Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos.
We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
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Besterman
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Schultz
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002970:Cleavage Stage, Ovum / Q000187:drug effects*; D046508:Culture Techniques; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D019833:Genistein; D006868:Hydrolysis; D007529:Isoflavones / Q000494:pharmacology*; D008715:Methionine; D051379:Mice; D010710:Phosphates; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D005419:Flavonoids; D007529:Isoflavones; D010710:Phosphates; D004247:DNA; D008715:Methionine; D019833:Genistein; D011505:Protein-Tyrosine Kinases