David J. Nishioka, PhD, University of California Berkeley - US grants

The control of the cell cycle in early sea urchin and starfish embryos will be studied in terms of nucleo-cytoplasmic interactions. Aphidicolin, a recently discovered drug which inhibits DNA replication specifically, will be applied to early embryos in connection with the recently developed technique of egg fusion. This experimental approach is designed to make several new determinations about cell cycle control in the early embryo: (1) whether or not the unreplicated nucleus exerts negative control over progression through the cell cycle; (2) whether or not the cytoplasm of the replicated cell can exert positive control over the unreplicated nucleus; and (3) whether or not any controlling factors inferred from these studies can cross taxonomic class barriers between sea urchins and starfish. Additional new determinations about necleocytoplasmic interactions in unfertilized eggs and immature oocytes using the egg fusion technique only include: (4) whether or not nuclear DNA replication is stimulated in an unfertilized sea urchin egg upon fusion with a fertilized egg; (5) whether or not germinal vesicle breakdown and maturation of an immature sea urchin oocyte is stimulated upon fusion with an unfertilized egg; (6) whether or not germinal vesicle breakdown and maturation of an immature starfish oocyte is stimulated upon fusion with a mature starfish oocyte; and (7) whether or not any controlling factors inferred from these studies can cross taxonomic class barriers between sea urchins and starfish. These determinations should provide a more complete understanding of how the nucleus and cytoplasm are related with one another during the production of the egg and through early cleavage of the embryo.

The general objectives of this research plan are to determine how the sperm surface differentiates through spermatogenesis and how sperm surface components disperse in the egg surface after fertilization. More specific aims include: (1) the use of monoclonal antibodies against site-specific sperm surface antigens to determine when during spermatogenesis and where in the testis these antigens are added to the surfaces of spermatogenic cells; (2) to clone the genes coding for these site-specific surface antigens and, using these cloned genes, (3) to determine when during spermatogenesis and where in the testis these genes are transcribed; (4) to prepare and screen appropriate expression libraries from which these genes may be isolated, (5) to subclone these genes into appropriate transcription vectors, and (6) to devise effective sectioning techniques to carry out definitive in situ hybridization and antibody binding studies; (7) to determine whether site-specific sperm surface antigens disperse laterally on the surfaces of polyspermically fertilized eggs and, if so, (8) to determine the effects of cytoskeletal and metabolic inhibitors on this lateral dispersal; and, using recently developed procedures for fusing eggs, (9) to determine if sperm surface antigens disperse evenly in unfertilized- fertilized and fertilized-fertilized egg fusions. Since the surface antigens that will be studied possess Ca2+ -channeling capabilities and are known to be involved in the sperm acrosome reaction and sperm-egg binding, these aims and determinations should provide closer understandings of spermatogenesis, sperm fertility and infertility, sperm-egg binding, these aims and determinations should provide closer understandings of spermatogenesis, sperm fertility and infertility, sperm-egg binding, the transfer of sperm components to the fertilized egg, and the functions of sperm components in the early embryo. In more general biological terms, they should provide additional basic information about the movement of plasma membrane proteins in differentiating cells, the relationship between the cytoskeleton and membrane fluidity, membrane fusion, and cellular derepression.