Protein phosphorylation and cell diversification in the mouse early embryo.
Bloom, Theodora Leah
University of Cambridge
Department of Anatomy.
Doctor of Philosophy (PhD)
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Bloom, T. L. (1990). Protein phosphorylation and cell diversification in the mouse early embryo. (Doctoral thesis). https://doi.org/10.17863/CAM.14332
This dissertation reports the results of studies into the control of compaction of the mouse preimplantation embryo. Compaction is a post-translationally controlled rearrangement of cell contacts and the cytoskeleton that occurs at the 8-cell stage of development. This re-arrangement seems to be necessary for the differentiation of the two cell types present in the blastocyst. Protein phosphorylation is a post-translational modification believed to be important in the modulation of cell shape and cytoskeletal assembly. It is therefore feasible to propose a role for protein phosphorylation in compaction. Two types of approach have been used to investigate the possible role of protein phosphorylation in compaction. Firstly, embryos have been treated with two drugs, 6-dimethylaminopurine (DMAP) and a phorbol ester (phorbol myristate acetate, PMA), each of which seems to affect both protein phosphorylation and compaction. DMAP is an adenine analogue and putative inhibitor of protein phosphorylation that was found to perturb the cell cycle of mouse embryos. In addition, DMAP caused rapid cellular flattening of 4-cell and 8-cell embryos. However, this flattening was not accompanied by cell polarisation and did not seem to be mediated by the cell adhesion molecule uvomorulin. It is therefore unlikely to be related directly to the flattening that occurs at compaction. Phorbol esters, such as PMA, are potent stimulators of the membrane-associated, Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC). Incubation in medium containing PMA had some effects on the cytoskeleton of oocytes and early embryos but caused severe, widespread disassembly of the cytoskeleton and reversal of flattening in 8-cell embryos. These effects of PMA, seen specifically at the 8-cell stage, may be related to the spatially restricted disassembly of the cytoskeleton that occurs naturally during compaction at the 8-cell stage. This interpretation provides indirect evidence for a possible role for PKC activity, and hence protein phosphorylation, in the process of compaction. The relationship between protein phosphorylation and the events occurring at the 8-cell stage has been examined more directly by labelling 4-cell and 8-cell embryos with [32P]orthophosphate and examining the phosphoproteins obtained by one and two-dimensional gel electrophoresis. By synchronising groups of embryos precisely to successive cleavage divisions prior to labelling, changes in phosphoprotein profile associated with passage through the 4-cell and 8-cell stages have been described. While many of the 32P-labelled phosphoproteins detectable after electrophoresis in one or two dimensions are similar at each stage examined, there are some changes associated specifically with passage through the 8-cell stage which may be related to the cell flattening and polarisation occurring at this time. In addition, the profile of 8-cell embryos differed according to the duration of pulse-labelling with [32Pjorthophosphate or the inclusion of "chase" periods. Finally, several treatments that affect features of compaction, including exposure to DMAP and PMA, have been used to assess the link between the observed changes in phosphoprotein profile and the events of compaction. Embryos were also incubated in protein synthesis inhibitors, which cause premature cell flattening in 4-cell embryos and in Ca2+-free medium, which prevents intercellular flattening and delays polarisation of 8-cell blastomeres. In each case, the relative labelling intensity of some of the phosphoproteins characteristic of untreated 8-cell embryos was altered. The behaviour of these phosphoproteins suggests that they may be important in the mechanism by which cells flatten and polarise or in the maintenance of flattened, polarised, cells; they now provide a focus for future study.
This record's DOI: https://doi.org/10.17863/CAM.14332