The expression and function of VEGF at embryo implantation “window” in the mouse

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays a critical role in angiogenesis. Recent reports indicated that VEGF was closely involved in embryo implantation and embryonic vasculogenesis. However, very little information is available about the detailed expression and function of VEGF at implantation “window”. In this work, VEGFs were primarily present on uterine epithelial cell monolayer and blastocysts including the outgrew trophoblasts at implantation window. VEGF antibodies decreased the number of mice embryos implanted and the percentage of blastocysts with attachment and outgrowth in a co-culture model in a dose-dependant manner. These findings demonstrate that VEGF is one of the essential cytokines for embryo implanta-tion in mouse. VEGF may act as a local mediator to regulate the maternal-fetal interaction, and facilitate blastocyst implantation.     

Role of αVβ3 integrin in embryo implantation in the mouse

Integrin, a heterodimeric adhesive molecule composed of α and β subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the “implantation window” stage, α Vβ 3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that α Vβ 3 integrin was clearly expressed in the mouse blastocyst. Injection of α Vβ 3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, α Vβ 3 integrin antisera at 1︰100 and 1︰200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of α Vβ 3 integrin antiserum was dosage/dilution-dependent. Thus, α Vβ 3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at “implantation” stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.     

Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction

Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

Effects of blocking LeY oligosaccharide on cell surface to MMPs secreted by blastocysts and epithelial cells in mousein vitro

In order to understand the role of Le+Y oligosaccharide antigen (Le+Y) during implantation, the relationship of Le+Y on the cell surface with matrix metalloproteinase (MMPs) secreted by blastocysts and monolayer epithelial cells during implantation in the mouse %in vitro% was studied by monoclonal antibody (mAb) AH-6, directed to Le+Y[Fuc α1-2 Gal β1-4 (Fuc α1-3) GlcNAc-], and gelatin zymography. The results showed that MMPs secretion was reduced after Le+Y on the cell surface of either epithelial cells or trophoblasts was blocked. It indicated that MMPs expression which played an important function during the process of implantation were regulated by Le+Y. Therefore, it was considered that Le+Y could regulate embryos invasion by some mechanism.     

IGF-Ⅱ and IGFBP-1 reversely regulate blastocyst implantation in mouse

Insulin-like growth factor (IGF)-Ⅱ and IGF binding protein (IGFBP)-1, members of IGF family, are important in the cyclic development of endometrium and the blastocyst implantation. In the present study, the indirect immunofluorescence showed that IGF-Ⅱ and IGFBP-1 were specifically expressed at the maternal-fetal interface. In a co-culture system, IGF-Ⅱ significantly enhanced the attachment and outgrowth of the blastocyst on monolayer of uterine epithelial cells, while IGFBP-1 did not affect the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography showed that IGF-Ⅱ enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not affect the activities of MMP-2 and MMP-9. In conclusion, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be regulated by the interaction between the IGF-Ⅱ-expressing invading cytotrophoblast and maternal deciduas-derived IGFBP-1.     

Interfamily pregnancy and expression of CD57, CD68 in deciduas between golden hamster and mouse

Pregnancy between different species is one of the key steps to interspecific somatic cell cloning. Although interspecific clone embryos have been constructed, they could not develop to birth after being transferred to recipi-ents. In order to clarify the mechanism of this phenomenon, interfamily pregnancy between golden hamste (Mesocricetus auratus) and mouse (Mus musculus) was studied. Co-culture results indicated that the adhesion ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 12, 24, 48 and 72 h after co-culture were all significantly lower than those of mouse blastocysts. The outgrowth ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 48, 72 h after co-culture were both significantly lower than those of mouse blastocysts (P < 0.01). Golden hamster抯 blastula could be implanted and develop to D 11 of pregnancy after being transferred to mouse uterus (the 7th day after embryo transfer). Compared to the transfer of mouse embryo to mouse uterus, the successful ratio of interfamily embryo transfer was lower and the bulk of fetus was smaller than that of intraspecific fetus. Compared to intraspecific preg-nancy of mouse, the remote decidual tissue of interfamily pregnancy on D8 is looser. At the same time, expressions of CD57 and CD 68 in remote deciduas were both higher than those in the secondary deciduas in both intraspecific and interfamily pregnancy. However, expressions of the two molecules in interfamily pregnancy were lower than those in intraspecific pregnancy. These results showed that interfam-ily pregnancy could be established between golden hamster and mouse. But the development of fetus in interfamily pregnancy was slower than that in intraspecific pregnancy. The expression difference of CD57 and CD68 indicates the difference of immunoreaction between interfamily and in-traspecific pregnancy, which may be one of the reasons lead-ing to interfamily pregnancy termination.     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

Regulation of embryo implantation by nitric oxide in mouse

Intrauterine injection and zymography were used to investigate the effect of nitric oxide (NO) on embryo implantation in mice. On day 3, one uterine horn of female pregnant mice was injected intraluminally with various doses of nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), while the contralateral horn served as control. Animals were sacrificed by cervical dislocation on day 7 of gestation, and the number of implanted embryos in each horn was calculated. The results showed that lower doses (0.05 mg L-NAME) did not inhibit implantation significantly (P > 0.05), but high doses (0.2 mg L- NAME) resulted in a significant reduction in the number of implanted embryos (P < 0.05). Co-administration of SNP, a generator of NO, with L-NAME would reverse the antiimplantation effect of L-NAME. To further understand the precise mechanism of NO in implantation, matrix metalloproteinase (MMPs) activities were detected by gelatin zymography. The reduction in the number of implanted embryos in 0.2 mg L-NAME treated group was associated with decreased MMP-9 activity but a stable MMP-2 activity. The activities of MMP-2 and MMP-9 were not changed in L-NAME and SNP treated group. These data suggest that NO acts as a mediator to regulate the activity of MMP-9, and facilitates embryo implantation.     

Expression, distribution and function of the focal adhesion kinase (pp125FAK) during murine ectoplacental cone outgrowthin vitro

Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.     

Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor.

A critical point during mammalian pregnancy is the implantation of the blastocyst when the embryo attaches to the wall of the uterus. The autonomously developing preimplantation embryo then becomes dependent on the maternal environment for its continued development. Little is known about the regulation of implantation, except that a complex interaction between peptide and steroid hormones synchronizes the preparation of the uterus for implantation with the development of the embryo. Whether the implantation event is under maternal or embryonic control is also unclear (reviewed in refs 1, 2). We have previously shown that a cytokine, leukaemia inhibitory factor (LIF), is expressed in the uterine endometrial glands specifically on the fourth day of pregnancy. This burst of expression is under maternal control and always precedes implantation of the blastocyst. Here we report that transient expression of LIF in mice is essential for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and do not develop. The blastocysts, however, are viable and, when transferred to wild-type pseudopregnant recipients, they can implant and develop to term.     

人Rab26基因的克隆和表达

以完整EST为参考序列设计引物,以人的胎脑cDNA为模板,用PCR方法筛选获得Rab26基因全长序列,并亚克隆到载体pGEM-T,真核表达载体pEGFP-N1和原核表达载体pET．32a（＋）中,RT-PCR显示该基因在不同组织的肿瘤细胞株中表达量有明显的差异．把Rab26基因转染入HeLa细胞中,通过与绿色荧光蛋白（GFP）基因融合表达,显示Rab26定位于胞内膜性细胞器上．在大肠杆菌表达系统获得Rab26基因的高表达．这些结果为进一步研究Rab26基因在细胞内吞和运输功能等方面打下了基础．     

Expression of matrix metalloproteinase-28 in human normal cytotrophoblast cells and a choriocarcinoma cell line,JEG-3

There are striking similiarities present between the behavior of invasive placental cells and that of invasive cancer cells. Matrix metalioproteinases (MMPs) are one of the most important mediators. MMP-28, the new member ofMMPs, was sequenced and identified recently. Expression of MMP-28 mRNA and protein in the cytotrophoblast cells anda choriocarcinoma cell line, JEG-3 cell, was conducted by zymography, RT-PCR and Northern blot. There is MMP-28mRNA expression in both the cytotrophoblast cells and JEG-3 cells by RT-PCR. The activity of MMP-28 in cytotrophoblast cells was significantly weaker than that in JEG-3 (P < 0.01) by zymography. Furthermore, mRNA expression of MMP-28 was significantly stronger (P < 0.001) in JEG-3than in human cytotrophoblast cells in a time-dependent way by Northern Blot. Our results suggest that MMP-28 may play a role in some of the tissue-remodeling events associated with normal pregnancy and tumor progression.``     

Replacement of inner cell mass in mouse

The intra- or inter-strain reconstituted blastocysts were produced by replacing the inner cell mass of Kunming mouse blastocysts with that of Kunming or C57BL/6 mouse strain blastocysts. A total of 192 intra-strain reconstituted blastocysts were transferred into 17 pseudopregnant Kunming mice, and 2 reconstituted embryos were developed into term: while 115 inter-strain reconstituted blastocysts were produced, analysis of the reconstituted blastocysts showed that the morphology and cytoskeleton srtucture of the blastocysts were not different from those of normal blastocysts, however, no viable offspring was obtained after embryo transfer for these inter-strain reconstituted blastocysts. The results demonstrated that the intra-strain reconstituted blastocysts could normally develop into term, whereas the inter-strain reconstituted blastocysts possessed less developmental potential as the intra-strain reconstituted blastocysts. This study may give light to solve the problem of low implantation rate and placenta abnormality in mammal cloning.     

P-5m八肽多克隆抗体的制备及应用

目的制备P-5m八肽的多克隆抗体并进行初步鉴定和应用,为研究P-5m八肽的功能及作用机制获得重要的实验工具.方法将9-氟甲氧羰基(Fmoc-)固相合成法合成的P-5m八肽纯化后与载体蛋白-血蓝素联接;取偶联后的多肽皮下注射免疫新西兰白兔,并加强免疫得到抗血清,以C18反相色谱分离柱分离纯化;通过间接ELISA方法测定血清效价;利用Transwell实验和Western blot方法初步鉴定多克隆抗体拮抗P-5m八肽抑制肿瘤细胞转移的效果.结果纯化后的抗体经间接ELISA测定效价均大于1∶160 000,表明获得高效价的多抗;通过Transwell实验证明该抗体能够封闭P-5m对肿瘤细胞侵袭的抑制能力;通过Western blot方法证实此抗体效价较高,特异性较强;该多克隆抗体能够封闭P-5m在SW620细胞中上调E-cadherin表达的作用,并阻遏P-5m对MMP-2和MMP-9表达的下调作用,与对照组比较差异具有统计学意义(P0.05).结论 P-5m多克隆抗体可用于Transwell和Western blot等实验,为研究P-5m八肽的功能及抗肿瘤转移作用机制提供实验依据.     

辛伐他汀对小鼠结肠癌细胞CT26凋亡的诱导作用及机理研究

考察了辛伐他汀对小鼠结肠癌细胞CT26生长率的影响,并探究其对CT26细胞凋亡的诱导作用及机理。以四甲基偶氮唑盐微量酶反应比色法测定辛伐他汀对CT26细胞生长的抑制情况;以蛋白质免疫印迹法测定细胞凋亡和增殖的标志蛋白PARP、Cleaved-PARP、P21和磷酸化的P53的蛋白表达水平;以AMPK的抑制剂Compound C来阻断AMPK信号途径后,观察辛伐他汀对CT26细胞中Cleaved-PARP表达的影响;以Compound C处理CT26细胞后,以RT-PCR和蛋白质免疫印迹法测定辛伐他汀在CT26细胞中诱导KLF2和KLF4的情况.辛伐他汀可以明显抑制CT26细胞生长,可以诱导CT26细胞中的凋亡相关的Cleaved-PARP蛋白上升,并且其作用机理可能和KLF4的上升有关.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.