Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

ROCK inhibition prevents early mouse embryo development

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.     

Cell surface changes on the mouse blastocyst at implantation

Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

不同发育时期小鼠胚泡表面Lewis寡糖抗原的表达

在胚泡表面表达的Lewis寡糖抗原 (LewisX ,LewisY)在胚胎发育以及着床过程中起重要作用 .应用免疫印迹和免疫荧光等方法对着床前小鼠胚泡表面的Lewis寡糖抗原进行分析 .结果发现 :小鼠胚泡LewisX寡糖蛋白有 2 7kD、2 9kD、6 8kD和 80kD 4种 ,LewisY寡糖蛋白有 70kD和 90kD 2种 ;2种寡糖抗原均在 8细胞时期开始表达 ,其中 ,LewisY寡糖抗原在胚泡表面的表达持续升高 ,直至胚泡着床 ;而LewisX寡糖抗原的表达则在桑椹期后逐渐降低 ,但仍在胚胎期的囊胚腔侧的顶端可见有部分表达 ;应用RT PCR的分析结果显示 ,LewisX合成的关键糖基转移酶FUT9基因在 4细胞及桑椹期高表达 ,到胚泡期虽然强度明显减弱 ,但仍有表达 ;而LewisY合成关键酶FUT1基因在 4细胞未见表达 ,在桑椹和胚泡阶段均有表达并逐渐升高 ,表达趋势与相应寡糖的表达趋势基本一致 .结果说明 ,在小鼠胚泡表面表达的Lewis寡糖抗原的表达受到相应糖基转移酶基因转录的调控     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

小鼠早期胚胎发育期间LIF基因表达的研究(简报)

白血病抑制因子(Leukemia inhibitory fac-tor,LIF)是近年来研究较为广泛的细胞生长调节因子之一。最初发现LIF能够在体外诱导小鼠髓样白血病细胞株M1细胞向正常细胞分化,进一步分离纯化蛋白以及克隆基因后发现LIF在体外还具有多种功能,作用于不同的靶细胞时引起的生理效应也各不相同。目前已知的功能有:刺激肝脏细胞急性期反应蛋白的     

Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.     

Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation

The mouse blastocyst forms during the 32-cell stage with the emergence of the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. This is a late event in the assembly of TJ-associated proteins within trophectoderm which, from our previous data, spans from 8- to 32-cell stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1(&agr;)+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to -insoluble pool, may regulate occludin association with ZO-1(&agr;)+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

The Bysl gene product, bystin, is essential for survival of mouse embryos

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin apparently disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.     

Association of collagen with preimplantation and peri-implantation mouse embryos

By immunofluorescence analyses, we have determined that Type III procollagen, Type III collagen, and B and C chains of basement membrane collagen are associated with preimplantation mouse embryos. Type III collagen and procollagen appear to be associated with embryos at the 4-cell stage and beyond, whereas antibodies to B and C collagen chains bind to 2-cell and later embryos. All of these collagen types are detected in increasing amounts as embryos develop in a defined medium, indicating that the embryo is capable of their synthesis. By the blastocyst stage, the collagens are primarily localized intercellularly. Cells of the inner cell mass (ICM) also bind collagen antibodies. When isolated ICMs become two-layered, both the inner presumptive ectoderm layer and the outer primitive endoderm layer react with antibodies to Type III collagen and procollagen. The endoderm cells also react avidly with antibodies to B- and C-chain collagens. Preimplantation embryos and ICMs fail to react with antibodies to Types I and II collagen. During peri-implantation stages, blastocysts continue to react with antibodies to Type III and basement membrane collagens. There is no obvious relationship between the intensity of immunofluorescence and the change in the blastocyst surface from nonadhesive to adhesive. Furthermore, blastocysts prevented from undergoing implantation-related events in utero and in vitro react extensively with collagen antibodies. Blastocyst surface collagens might, nevertheless, play a role in implantation by undergoing organizational changes.