层粘连蛋白及其肽段对小鼠胚泡粘附和扩展的作用

作为细胞外基质的主要成分之一的层粘连蛋白（ＬＮ）,对小鼠胚泡的粘附、扩展有显著促进作用。ＬＮ分子上的一些活性位点对胚泡的粘附和扩展也具有一定的作用,含ＲＧＤ位点序列的合成肽段ＲＧＤＳ对胚泡的粘附有促进作用;含ＹＩＧＳＲ位点序列的合成肽段ｃＹＩＧＳＲ对胚泡的粘附和扩展均有促进作用;且ＲＧＤＳ和ｃＹＩＧＳＲ可以竞争性抑制ＬＮ对胚泡粘附和扩展的促进作用。以上结果表明ＬＮ对胚泡的作用是通过胚泡上不同的ＬＮ     

小鼠外胎盘锥次级滋养层巨细胞的分离、培养与鉴定

目的：建立小鼠外胎盘锥次级滋养层巨细胞的分离、培养方法.方法：在小鼠妊娠第8 d,通过机械分离和组织块翻转干涸法分离、培养小鼠外胎盘锥次级滋养层巨细胞;免疫细胞化学鉴定细胞来源与纯度;酶谱方法检测其所分泌基质金属蛋白酶2和9（matrix metalloproteinase-2,9,MMP-2,9）结果：利用机械分离法分离外胎盘锥可生长于Matrigel包被的培养板上,并生长出次级滋养层巨细胞,表达特异性标志物Cytokeratin;体外培养24 h、48 h后,外胎盘锥滋养层巨细胞的粘附率分别为（83.3±9.1）%和（92.4±7.3）%;扩展率分别为（45.5±5.3）%和（56.4±6.8）%;及其所分泌的MMP-2,9的24 h光密度值分别为（87±4.7）和（351.5±25.2）;48 h分别为（186±40.2）和（556.5±61.5）.结论：采用机械分离和组织块翻转干涸法,可简单、快捷地获得高纯度小鼠外胎盘锥次级滋养层巨细胞.     

白血病抑制因子与哺乳动物的胚胎着床(英文)

胚胎着床是处于活化状态的胚泡与处于接受态的子宫相互作用,最后导致胚胎滋养层与子宫内膜建立紧密联系的过程。已证实白血病抑制因子(LIF)在哺乳动物胚胎着床过程中起着十分重要的调节作用。LIF通过其受体及信号传递亚单位gp130发挥其生物学功能。LIF对胚胎发育到胚泡阶段及以后内细胞团和滋养层细胞的生长和分化有明显的促进作用。 在小鼠中,LIF及其受体和gp130在着床期小鼠子宫内表达量最高,因此LIF可能在小鼠胚胎着床过程中起重要作用。在人中,LIF在子宫内膜中的表达与人胚胎着床的时间一致,提示LIF可能与人的胚胎着床紧密相关。此外,LIF在猪、羊、水貂、兔和臭鼬等动物胚泡着床前和着床期的子宫中也都有表达,并在着床期出现峰值。因此,LIF也可能在这些动物的胚胎发育和着床过程中有重要作用。LIF受体基因敲除小鼠表现为胎盘发育不全,这说明LIF对小鼠胎盘形成和胎盘的功能维持起重要作用。 小鼠子宫中LIF的表达可能受雌激素而上调。美洲长尾猴(绒)及兔子宫中LIF的表达则呈孕酮依赖性。然而孕酮可抑制人着床期子宫内膜腺上皮和蜕膜组织内LIF的表达。在不同种类的动物中,LIF在子宫中的表达有不同的调节机制。 胚泡在LIF基因敲除的雌鼠子宫内不能着床的原因并不是由于胚泡发育异常,而是由于雌鼠不能表     

整合素及其在胚泡植入中的作用

整合素是一类由α、β亚基构成的异二聚体粘附分子,能够与胶原蛋白、纤连蛋白和玻连蛋白等细胞外基质组分相互作用,调节细胞粘附和通讯.作为双向传递分子,整合素通过“胞内→胞外”和“胞外→胞内”两种方式介导细胞信号传递.成功的植入是侵入性的胚泡和接受性的子宫内膜相互作用的结果,整合素能够调节胚泡滋养层与子宫内膜之间的细胞-细胞及细胞-细胞外基质相互作用,是“植入窗口”期子宫内膜接受性的标记分子.     

小鼠胚胎与子宫单层上皮细胞共培养的研究

本文报道建立了小鼠胚胎与小鼠子宫单层上皮细胞体外共培养系统。结果揭示;小鼠胚胎与 子宫单层上皮细胞共培养可以促进胚胎的发育、粘附和扩展;如果培养液中加入 ３、６７ × １０－６ｍｏｌ／Ｌ １７β－雌二醇,可以显著提高胚胎在共培养系统中的发育率、粘附率和扩展率。以上结果表明：小鼠 胚胎与小鼠子宫单层上皮细胞共培养系统是研究胚泡着床机理较理想的研究手段。     

氧对滋养细胞侵入的调节作用

胚胎在子宫内的植入过程包括粘附、迁移和侵入等步骤 ,其中源自胚胎滋养外胚层的绒毛外滋养层细胞对子宫基质的合理侵入 ,是决定能否正常妊娠的关键。人类胚胎植入部位及胎盘组织内氧含量的变化持续从受孕至分娩的全过程 ,在此期间与滋养细胞侵入相关的一些指标随氧环境的变化而改变 ,提示氧对滋养细胞的侵入过程起一定调节作用。1.早孕组织的氧含量变化在人类胎盘发育的早期 ,来自胚泡的滋养细胞突破子宫上皮细胞及其基底膜 ,侵入下层的子宫基质 ,逐步形成绒毛。根据其位置及功能的不同 ,绒毛可分为漂浮绒毛和固定绒毛两种 ,前者负责胚胎与…     

哺乳动物胚泡着床及影响因素

哺乳动物胚泡着床是生殖过程中的关键环节。受精卵经过早期发育形成了胚泡，胚泡脱去透明带后，经定位、粘附、滋养层侵入，植入到子宫内膜中，同时母体子宫内膜发生蜕膜化控制植入的程度，最终完成着床过程。着床过程受多种因素的影响，主要因素有：母体子宫内膜和胚泡发育的同步化，母体的激素环境，胚泡分泌的激素，母体子宫的接受性及局部免疫保护作用。     

天花粉蛋白对胚泡影响的超显微观察

天花粉蛋白引起中期流产的机制,在于它对胎盘滋养层细胞具有一定的损伤专一性;这种专一性也表现在离体培养的实验。天花粉蛋白能否用于抗早孕,是一个值得探讨的问题。早期胚泡由围绕囊胚     

滋养层模型研究进展

滋养层细胞是胎盘的主要组成成分,滋养层发育不良会导致胎盘缺陷,进而引发相关妊娠疾病。为阐明疾病致病机制及治疗相关疾病,有必要了解滋养层发育机制。鉴于伦理限制,人类滋养层研究并没有像小鼠那样成熟,合适的体外模型是目前研究的重点和难点。干细胞、类器官、材料科学及测序技术的快速发展极大地促进了体外滋养层发育模型的建立与研究。近年来,国内外研究团队陆续报道了一系列二维(2D)和三维(3D)滋养层发育模型,用于模拟体内胎盘发育和功能。本文主要围绕滋养层的发育、体外滋养层发育模型的建立及其应用等进行综述,为相关研究提供参考。     

γ-氨基丁酸信号对小鼠胎盘早中期建立的影响

为了观察γ-氨基丁酸B型受体R1亚基(GABAB1R,GB1)在妊娠小鼠第1天至第8天(D1～D8)子宫中的表达规律,探讨γ-氨基丁酸(GABA)信号是否参与了胎盘早中期建立.应用半定量RT-PCR、免疫组织化学及Western blotting检测GB1在妊娠小鼠D1～D8子宫中的表达情况,原代分离D8.5的绒毛膜锥(EPCs)及蜕膜细胞,检测GABA及GABA B型受体激动剂baclofen、拮抗剂2-hydroxysaclofen对EPCs黏附及外延生长以及对蜕膜细胞侵袭能力的影响.D8时体内注射0.05 g/kg、0.5 g/kg及5 g/kg的GABA,于D14收集胎盘,制作石蜡切片.结果显示,GB1 mRNA及蛋白动态表达于D1～D8子宫.100μmol/L GABA及5μmol/L Baclofen促进EPCs的外延生长,抑制蜕膜细胞的侵袭,同时,20μmol/L 2-hydroxysaclofen成功逆转GABA对EPCs及蜕膜细胞的侵袭调节作用.5 g/kg的GABA可导致D14小鼠胎盘的迷路层滋养层细胞增多,母鼠血窦及胎鼠血管减少或发育不良,海绵滋养层的细胞滋养层细胞变小,糖原细胞减少或消失.结果提示,在小鼠早中期胎盘建立过程中,GABA信号促进滋养层侵袭至母体蜕膜组织,而抑制蜕膜细胞积极主动的侵袭行为,并可损害胎盘的结构.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre‐ and peri‐implantation embryonic development in mice

Recently, many studies have investigated the role of extracellular vesicles (EVs) on reproductive events, including embryo development and death, oviduct–embryo crosstalk, in vitro fertilization and others. The aim of this study was to demonstrate whether outgrowth embryo–derived EVs function as bioactive molecules and regulate mouse embryonic developmental competence in vitro and implantation potential in utero. The EVs from mouse outgrowth embryos on 7.5 days postcoitum were detected and selectively isolated to evaluate the embryotrophic functions on preimplantation embryos. Developmental outcomes such as the percentage of blastocyst formation, hatching, and trophoblastic outgrowth were assessed. Furthermore, the total cell number and apoptotic index of blastocysts, which were incubated with EVs during the culture period, were evaluated by fluorescence microscopy. Implantation potential in utero was investigated following embryo transfer. The EVs from outgrowth embryo–conditioned media have rounded membrane structures that range in diameter from 20 to 225 nm. Incubation with EVs improved preimplantation embryonic development by increasing cell proliferation and decreasing apoptosis in blastocysts. Moreover, the implantation rates following embryo transfer were significantly higher in EV–supplemented embryos compared with the control. Collectively, EVs from outgrowth embryo could enhance the embryonic developmental competence and even implantation potential in mice.     

Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.     

Effects of leukaemia inhibitory factor on embryo implantation in the mouse

Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine. Recent reports indicate that LIF is relevant to murine embryo implantation. In this work, results of indirect immunofluorescence under a confocal microscope illustrated that LIF was mainly located in the uterine lumen and uterine epithelial cells in pregnant mice on day 4. The number of embryos implanted in pregnant mice on day 8 decreased significantly after injection of 3 microg LIF antibodies into a uterine horn (P<0.001), which demonstrated again that LIF is a critical factor for embryo implantation. In a co-culture system, LIF (0.1 ng/ml, 1 ng/ml, 10 ng/ml and 100 ng/ml) significantly enhanced the blastocyst outgrowth after 24, 48 or 72 h of co-culture, and outgrowth areas after 72 h of co-culture. Conversely, 5 microg/ml and 10 microg/ml, but not 1 microg/ml, LIF antibodies decreased the percentage of blastocysts with outgrowth; only 10 microg/ml LIF antibody inhibited blastocyst outgrowth area significantly (P<0.001). However, neither LIF nor its antibodies changed embryo attachment. Analysis of correlation showed that the effects of LIF or its antibodies on the blastocyst outgrowth were dose-dependent. In summary, different pathways may exist to regulate the blastocyst attachment and outgrowth on a monolayer of uterine epithelial cells. LIF protein from the maternal uterus exerts an essential role in embryo implantation in the mouse, which is mediated by stimulating trophoblast outgrowth, but not by promoting the attachment.     

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

Effects of anandamide on embryo implantation in the mouse

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.     

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.