胚胎着床点高表达,在组织及体内外细胞蜕膜化诱导模型中高表达,在小鼠妊娠早期子宫内膜蜕膜化过程中可能发挥作用,但具体机制有待进一步研究。     

蛋白激酶H11基因在小鼠子宫中的表达及其性激素调节

为研究蛋白激酶H11基因在生殖系统中的作用,我们采用半定量RT-PCR和原位杂交方法,研究了蛋白激酶H11基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和胎盘以及正常动情周期子宫中的表达及其受性激素的调节。结果发现:蛋白激酶H11基因在小鼠多种组织中都有表达,在卵巢及子宫等一些生殖相关的组织中表达水平较高;妊娠初始期,蛋白激酶H11基因在小鼠子宫内膜植入位点处有明显的高表达,其mRNA定位于腔上皮细胞和基质细胞中。在动情周期中,蛋白激酶H11基因在动情前期子宫中表达水平较低;卵巢切除模型显示雌激素和孕激素均可显著上调蛋白激酶H11基因的表达。以上结果提示蛋白激酶H11可能参与了胚胎植入过程中腔上皮细胞凋亡和基质细胞增殖与蜕膜化以及动情周期小鼠子宫内膜细胞的功能调节[动物学报51(3):462-468,2005]。     

前蛋白转换酶Furin和PC7在小鼠母胎界面的动态表达及其在胚胎粘附和扩展中的作用

通过免疫组化、免疫荧光和小鼠胚胎-子宫内膜上皮细胞共培养体系,研究了前蛋白转换酶Proprotein Convertases (PCs)家族中的Furin和PC7在小鼠妊娠早期子宫和胚胎中的表达及对胚胎植入的影响.结果显示:Furin和PC7在妊娠D1-D4小鼠子宫的腺上皮和D5-D7小鼠子宫的蜕膜、腺上皮高表达;PC7在植入前胚胎的2-细胞和4-细胞期表达很低,8-细胞期表达开始增加,囊胚期滋养外胚层有显著的高表达.Furin的抑制剂Dec-RVKR-CMK可显著抑制共培养体系中胚胎的粘附和扩展.以上结果表明,Furin在植入期胚胎的粘附和扩展中发挥重要作用.此外,Furin和PC7可能参与子宫内膜蜕膜化和早期胚胎发育.     

早孕小鼠子宫内膜钙网蛋白的表达规律

采用RT-PCR、间接免疫荧光组织化学、Western 印迹及原位杂交技术分别检测未孕(d0)和妊娠d1、d2、d3、d4、d5、d6、d7天小鼠子宫内膜中钙网蛋白(calreticulin, CRT)的表达规律, 探讨CRT在胚胎着床中的作用.结果显示CRT mRNA在妊娠小鼠子宫内膜中的表达明显高于未孕小鼠(P <0.05), 且随着妊娠天数的增加呈逐渐增强的趋势.间接免役荧光组织化学结果显示CRT表达于子宫内膜基质细胞、腺上皮以及腔上皮, 并在妊娠第4、5天基质细胞的胞浆中呈现高峰.实验结果提示, CRT在妊娠早期子宫内膜的持续表达, 可能通过调节整合素介导的细胞信号通路而调节胚胎滋养层细胞的黏附、侵袭, 参与胚胎着床.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

沙眼衣原体感染后妊娠大鼠子宫组织C3 和Crry mRNA的表达变化

目的通过大鼠生殖道沙眼衣原体(Chlamydia trachomatis,CT)感染模型研究CT初次感染后妊娠大鼠在胚胎着床期子宫局部补体C3和补体调节蛋白Crry的mRNA表达量的变化。方法选择成年雌性SD大鼠36只,随机均分为正常组和感染组,感染组通过阴道接种CT D型株。而后在妊娠大鼠的胚胎植入期(即妊娠第5、6、7d)分别处死大鼠,取子宫组织计数胚胎植入数,通过逆转录扩增(RT-PCR)方法半定量检测在胚胎植入期C3和Crry的mRNA的表达量的变化,并通过SPSS软件对数据差异显著性进行分析。结果1.感染组在植入期C3的mRNA比正常组高表达,差异有显著性(P<0.001);2.Crry的mRNA虽比正常组表达有所增加,但两组数据无显著性差异(P>0.05);3.感染组较正常组平均胚胎植入数下降(P<0.01),并且感染组C3mRNA的表达量与对应的胚胎植入数存在显著的负相关(r=(0.638,P<0.05)。结论大鼠生殖道感染CT后,妊娠大鼠胚胎植入数降低,而这种变化很可能与子宫局部补体C3的高表达及过量的补体活化有关。     

CT感染大鼠子宫内膜植入窗期基质金属蛋白酶的表达

目的 通过大鼠生殖道沙眼衣原体（chlamydia trachomatis,CT）感染模型研究CT初次感染后妊娠大鼠在子宫内膜植入窗期基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制物-1（TIMP-1）的表达对胚胎着床的影响。方法选择成年雌性SD大鼠30只,随机均分为对照组和感染组,感染组通过阴道接种CTD型株,而后在妊娠大鼠的植入窗期（即妊娠第5、6、7天）分别处死大鼠,采用链菌生物素蛋白一过氧化酶连接法和免疫组化测定两组子宫内膜种植窗期MMP-9及TIMP-1。采用HPIAS-1000高清晰度彩色病理图文小报告管理系统测定感染组和对照组中MMP-9＼T1MP-1在单位面积中各项免疫组织化学反应阳性颗粒的平均光密度值,并通过SPSS软件对数据差异显著性进行分析。结果在两组大鼠的子宫内膜中MMP-9均呈阳性表达。腺上皮细胞、腔上皮细胞和基质细胞胞浆内均可见棕黄色颗粒沉着,上皮细胞染色强度大于基质;感染组的表达较对照组弱,差异有显著性（P〈0．05）。TIMP-1表达特点和MMP-9相似,感染组的表达也较对照组弱,差异有显著性（P〈0．05）。结论生殖道感染CT后,妊娠大鼠子宫内膜植入窗期MMP-9及TIMP-1的低表达,可能是影响胚胎着床、导致不孕的重要因素。     

Wnt3a在小鼠孤雌胚胎及正常胚胎早期着床部位的表达变化

目的研究小鼠孤雌激活胚胎和体内正常发育胚胎在子宫中发生着床过程时,在早期着床部位上Wnt3a信号分子的表达变化。方法运用免疫组织化学染色法比较受体子宫中的孤雌胚胎着床位点和体内正常发育胚胎着床位点上Wnt3a的表达状况。结果免疫组织化学染色结果发现:(1)在着床期怀孕第5.5天和6.5天,受体子宫中孤雌胚胎的着床位点处和体内正常发育胚胎的着床位点处,Wnt3a在两种子宫上的表达情况相似,而在两种胚胎上的表达情况不同;(2)Wnt3a信号在空怀假孕母鼠子宫上表达情况与相同怀孕期有正常胚胎着床的子宫上的表达情况相似。结论胚胎着床与否及胚胎正常与否均不影响Wnt3a在小鼠早期着床期子宫上的表达,但在怀孕第5.5天和6.5天时孤雌激活胚胎和体内正常发育胚胎上Wnt3a的表达有差异。     

一氧化氮对小鼠胚胎围植入期子宫VEGF及其受体表达的调节

为研究NO在胚胎植入中的作用机理 ,本文采用子宫角注射、原位杂交及Westernblot方法研究了一氧化氮 (NO)在小鼠胚胎植入过程中对血管内皮生长因子 (VEGF)及其受体表达的调节。受试小鼠于妊娠第三天 (D3 )在一侧子宫角内注射一氧化氮合酶 (NOS)抑制剂N 硝基 L 精氨酸甲酯 (L NAME)或者L NAME与NO的供体硝普钠 (SNP)合用 ,另一侧子宫角为对照侧 ;收集并分别检测了D5,D6和D7天小鼠子宫中VEGF及其受体mRNA和蛋白的表达情况。结果显示 :与对照侧相比 ,L NAME处理后小鼠胚胎围植入期子宫中VEGF及其受体mRNA的表达有不同程度的下降 ;对VEGF及其受体蛋白表达水平检测表明 ,抑制的NO产生也使VEGF及其受体蛋白在小鼠围植入期子宫中的表达有不同程度的降低。当NOS抑制剂和NO的供体SNP同时注射小鼠时 ,VEGF及其受体mRNA和蛋白表达都恢复到正常水平。以上结果表明 ,在小鼠胚胎植入中NO可通过调节VEGF及其受体的表达参与血管新生 ,从而对胚胎植入起到调节作用     

妊娠大鼠子宫和胎盘IL-1 β表达及IFN-γ对其表达的影响

为探讨妊娠过程中白介素 1β (IL 1β)表达的变化及干扰素 γ (IFN γ)对其表达的影响 ,以妊娠Spre qne Dawley大鼠为模型 ,采用逆转录 -聚合酶链反应的方法检测了妊娠不同时期子宫和胎盘IL 1βmRNA表达的动态变化及IFN γ对子宫和胎盘IL 1βmRNA表达的影响。实验结果表明 ,在胚泡植入子宫内膜前 ,子宫组织内IL 1βmRNA的表达较高 ,胚泡刚植入时 ,IL 1βmRNA的表达显著降低 ,之后 ,随着胚胎的发育和胎盘的增生 ,子宫组织内IL 1βmRNA的表达逐渐升高。妊娠早、中、晚期胎盘IL 1βmRNA没有显著性差异。注射超生理剂量的IFN γ后 ,植入前期、植入后期及妊娠中期和妊娠晚期IL 1βmRNA的表达均显著减少 ,胎盘IL 1βmRNA的表达在妊娠晚期出现下降。上述结果提示 ,高剂量的IFN γ对妊娠一定时期的大鼠子宫和胎盘IL 1βmRNA的表达有抑制作用     

Expression of maspin in the early pregnant mouse endometrium and its role during embryonic implantation

Maspin (serpin B5), a tumor-suppressing member of the serine protease inhibitor family, participates in cell migration, adhesion, invasion, and apoptosis. These processes are also critical for embryo implantation, but the role of maspin in embryo implantation remains poorly understood. The aim of the present study was to investigate the spatiotemporal expression of maspin in early pregnant mouse endometrium and its role in embryo implantation. Real-time quantitative PCR analysis, immunohistochemistry, and Western blotting were used to detect mRNA and protein expression of maspin in the endometria of nonpregnant and early pregnant (days 0 to 7) mice. On day 3 of pregnancy, mice in the treated group (n = 20) were injected in the left uterine horn with antimaspin polyclonal antibody and in the right horn with purified rabbit IgG; control mice (n = 20) were injected only with purified rabbit IgG in the right uterine horn. Implanted embryos were counted on pregnant day 8. The mRNA and protein expressions of maspin were higher in the endometria of pregnant mice than nonpregnant mice; these levels gradually increased from day 1 of pregnancy, peaked on day 5, and then decreased on days 6 and 7. The mice treated with antimaspin polyclonal antibody group had far fewer implanted embryos than did the control group. Taken together, these results suggest that maspin, a tumor suppressor, may play an important role in embryo implantation.     

Dissection of 6.5 dpc mouse embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking. Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.     

电磁脉冲对孕鼠及其胚胎的影响

目的：探讨电磁脉冲（electromagneticpulses,E脚）对孕期小鼠及其胚胎发育的影响。方法：采用不同场强的EMP（分别为0、50、100、200、400kV·m-1）辐照器官形成期的BALB／c孕鼠,于孕18天解剖小鼠,测量孕鼠体重增长值、脏器／体重,胎盘重、胎鼠体重、身长、尾长,并记录吸收胎、死胎、生长发育迟缓及畸胎的数量。结果：各辐照剂量组孕鼠体重增长值、脏器／体重与对照组相比均无显著性差异（P〉0．05）;胎盘重、胎鼠体重、身长、尾长数值明显低于对照组（P〈O．01）。50、400kV·m-1和100、200、400kV·m-1辐照组可分别导致死胎率和生长发育迟缓率增加（P〈0．05）,在400kV·m-1的EMP辐照组中,畸胎数也有升高的趋势,其中,畸胎主要表现为肢体和骨发育异常。结论：本实验条件下,不同场强的EMP辐照可对器官形成期小鼠胚胎的生长发育产生一定的影响,胚胎肢芽及骨发育可能是EMP作用的特殊靶点。     

Expression and localization of SerpinB11 in mouse uteri during peri-implantation and the estrous cycle

Serine protease inhibitor (Serpin) B11 has been identified as a novel serine protease inhibitor but the biological functions of SerpinB11 in female reproduction are unknown. Therefore, we investigate the spatiotemporal expression and regulation of SerpinB11 during the peri-implantation period. SerpinB11 mRNA and protein were detected in the uteri of pregnant mice on days 1–8 (day 1?=?presence of a vaginal plug). SerpinB11 protein was localized in the embryonic implantation site on day 5 when embryo implantation occurred and was also strongly expressed in the primary decidual zone on day 6 and secondary decidual zone on days 7 and 8. The expression of SerpinB11 was induced by the activated blastocyst (based on patterns of expression during pseudopregnancy and delayed implantation) and by artificially induced decidualization. Moreover, expression of SerpinB11 was regulated by estradiol and progesterone in ovariectomized mice. The results were further supported by data from the estrous cycle. Thus, SerpinB11 is probably involved in embryo implantation and decidualization.     

Galactose-binding lectins as markers of pregnancy-related glycoproteins

Protein extracts from pregnant mouse endometria were compared with those obtained from non-pregnant and pseudopregnant mice to detect early pregnancy-specific galactose-rich glycoproteins. Gradient gel electrophoresis combined with lectin overlay and lectin histochemistry were used to identify Ricinus communis I (RCA-I), R. communis II (RCA-II) and Cytisus scoparius (CSA) lectin binding glycoproteins. Using this approach, galactose-rich glycoproteins were identified that were maximally expressed in the estrus phase of non-pregnant endometria and also those that had peak expression in pregnancy. Lectin histochemistry revealed pregnancy related changes in three portions of mouse endometrium: endometrial glands, luminal epithelium and its basement membrane. Two major glycoproteins (RCA-I reactive 64 kDa and RCA-II reactive 35 kDa) were specifically expressed in peri-implantation endometrium on days 3 and 4 of pregnancy. The appearance of these glycoproteins during the period of the implantation window in mouse suggests that they could serve as markers of uterine receptivity to the implanting blastocyst.     

Significant differences between mouse and human trophinins are revealed by their expression patterns and targeted disruption of mouse trophinin gene.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.     

Relationship between macrophages in mouse uteri and angiogenesis in endometrium during the peri-implantation period

The objective of this study is to examine the change in macrophage numbers, inducible form of NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expression both before and after embryo implantation in the uterine tissue of mice. In order to explore the mechanism of macrophages in endometrial angiogenesis, 8-week-old female mice were divided into three groups: pregnant group, pseudopregnant group (mated to male mice that had been vasectomized), and estrous group (unmated). Individuals from these three groups were sacrificed at time intervals D1.5 to D6.5. Formalin-fixed paraffin-embedded tissue was used for immunocytochemical localization of Mφ, iNOS, and VEGF utilizing standard methodology. The proportion of macrophages in the peripheral blood was determined by flow cytometry, and the relationship between macrophage, iNOS, and VEGF expression was analyzed. The proportion of peripheral blood macrophages in the pregnancy group was significantly higher than that in the other groups. The results of immunohistochemistry determined that the macrophages exhibited changes in both numbers and distribution. The number of macrophages in the endometrium of the pregnancy and pseudopregnancy groups was significantly higher than that in the control (estrous) group. In the pregnancy group, macrophage numbers dramatically decreased and gradually transferred to the perimetrium on D4.5. Immunostaining revealed strong staining in the pregnancy group and weaker staining in the pseudopregnant and control groups for both iNOS and VEGF. There was strong, dense immunostaining at the implantation site for both iNOS and VEGF, whereas light immunostaining was seen in interimplantation tissues on D5.5 to D6.5. In the pregnant group, peripheral blood and uterine macrophage proportions were negatively correlated, whereas the amount of macrophages, iNOS, and VEGF expression in the endometrium were positively correlated. The expression of iNOS and VEGF in the endometrium also displayed a strong positive correlation. In conclusion, during embryo implantation, macrophages levels decreased in the uterus, whereas the number of peripheral macrophages increased, suggesting that macrophages may migrate into the peripheral blood and uterus to adapt for pregnancy. Additionally, an increase in the expression of iNOS and VEGF was observed during the implantation window, implying that iNOS and VEGF may play an important role in promoting embryo implantation. The positive correlation between macrophages, iNOS, and VEGF in the implanting uterus implied that macrophages might regulate iNOS and VEGF during the implantation process.