This proposal will test two working models which explain the relationship between phosphorylation of the two sperm specific basic nuclear proteins (Sp histones) of the sea urchin, chromatin structural alterations and male nuclear decondensation. The first model postulates that the phosphorylation of specific tetrapeptide sites in these proteins alters physical properties such as thermal stability, nuclease accessibility, average nucleosomal repeat length and histone DNA binding affinities that reveal how the histones and DNA are arranged. The experiments are designed to assess the contribution of each Sp histone separately. Measurements will be made on chromatin reconstituted from purified histones and DNA in the absence of complicating factors present in the living cell. The results will elucidate some of the alterations on first order chromatin structure observed in vivo. The second model addresses the potential consequences of Sp histone phosphorylation upon male chromatin decondensation and postulates at least two requirements for decondensation: phosphorylation and activation of a factor(s) sensitive to the drug 6-dimethylaminopurine. Using microinjection of condensed nuclei differing in their degree of phosphorylation, several factors will be evaluated including the role of Sp histone phosphorylation in decondensation, and the role of other metabolic activities. The factors absent in immature oocytes will be investigated, and an assay will be developed for the decondensation factor(s) which are sensitive to the drug. The consequences of phosphorylation at the specific tetrapeptide sites are unlikely to be restricted to sea urchin male germ cells. Similar sites are known in most H1 histones and in several nucleic acid packaging proteins and regulators, emphasizing the general importance of studying their phosphorylation. %%% The long term objective of this research is an understanding of the inactivation/activation of the sperm nucleus which occurs during spermatogenesis and following fertilization. The successful genetic reactivation of the dormant sperm nucleus by egg cytoplasm, following fertilization, is essential to successful biparental embryonic development.
