白细胞介素8在着床前小鼠子宫及胚卵的表达

目的:观察着床前小鼠子宫及胚卵白细胞介素8(IL-8)的表达.方法:免疫组织化学显色及图像分析技术,对IL-8蛋白在妊娠1～4 d小鼠子宫及胚卵的表达进行定位及半定量分析;用RT-PCR技术检测妊娠1～4 d小鼠子宫及胚卵IL-8mRNA的表达情况.结果:与未孕小鼠相比,妊娠各天小鼠子宫中IL-8蛋白和mRNA表达明显升高,于妊娠4d表达最强.IL-8蛋白和mRNA均在妊娠2 d(Ⅱ细胞期)的胚卵表达最弱,妊娠4d(胚泡期)的胚卵表达最强.结论:妊娠1～4 d小鼠子宫及胚卵持续表达IL-8蛋白和mRNA,尤其是在着床前表达量升高,提示它们可能参与小鼠胚泡的着床过程.     

ATF4基因在小鼠胚胎围着床期子宫内膜的表达

目的 探讨转录激活因子4(ATF4)在小鼠胚胎围着床期子宫内膜中的表达规律及在胚胎植入过程中的作用.方法 妊娠0～7d小鼠子宫内膜,用RT-PCR、免疫组织化学法和Western blot分别检测小鼠内膜ATF4 mRNA及蛋白的表达;用子宫角内注射ATF4抗体进行干预.结果 1)妊娠各组小鼠子宫内膜ATF4 mRNA的表达量均显著高于未孕组d0(P ＜0.0S),且随妊娠天数的增加其表达量逐渐增高,到d4达到峰值,之后逐渐下降;2)ATF4蛋白主要在基质细胞胞质表达,其表达规律与mRNA表达规律基本一致.3)子宫角ATF4抗体注射后与对照组相比着床数明显减少.结论 ATF4在小鼠妊娠早期可能参与子宫内膜蜕膜化过程,有利于着床.     

整合素α4、β1 mRNA在着床前小鼠子宫内膜及胚卵的表达

目的 观察着床前小鼠子宫内膜及胚卵整合素α4、β1mRNA的表达。方法 用原位杂交技术检测妊娠1～4d小鼠子宫内膜及胚卵整合素α4、β1mRNA的表达。结果 整合素α4、β1mRNA主要表达在子宫内膜腔上皮，腺上皮，表达强弱不等。妊娠3d基质细胞出现阳性信号，4d增强；胚卵整合素α4、β1mRNA仅在Ⅱ细胞期弱表达，在其他时期表达较强。结论 妊娠1～4d小鼠子宫内膜及胚卵持续表达整合素α4、β1mRNA，提示它们可能参与小鼠胚泡的着床过程。     

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.     

HOXa-10基因在小鼠胚胎着床过程中的作用

目的探讨HOXa-10基因在小鼠胚胎着床过程中的作用。方法应用实时荧光定量PCR(FQ-PCR)分别检测未孕及早孕小鼠第1、2、3、4、57、d HOXa-10基因表达量的变化规律;同时用可以持续表达HOXa-10的DNA/脂质体复合物质粒和HOXa-10反义寡脱氧核糖核酸分别转染到受孕2d小鼠子宫角内,观察胚泡着床数。结果FQ-PCR结果显示,随着小鼠妊娠天数的增加,HOXa-10基因表达量也逐渐增高,在孕4d达到峰值,孕5d开始下降,孕7d时表达量已接近未孕状态;在用HOXa-10 DNA质粒/脂质体转染孕2d小鼠子宫角后,小鼠的胚泡着床数明显增加;用HOXa-10反义寡脱氧核糖核酸/脂质体转染孕2d小鼠子宫角后,小鼠的胚泡着床数明显减少(P<0.05)。结论在胚泡植入窗口期,子宫内膜HOXa-10基因表达上调,其可能参与了胚泡着床过程。     

小鼠胚泡植入早期FucT-Ⅶ和sLe(X)寡糖抗原表达

目的探讨妊娠第1～5天(d1～5)小鼠子宫内膜唾液酸化的路易斯寡糖[sLe(X)]合成关键酶α-1,3岩藻糖基转移酶基因(FucT-Ⅶ)的表达和其表面sLe(X)寡糖抗原表达对胚胎植入的影响。方法应用RT-PCR和免疫组化方法检测妊娠第1～5天小鼠子宫内膜FucT-Ⅶ及寡糖抗原的表达规律。用子宫角内注射sLe(X)单克隆抗体的方法检测小鼠胚胎着床数。结果妊娠第1～5天小鼠子宫组织均有FucT-Ⅶ的表达,且在妊娠d4表达更强(P<0.05)。妊娠第1～5天sLe(X)寡糖抗原表达在子宫内膜的腔上皮和腺上皮。单侧子宫角sLe(X)抗体注射后胚胎的着床数量明显较对侧(注射等体积生理盐水)减少。结论在小鼠胚胎着床过程中,FucT-Ⅶ及sLe(X)寡糖抗原可能参与了胚泡的定位、黏附及侵袭过程,在胚泡植入的早期发挥作用。     

T淋巴细胞侵袭转移诱导蛋白1在小鼠子宫内膜基质细胞-胚泡共培养体系中对胚泡黏附的影响

目的:检测不同浓度T淋巴细胞侵袭转移诱导蛋白1 (Tiaml)抗体下,小鼠着床窗子宫内膜基质细胞内的Tiaml蛋白表达及其对基质细胞-胚泡共培养体系中胚泡黏附的影响,探讨Tiaml对小鼠胚胎着床的影响.方法:提取着床窗小鼠子宫内膜基质细胞并构建基质细胞-胚泡共培养体系;分别用免疫细胞化学和免疫印迹法检测不同浓度抗Tiaml抗体时着床窗基质细胞Tiaml蛋白的表达及其对胚泡黏附的影响.结果:抗体干预后着床窗基质细胞内的Tiaml蛋白表达水平降低;在不同抗体浓度下胚泡黏附率有差异.结论:随Tiaml抗体浓度增加,着床窗小鼠子宫内膜基质细胞Tiaml蛋白表达量及胚泡黏附率降低.表明Tiarnl可能在胚泡植入过程起重要的作用.     

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。     

Effect of estrogen and progestogen on the expression of interleukin-8 in mouse endometrium and blastocyst during preimplantation

目的:研究雌、孕激素对着床前小鼠子宫内膜及胚泡白细胞介素8(IL-8)表达的影响.方法:利用经典小鼠胚泡延迟着床模型,应用免疫组织化学显色、免疫印迹及图像分析技术,对着床前小鼠子宫内膜及胚泡IL-8的蛋白表达进行定位及半定量分析.结果:IL-8主要表达于妊娠小鼠子宫内膜腔上皮细胞及胚泡内细胞群和滋养层细胞的细胞质内.在子宫内膜组织中,切除双侧卵巢后单独给予孕酮组(P4组),IL-8表达低于联合应用雌二醇和孕酮组(E2+P4组)和对照组;E2+P4组IL-8表达上调,但低于对照组.在各组胚泡中,单独给予孕酮获得的静止胚泡、IL-8蛋白含量低于正常胚泡;联合应用雌二醇、孕酮获得的激活胚泡、IL-8的蛋白表达高于静止胚泡.结论:切除卵巢后,联合应用雌、孕激素可上调着床前小鼠子宫内膜及胚泡IL-8蛋白的表达.     

宫腔注射PBMCs改善小鼠胚泡着床障碍及其机制初探

目的:探讨宫腔注射外周血单个核细胞(PBMCs)对着床窗期着床障碍模型小鼠胚泡着床的影响及其机制。方法:选取SPF级性成熟昆明雌性小鼠,将与同批雄性小鼠交配后发现阴栓者(妊娠第一天,Pd1)分为对照组、着床障碍组和PBMCs处理组,每组28只。PBMCs处理组于Pd2宫腔内注射PBMCs 2.5μl(1-2×10~7个/ml);着床障碍组和PBMCs处理组于Pd2早9∶00皮下注射米非司酮(0.08mg/0.1ml)复制着床障碍模型。对照组注射等量磷酸盐缓冲液(PBS)。Pd4晚10∶00处死各组部分小鼠,采用免疫组化染色法和Real Time-PCR检测着床窗期子宫内膜血管内皮生长因子(VEGF)及整合素β_3蛋白和mRNA表达水平,Pd8处死各组其余小鼠,观察胎泡着床情况,计算各组着床率。结果:与对照组相比,着床障碍组小鼠的着床率和着床窗期VEGF及整合素β_3蛋白和mRNA表达明显降低(P<0.01);PBMCs处理组小鼠着床率和着床窗期VEGF及整合素β3蛋白和mRNA表达较着床障碍组显著增加(P<0.01),且接近对照组水平(P>0.05)。结论:PBMCs宫腔内注射能提高着床障碍模型小鼠妊娠率,其机制可能与其增加着床窗期子宫内膜VEGF和整合素β_3表达,从而改善小鼠子宫内膜容受性有关。     

GRIM-19在小鼠植入前胚胎中的表达及其作用

目的 通过研究干扰素/维甲酸联合应用诱导细胞凋亡相关基因 (GRIM-19)在小鼠植入前胚胎中的表达及其与胚胎质量之间的相关性,探讨GRIM-19在小鼠植入前胚胎发育中的作用。方法 采用实时定量PCR,检测小鼠植入前胚胎（2-细胞期、4-细胞期、8-细胞期、桑葚胚和囊胚）中GRIM-19 mRNA水平 (n =16);将小鼠8 细胞期胚胎按卵裂球大小、形态、透明带、胞质碎片分为优胚组和非优胚组,分别检测两组胚胎GRIM-19 mRNA水平,并分析其相关性 (n =13);制作假孕鼠（23只）,并随机分为A组（12只）和B组（11只）,采用移植器将优质和非优质8-细胞期胚胎移入假孕鼠的子宫内,观察两组雌鼠妊娠率及产仔率。结果 GRIM-19在小鼠植入前各期胚胎中均有表达,其mRNA水平从2-细胞期逐渐增高,至8-细胞期达高峰,随后呈下降趋势。优质8-细胞胚胎的GRIM-19 mRNA水平明显高于非优胚组（P <0.05）。两组8-细胞胚胎移植后,A组雌鼠妊娠率及产仔率显著高于B组雌鼠（P<0.05）。 结论 小鼠植入前胚胎中GRIM-19的表达量随发育时程的改变而变化,且与胚胎质量密切相关。     

Possible involvement of crosstalk cell-adhesion mechanism by endometrial CD26/dipeptidyl peptidase IV and embryonal fibronectin in human blastocyst implantation

When human blastocysts hatch through the zona pellucida, gaining the ability to adhere to the endometrium, crosstalk between the embryo and the uterus may represent a successful outcome of their synchronized development and differentiation. CD26/dipeptidyl peptidase IV is known as a marker molecule of the implantation phase endometrium. To study the role of CD26 in implantation, 35 human hatched blastocysts were prepared by enzymatic treatment of expanded blastocysts that had been grown on schedule from frozen-thawed surplus embryos at the 2- or 4-cell stage. The blastocysts were placed on CD26-overexpressing or mock-transfected control monolayer cell cultures. The CD26-overexpression caused significantly higher blastocyst adhesion rate (53.3% versus 25.0%, P < 0.05) and significantly larger outgrowth area of trophectoderm (1.7-fold, P < 0.05). The second part of the present study was to show the expression of fibronectin, a CD26 ligand, in human preimplantation embryos, using the same donated resources. Fibronectin mRNA was detected by RT-PCR from the single hatched blastocyst (2/2) and from the single early blastocyst (3/6) but not from the single morula (0/5) samples. An indirect immunofluorescence technique verified the localization of fibronectin on the surface of the blastocyst. These results indicate that the adhesion mechanism by endometrial CD26 and embryonal fibronectin may be involved in human blastocyst implantation.     

小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶1表达的影响

目的 探讨小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶1（DNA methyltransferase 1，Dnmt1）表达的影响。方法 应用免疫组织化学方法确定Dnmt1蛋白在小鼠输卵管的组织学定位；选取植入前胚胎的2细胞期、4细胞期和8细胞期作为3个观察点，采用实时逆转录-聚合酶链反应和Western印迹分别检测正常妊娠和假孕小鼠输卵管上皮Dnmt1 mRNA和蛋白表达水平的变化。结果 Dnmt1蛋白主要表达于上皮层；妊娠组小鼠各期Dnmt1 mRNA表达水平均显著性低于同期假孕组（P〈0．05），妊娠组4细胞期Dnmt1蛋白表达水平显著性低于同期假孕组（P〈0．05）。结论 小鼠植入前期的胚胎对母体输卵管上皮Dnmt1 mRNA和蛋白表达存在调节作用，提示胚胎可能通过DNA甲基化间接调控输卵管上皮某些基因的表达，Dnmt1可能参与胚胎-母体交流的部分机制。     

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel and embryoblast. GLUT3 was exclusively localised in the apical membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.     

Expression and implications of tissue inhibitor of metalloproteinases-4 in mouse embryo

Matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), and MMP-TIMP interactions may contribute to the highly programmed process of embryo implantation. The loss of the delicate MMP-TIMP balance may lead to abnormal implantation. The role of TIMP-4 in mouse implantation has not been reported. This study examined mRNA and protein expression levels of TIMP-4 in the blastocyst and uteri of pregnant mice. We also investigated the effects of a specific TIMP-4 antibody on embryo outgrowth and on the gene and protein expression levels of two gelatinases. High levels of TIMP-4 mRNA and protein were detected in day 3-5 embryos and in the trophoblast cells of mice blastocysts, suggesting that TIMP-4 may be involved in embryo implantation. Furthermore, TIMP-4 antibody promoted blastocyst outgrowth in a dose-dependent manner, but had no effect on blastocyst adhesion to extracellular matrix. A specific TIMP-4 antibody also increased mRNA and protein expression levels and the enzymatic activities of gelatinase A (MMP-2) and gelatinase B (MMP-9). This study suggests that TIMP-4 may restrict mouse blastocyst outgrowth and embryo implantation by inhibiting the activities of MMP-2 and -9.     

Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development

The developmental potential in vitro and in vivo of preimplantation mouse embryos biopsied at the 4-cell, 8-cell and morula stages were investigated. Biopsy had the least impact when performed at the 8-cell stage. There was no effect of biopsy on the development of 8-cells of blastocysts in vitro (95% compared with 99% of controls) or the implantation rate after transfers (82 versus 87%, P greater than 0.05); however, fewer embryos (52 versus 71%, P less than 0.05) resulted in viable fetuses. There was no effect of biopsy at the 8-cell stage on fetal weight on day 17. Blastocyst formation in vitro was significantly less for 4-cell biopsies compared with their controls (76 versus 90%, P less than 0.001) and biopsy also affected the implantation rate (44 versus 59%, P less than 0.01). Biopsy was most detrimental when performed on morulae, reducing the implantation rate from 65% for controls to 21% for biopsies (P less than 0.001). Fetal viability was also markedly affected with a reduction on day 17 from 42 to 26% accompanied by a significant reduction (24%, P = 0.02) of the mean fetal weight. Handling of embryos for biopsy at the morula stage, which involved removal of the zona pellucida, was a significant but not complete cause of the reduced implantation potential observed (sham-controls and intact-controls: 34 and 65%, P less than 0.001), while puncture of the zona during the biopsy of 4-cell and 8-cell embryos had no effect. Therefore, the 8-cell mouse embryo is the most suitable state for embryo biopsy.     

Fertilization and early embryology: Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium

The formulation of chemically defined culture media that supportprimate embryo development would facilitate studies on primatepreimplantation embryogenesis. The specific aims of this studywere (I) to evaluate the development of macaque embryos in asimple, chemically defined, protein-free medium developed fora rodent embryo model, and (ii) to determine if a two-step progressiveculture system could enhance blastocyst development and zonaescape. In experiment 1, in-vitro-fertilized pronucleate stageembryos (n=81) from nine monkeys were randomly allocated toone of three treatments: (a) hamster embryo culture medium-6(HECM-6; chemically defined, protein- free medium), (b) CMRL-BCSmedium (modified CMRL 106 medium containing 20% bovine callserum; BCS), and (c) a two-step culture procedure (HECM-6 throughto the 8- to 12-cell stage, and CMRL-BCS medium beyond thatstage). Optimal development was attained equally (P0.05) withembryos cultured in CMRL-BCS medium or the two-step procedure(48 and 61% blastocysts respect ively). HECM-6 alone supporteddevelopment to the morula stage (72%) equally as well as CMRL-BCSmedium (80%) or the two-step procedure (69%), but not to theblastocyst stage (22 versus 48 and 61% respectively). Hatchingof the blastocysts was essentially limited to the serum-containingmedia (CMRL-BCS medium, 31% two-step procedure, 44%). In experiment2, in-vitro-fertilized pronucleate-stage embryos (n=87) fromnine monkeys were randomly placed in each of four two-step treatments:(a) HECM-6 through to the 8- to 12-cell stage and CMIRL-BCSmedium beyond that stage, (b) HIECM-6 through to the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HIECM 6 throughto the morula stage and CMIRL-BCS medium beyond that stage,and (d) HECM-6 through to the morula stage and HECM-6-BCS beyondthat stage. Greater (P 0.05) percentages of embryos developedinto blastocysts, expanded blastocysts and hatched blastocystswhen switched at the 8- to 12-cell versus the morula stage inthe second step medium. When transferred into BCS containingmedium at either the 8- to 12-cell or morula stage, embryosunderwent blastulation and expansion equally well in CMIRL-BCSmedium versus HECM-6-BCS. However, when embryos were switchedto the second step medium at the 8- to 12-cell stage, hatchedblastocysts 1690 were obtained more (P0.05) frequently in CMRL-BCSmedium (50.9%) than in HECM-6-BCS (37%). This work is the firstto produce in-vitro-fertilized primate blastocysts culturedfrom the pronucleate stage in chemically defined, protein-freemedium, and demonstrates that while primate embryos can formmorulae in such a medium, their requireents for blastocoel formationand zona escape appear to be more demanding, and may be acquiredas early as the 8-cell stag.