Our long-term objectives are to understand the molecular details of the normal developmental steps in the formation of the nucleus of a spermatozoon that is capable of fertilization of an ovum and transmitting its genetic material. During the development of mammalian spermatids into spermatozoa (spermiogenesis), histones are displaced, at least two basic transition proteins (TP1 and TP2) become prominent in the nucleus; these are in turn displaced, and essentially only protamines remain in the sperm nucleus. The role of these TPs in histone displacement or chromatin condensation during spermiogenesis is not known. For the proposal, we plan to test the hypothesis that TP1 and TP2 are essential for the steps of histone displacement and proper nuclear condensation involved in the formation of normal spermatozoa. We will do this by generating 'knockout' mice with null mutations of the genes for TP1 and TP2 using homologous recombination to delete the genes in embryonic stem cells and formation of chimeras that transmit the mutated allele to their offspring. Using these mice, we will examine the effects of null mutations for TP1, TP2, and both genes, either in the heterozygous state, on male fertility and on specific events in spermiogenesis. Male mice with various combinations of null mutations for these genes will be examined by the following means and compared to normal mice. Fertility and sperm production will be quantified. The steps of spermatid development at which abnormalities and condensation occur will be determined by light and electron microscopy. 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 post-translational modifications of the nonmutated TP and the protamines, their affinity for DNA, their association with different chromatin fractions, and the relationship of the proteins with each other will be determined. If the mutation affects the deposition of the nonmutated TP or protamines, alterations in transcription, translation, and nuclear transport of these gene products will also be measured, as there might be some coordinate regulation within this group of proteins. The results will determine whether or not TP1 and TP2 have essential roles in spermiogenesis. If they are essential for sperm production, the defects observed in the mutants will provide directions for further biochemical and in vitro studies of their roles in normal spermatid development. If their effect is more subtle and spermatozoa are produced in TP-mutant mice but they are subfertile or infertile, analysis of abnormalities in chromatin condensation, headshape, or genomic imprinting may provide insight into possible causes of human infertility.
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