小鼠胚胎致密化起始的调控机制

植入前小鼠胚胎的发育事件包括第一次卵裂、胚胎基因组激活、桑椹胚致密、囊胚形成。小鼠受精卵胚胎的致密化发生在8-细胞阶段晚期, 致密过程中, 胚胎卵裂球本身以及卵裂球之间发生了一系列的变化。这些变化包括卵裂球微绒毛以及胞质成分的极性化分布, 卵裂球之间形成特殊的胞间连接。致密化是哺乳动物胚胎发育过程中的第一个细胞分化事件, 即导致了内细胞团以及滋养外胚层的产生。植入后, 内细胞团将发育成为胚体, 滋养外胚层将发育成为胎盘等胚外组织。细胞粘附分子E-cadherin介导的胞间粘附起始了致密化。卵裂球发生粘附所需的组分在致密前已经存在, 但是直至8-细胞阶段晚期连接复合体才表现出明显的粘附活性。敲除E-cadherin基因, 发现母源性的E-cadherin足以介导致密。E-cadherin介导的胞间粘附是细胞粘附的第一步。文章综述了E-cadherin介导胞间粘附的具体过程以及蛋白激酶C(Protein kinase C, PKC)调控该过程的相关 机制。     

Regulation of S-adenosyl methionine synthesis in the mouse embryo

In early embryos, methylation is involved in "gamete imprinting" and inactivation of artificially introduced foreign genes. We studied the biosynthesis of the universal methylation cofactor: S-Adenosyl methionine (SAM). In the mouse, SAM conversion from methionine is limited by saturation of the methionine endogenous pool. SAM is present at a practically unchanged level from the unfertilized oocyte to early morula. SAM synthesis is increased at the time of compaction. In blastocysts, although methionine uptake is increased, the conversion rate from methionine is lowered. We observed no differences between C57 Black and Swiss albino random bred strains. In few experiments with human unfertilized oocytes and spared embryos, we observed higher methionine incorporation, and higher conversion to SAM. Next, the effect of two methylation inhibitors was tested, on early mouse embryonic development, at the one-cell and the two-cell stage. We found that ethionine is very toxic, even at the lowest tested concentration of 25 microM. Homocysteine is more potent at the one-cell stage than at the 2-cell stage, and it only partially blocks blastocyst formation from the 2-cell stage even at a concentration of 500 microM. It clearly acts as a methylation inhibitor; it lowers the SAM pool and the methylation index, SAH/SAM ratio (SAH: S-Adenosyl Homocysteine). We also found that homocysteine is an unexpected competitor for methionine influx and efflux.     

Regulation of redox metabolism in the mouse oocyte and embryo

Energy homeostasis of the oocyte is a crucial determinant of fertility. Following ovulation, the oocyte is exposed to the unique environment of the Fallopian tube, and this is reflected in a highly specialised biochemistry. The minute amounts of tissue available have made the physiological analysis of oocyte intermediary metabolism almost impossible. We have therefore used confocal imaging of mitochondrial and cytosolic redox state under a range of conditions to explore the oxidative metabolism of intermediary substrates. It has been known for some time that the early mouse embryo metabolises external pyruvate and lactate but not glucose to produce ATP. We now show at the level of single oocytes, that supplied glucose has no effect on the redox potential of the oocyte. Pyruvate is a cytosolic oxidant but a mitochondrial reductant, while lactate is a strong cytosolic reductant via the activity of lactate dehydrogenase. Unexpectedly, lactate-derived pyruvate appears to be diverted from mitochondrial oxidation. Our approach also reveals that the level of reduced glutathione (GSH) in the oocyte is maintained by glutathione reductase, which oxidises intracellular NADPH to reduce oxidised glutathione. Surprisingly, NADPH does not seem to be supplied by the pentose phosphate pathway in the unfertilised oocyte but rather by cytosolic NADP-dependent isocitrate dehydrogenase. Remarkably, we also found that the oxidant action of pyruvate impairs development, demonstrating the fundamental importance of redox state on early development.     

Immunohistochemical distribution of epimorphin in human and mouse tissues

A novel protein epimorphin has been identified as a mesenchymal signal factor. We reported previously ubiquitous expression of epimorphin in normal skin and a significant increased expression in diseased human skin. The present immunofluorescence study was conducted to determine systematically the distribution of epimorphin in adult human organs with an anti-epimorphin monoclonal antibody. Epimorphin was found to be widely distributed in all human organs examined. It was present in the connective tissue adjacent to or around various epithelial tissues, muscles and vessels. In particular, strong staining was present on the endomysium of muscles, the adventitia of blood vessels, along the sinusoidal lining of hepatocytes and connective tissue around epithelial cells, exocrine and endocrine glands. The results suggest that epimorphin may play a key role in maintaining normal tissue structure and interaction between mesenchymal tissue and epithelial tissue in vivo. ©; 1998 Chapman & Hall     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.     

Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation

During mouse oocyte maturation the regulation of the activity of a cytoplasmic maturation-promoting factor (MPF) was examined. The mouse MPF activity was determined based on its ability to induce maturation in immature starfish oocytes after microinjection with the cytoplasm from mouse oocytes. MPF appeared initially at germinal vesicle breakdown (GVBD), and its activity fluctuated in exact correspondence with meiotic cycles, reaching a peak at each metaphase and almost disappearing at the time of emission of the first polar body. Cycloheximide affected neither the initial MPF appearance nor GVBD. Thereafter, however, in the presence of cycloheximide the meiotic spindle was not formed and MPF disappeared, although the chromosomes remained condensed. After removing cycloheximide, MPF reappeared and was followed by the first metaphase and subsequently by polar body emission. Finally the meiotic cycle progressed to the second metaphase. Thus, for the appearance of MPF, there is a critical period shortly before the first metaphase, after which protein synthesis is required. In the presence of either cytochalasin D or colcemid, MPF activity remained at elevated levels. Addition of cycloheximide to such cytochalasin-treated oocytes, in which the meiotic cycle was arrested at the first metaphase, caused the MPF levels to decrease and was followed by movement of chromosomes to both poles where they decondensed and two nucleus-like structures were formed. Thus, the disappearance of MPF may initiate the metaphase-anaphase transition. Furthermore, detailed cytological examination revealed that chromosomes in cytochalasin-treated oocytes were monovalent while those treated only with cycloheximide were divalent, suggesting that dissociation of the synapsis is a prerequisite for chromosome decondensation after the disappearance of MPF. In all these respects, MPF seems to be a metaphase-promoting factor rather than just a maturation-promoting factor.     

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.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.     

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.     

Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions

Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.     

Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo.

The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure-eight shape. Seventy-two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P less than 0.001) more AT-blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT-embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD-embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.     

A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo

Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the A-P axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning including Distalless, Hoxd and Fgf family members was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, our data indicates that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells.     

Regulation of nerve growth factor synthesis in mouse submaxillary glands by testosterone.

—It has long been known that the activity of nerve growth factor (NGF) in extracts obtained from the male mouse submaxillary gland is higher than in extracts from the female gland, and that the activity present in female glands can be increased by testosterone treatment. This communication presents a study of the mechanism of the testosterone effect. Of several different steroids administered to female Swiss–Webster mice only testosterone propionate led to increased gland NGF activity. The increase did not appear to be due to an enhancement of the activity of pre-existing molecules on sympathetic nerve fiber outgrowth, or due to an altered affinity for the specific antibodies used in the estimation of NGF content, but appeared rather to be due to an accumulation of NFG molecules. The kinetics of change in the male gland NGF content upon castration and secondary testosterone propionate stimulation was analyzed by application of the plateau principle. The rate of loss of NGF from this organ was not measureably different between the castrate and testosterone propionate stimulated state. On the other hand, there was estimated to be a 10-fold difference in the rate of input between the basal and steroid stimulated state. Tracer amounts of radioiodine labelled NGF administered i.v. was not accumulated by the gland, and there is no evidence for uptake of this protein from the circulation. We, therefore, infer that the increased NGF concentration in male submaxillary glands is due to a 10-fold increase in the rate constant of synthesis.