Differentiation Of Blastomeres Research Articles

The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.

Governing cell lineage formation in cloned mouse embryos

Blastomeres of the pre-implantation mouse embryo form trophectoderm and inner cell mass via a process that requires the transcription factors Tead4, Cdx2, Oct4 and Nanog. In mouse morulae cloned by somatic cell nuclear transfer, we observed that the trophectoderm transcription factor Cdx2 is expressed very differently at the protein level compared to time- and stage-matched fertilized counterparts. Protein levels of Cdx2 in cloned embryos appear ‘erratic,’ i.e. are widely distributed, when plotted as histograms. In contrast to Cdx2, protein levels of the upstream factor Tead4 and of inner cell mass transcription factors Oct4 and Nanog are similar in cloned and fertilized embryos. These observations suggest that trophectoderm formation is initiated but not maintained correctly in cloned mouse morulae, which is consistent with cloned blastocysts' limited implantation and post-implantation success. Because a cell's ability to differentiate is greatly enhanced if it is surrounded by more cells differentiating the same way, a concept designated community effect by Gurdon, we reasoned that the insufficient cell numbers often observed in cloned embryos might lead to premature Cdx2 expression and differentiation of blastomeres into trophectoderm. Therefore, we created larger cloned embryos by aggregating them at the 4-cell stage. Homologous aggregation stimulates expression of multiple signaling pathways' components and results in cloned embryos with levels of Cdx2 similar to fertilized embryos. Most of the resultant morulae and blastocysts consist of cells of all three founders, indicating that aggregation increases stability of all of the individual components. We conclude that the induction of pluripotency in cloned embryos is more efficient than previously assumed, and we propose that a minimum cell number is necessary to stabilize pluripotency and inhibit premature expression of Cdx2 in cloned mouse embryos.

Distributional Changes in Lysosome-Like Bodies in Mammalian Embryos during the Course of Blastocyst Formation.

Distributional changes in lysosome-like bodies (LBs) in mouse, rat, rabbit and bovine embryos during the course of blastocyst formation were studied with acridine orange (AO) staining. AO-stained LBs were distributed throughout the cytoplasm of all round blastomeres in untransformed 8- and 16-cell mouse embryos, 32-cell rabbit embryos and 16-cell bovine embryos, whereas they were observed from the nucleus to the apical cytoplasm of all round blastomeres in 8-cell rat embryos. In morulae of these animals, LBs were present in the entire cytoplasm of inner round blastomeres, and were localized around the nucleus in outer flattened (mouse) and cuboidal (rabbit and cow) blastomeres or the apical cytoplasm in outer flattened (rat) blastomeres.In blastocysts, LBs were present in the entire cytoplasm of inner-cell-mass cells and around the nucleus of trophoblast cells. Such distribution of LBs in mouse embryos was also observed in those developed in vitro. From these results, it was suggested that there is a close relationship between distributional changes in LBs and the transformation and differentiation of blastomeres in mouse, rabbit and bovine embryos.

Distributional Changes of Acridine Orange-Stained Lysosomes in Hamster Eggs and Embryos during the Folliculogenesis and Early Development.

Distributional changes in lysosomes in hamster eggs and embryos during the folliculogenesis and early development were observed using the acridine orange (AO) staining method. AO-stained lysosomes were densely observed all over the cytoplasm of eggs in secondary follicles and of eggs in antral follicles until 13 hrs before ovulation, but were sparsely observed in the cytoplasm of eggs in primordial follicles. The lyso-somes were distributed throughout the cytoplasm in about a half of eggs in antral follicles at 12 and 10 hrs before ovulation, while they gathered around nuclei in the remaining half of the eggs. The lysosomes were distributed all over the cytoplasmic region of all eggs from 8 to 3 hrs before ovulation and of unfertilized eggs 2 hrs after ovulation. Such a distributional pattern remained unchanged until uncompacted 8-cell embryos. In compacted 8-cell embryos and blastocysts, the lysosomes were densely observed all over the cytoplasm in round blastomeres or inner-cell-mass cells, whereas they were sparsely observed in flattened blastomeres or trophoblast cells, mainly around nuclei. From the results on 8-cell embryos and blastocysts, it was suggested that there is the close relation between distributional changes of lysosomes and differentiation of blastomeres.

Cytogenetic analysis of the oocyte and embryo after removal of the zona pellucida following failed fertilization

Cytogenetic analysis of the oocyte and embryo after removal of the zona pellucida provides a simple screening method for examining the oocyte and embryo and considering the morphological abnormalities of the zona and adjacent formations, cytoplasm and nucleus. Seventy-one unfertilized oocytes and 27 embryos with poor morphology or cleavage arrest were studied after fixation, staining and mechanical removal of the zona. Fixation was done with a glutaraldehyde solution (1%). The oocytes and embryos were then stained, causing weakening of the zona. A slight pressure on the cover slide led to rupture of the zona and the exposure of the oolema. The oocytes and embryos were analyzed for sperm attachment to and penetration through the zona, appearance of the polar bodies, differentiation of blastomeres from cytoplasmic fragments, nuclear status — pyknotic non-analyzable form, chromosomes or different degenerative appearances of DNA. Peeling of the zona is an easy and reliable screening technique needing only an ordinary light microscope. It provides the advantage of observing the whole structure of the oocyte-embryo, not just the chromosomes as in classical cytogentic methods.

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [ 3H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.