In mammals, the development of spermatozoa from spermatids (spermiogenesis) is characterized by post-translational modification and then displacement of histones, at which time two major basic transition nuclear proteins (TP1 and TP2) become predominant in the nucleus. These are in turn replaced by protamines. The long-term objective is to determine which of these molecular changes are essential for the formation of a spermatozoon nucleus that is capable of efficient fertilization and transmission of genetic material. The first hypothesis to be tested is that both TP1 and TP2 have roles in histone displacement and proper nuclear condensation. This will be tested using mice with knockout mutations of the genes Tnp1 and Tnp2, for TP1 and TP2, respectively. The effects of null mutations for Tnp1, Tnp2, and both genes will be compared in mutant, wild-type, and heterozygous littermates in the following ways: Sperm production and quality and fertility will be quantified. Light and electron microscopy will be used to determine the steps of spermiogenesis at which abnormalities in nuclear shape and condensation occur. The loss of histones and the deposition of the nonmutated TP and protamines will be monitored by immunochemical methods and biochemical analysis of nuclei from whole testes and from separated cells. The sequence of biosynthesis, transport, and metabolism of the nonmutated TP and the protamines, their association with different chromatin fractions, and the relationship of the proteins with each other will be determined. If the effects of TP knockout mutations are subtle and subfertile spermatozoa are produced in TP-mutant mice, analysis of abnormalities in chromatin condensation, sperm morphology, or motility may provide insight into possible causes of human infertility. The second hypothesis is that posttranslational modifications of the histones, such as hyperacetylation of H4, are important for their displacement either by the TPs or other mechanisms. In this proposal, the nuclear histone acetyltransferases (HATs) in populations enriched in elongating spermatids will be characterized. The presence and effects of germ cell specific isoforms of known HATs will be evaluated by northern- or immuno-blotting or by examining testicular phenotypes, if any, of knockout mutations. The presence of novel HATs in elongating spermatids will be pursued by in-gel and solution enzymatic assays. Future studies will be directed towards genetic manipulation of HATs that are candidates for the hyperacetylation of H4.
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