Evidence linking programmed cell death in the blastocyst to polyamine oxidation

Programmed cell death occurs in the inner cell mass during blastulation concomitant with the loss of its trophectodermal potential, and blastocele fluid kills malignant inner cell mass cells with trophectodermal potential (ECa 247) but spares those with embryonic potential (P19). A previous study had shown that blastocele-like fluid from embryoid bodies of the teratocarcinoma C44 contains a low-molecular-weight cytotoxin that exhibits the same target-cell selectivity as normal blastocele fluid. The current paper shows that the preferential killing of cells with trophectodermal potential is caused by hydrogen peroxide generated during the oxidation of polyamines in the cyst fluid by amine oxidases. The greater resistance of cells with embryonic potential to hydrogen peroxide is due to glutathione-dependent mechanisms. These data lead to the conclusion that an amine oxidase in the blastocyst oxidizes polyamines in blastocele fluid, generating hydrogen peroxide which causes programmed cell death of normal and malignant cells with trophectodermal potential.     

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.     

Imaging filopodia dynamics in the mouse blastocyst

During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.     

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid

Abstract. There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential [26]. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential [5, 8–10]. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No troph-ectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.     

Ultrastructure of mouse blastocysts during blastocoele expansion

Summary The ultrastructure of mouse blastocysts with nascent and expanded blastocoele is described. In the early blastocyst cells adhere tightly and the blastocoele is often limited at its apex by cells containing a midbody. The expanding blastocyst exhibits a loose cell arrangement due to the presence of intercellular spaces and a cortical layer of filaments develops in cells enclosing the expanded blastocoele. When the blastocoele exceeds 1/2 the embryo diameter desmosomes appear between trophectoderm cells. Possible factors essential for blastocoele formation are discussed.     

Mosaicism in the mouse trophectoderm

The issue of mosaicism in the mouse trophectoderm is examined by reviewing two sets of evidence: one arguing for a mosaic, the other for a non-mosaic character. Evidence for mosaicism includes documented cellular contribution from the inner cell mass to the trophectoderm, and data that reveal the gradual pace of the allocation process that separates the inner cell mass and trophectoderm lineages. Evidence suggesting a non-mosaic character for the trophectoderm is based on the polarization process undergone by exterior cells in the eight-celled embryo, the heritability of the changes brought about by this process, and the formation of gap junctions between the resulting apolar, trophectoderm progenitor cells. Since inner-cell-mass cells are developmentally labile, spatially heterogeneous and translocate to the polar trophectoderm, it is concluded that the polar trophectoderm is a mosaic tissue.     

Phagocytosis reveals a reversible differentiated state early in the development of the mouse embryo

Mural trophectoderm cells of the mouse embryo possess a phagocytic potential as early as 3.5 days post coitum (d.p.c.). This first differentiated function shows a graded variation along the embryonic-abembryonic axis, from a maximal activity in the non-dividing cells of the abembryonic pole to a complete lack of activity in the replicating polar trophectoderm overlying the inner cell mass (ICM). This pattern can be explained by a negative control exerted by the ICM. Addition of FGF4, a factor secreted by ICM cells, strongly inhibited phagocytosis while inducing resumption of DNA synthesis in mural trophectoderm cells, revealing a reversible, FGF4-dependent differentiation state. Under conditions in which a small cluster of mural trophectoderm cells (<10) had internalized large particles, these otherwise morphologically normal embryos could not implant in the uterus, indicating that cells at the abembryonic pole have a critical role in initiating the implantation process. At post-implantation stages (6.5-8.5 d.p.c.), the ectoplacental cone and secondary giant cells derived from the polar trophectoderm also contained active phagocytes, but at that stage, differentiation was not reversed by FGF4.     

Integrity of the preimplantation pig blastocyst during expansion and loss of polar trophectoderm (Rauber cells) and the morphology of the embryoblast as an indicator for developmental stage

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.     

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.     

Hydrogen peroxide as a mediator of programmed cell death in the blastocyst

Previous work identified in blastocele fluid a soluble activity which killed embryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H2O2 [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H2O2 capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H2O2 toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H2O2 production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.     

Ability of outside cells from preimplantation mouse embryos to form inner cell mass derivatives

Previous studies have shown that inside cells in the preimplantation mouse embryo do not become committed to the formation of inner cell mass until after blastocyst formation. However, it is not yet clear whether outside cells are also labile late in preimplantation development or whether they become restricted to trophectoderm development at an earlier stage. The present study investigates the potency of outside cells isolated from late morulae just prior to blastocyst formation and shows that some, if not all, outside cells retain the potential to form inner cell mass derivatives in vitro and in vivo. This suggests that trophectoderm cells are not restricted in potential earlier than ICM cells and that all cells of the early embryo may be labile at least until blastulation.     

Cell allocation to the inner cell mass and the trophectoderm in rat embryos during in vivo preimplantation development

Summary The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.     

异源Oct-4基因启动子在猪、兔和小鼠早期胚胎中的时空表达活性

Oct-4是一种哺乳动物早期胚胎中特异表达的转录因子,它与细胞多能性的维持有关．异源Oct-4基因在早期胚胎中的表达模式尚不明确．构建了一个以完整的牛Oct-4调控区指导GFP表达的转基因结构pOct-4(p)-GFP,通过单精子注射的方法将其导入猪、兔和小鼠的受精卵中,分析其在胚胎发育过程中的表达情况．结果显示：牛Oct-4启动子驱动的GFP基因在3个物种的2-细胞胚胎就已经开始表达,在囊胚期表达加强且只特异表达于内细胞团中,而不表达于滋养层．研究表明：牛的Oct-4启动子在其他物种中也具有表达活性,异源性Oct-4启动子在不同物种的早期胚胎中具有相似的表达模式．     

囊胚形成的基因表达与调控(英文)

囊胚形成是胚胎早期发育过程中一个重要阶段 ,涉及几个重要的生理事件 ,即细胞融合 (compaction ,亦称致密化作用 )、囊胚腔出现、囊胚腔扩张及滋养层和内细胞团的分化。在细胞间连接蛋白的作用下 ,各种细胞间连接方式逐步建立起来 ,在合子型基因组表达调控下 ,促进了最终囊胚的形成。细胞间连接蛋白和细胞粘附相关蛋白参与组建各种细胞间连接 ,参与细胞融合、囊胚腔形成、滋养层分化和囊胚扩张等过程。通过顶部的紧密连接、侧部的缝隙连接和桥粒 ,建立起细胞的连接复合体。在人胚胎 8 细胞之前 ,卵裂球细胞界限明显 ,可能以中间连接方式相互作用 ;8 细胞期发生致密化作用 ,通过紧密连接将细胞分成顶部和基部 ,使得胚胎处于半封闭状态 ,促进胚胎内部积液 ,形成囊胚腔。细胞融合的同时也产生缝隙连接。桥粒最初出现在人胚胎达到 3 2 细胞阶段 ,桥粒连接参与囊胚腔形成以及在囊胚扩张时维持滋养层的稳定性。桥粒由一些跨膜粘蛋白组成 ,包括参与细胞内粘附的桥粒子和桥粒球以及一些细胞质内蛋白 (如desmoplakins,plakoglobin ,plakophilin) ,由细胞内蛋白质形成空斑结构并介导细胞角蛋白丝固定。对植入前牛胚胎的研究表明 ,只有DcII,DcIII和plako三种桥粒蛋白参与桥粒组建。在鼠囊胚中DcII的表达部位位于     

Trophectoderm cell subpopulations in the periimplantation mouse blastocyst

Trophectoderm of the preimplantation mouse blastocyst is composed of two cell subpopulations relative to their proximity to the inner cell mass. The polar trophectoderm overlying the inner cell mass proliferates to form the ectoplacental cone, and the mural trophectoderm endoreplicates and gives rise to giant cells. We examined specific differences in the two trophectoderm cell populations using a lectin (Dolichos biflorus) to detect cell surface characteristics and a simple sugar (D-Gal) to detect differences in incorporation. During the first day of delayed implantation, the mural trophectoderm presented twice as many lectin binding sites as did the polar trophectoderm. The mural trophectoderm of both nondelaying and delayed implantation blastocysts showed a greater rate of incorporation of the tritiated sugar by presenting more reduced silver grains in radioautograms. These results indicate that the mural trophectoderm and polar trophectoderm are two distinct cell types in the periimplantation blastocyst.