The long term objective of this study is to examine the roles that extragenetic components and sperm structural organization play in regulating the formation of functional spermatozoa and viable embryos. Special emphasis will be given to the structural organization of the nucleus of sperm, the role of non-mitochondrial RNA in spermatozoa, and the mechanisms regulating differential expression of a testis-specific mitochondrial isozyme system during the late stages of male germ cell differentiation. The sperm chromatin studies seek to identify genes that are expressed late in spermatogenesis and/or early in embryogenesis. The current availability of technologies that allow detection of single molecules will provide a means to investigate whether non-mitochondrial RNAs are present in spermatozoa. The isozyme studies are designed to define the mechanisms controlling isozyme switching in which an ubiquitous mitochondrial protein is replaced by a sperm-specific variant in the mammalian testis. The events under investigation are likely to manifest paternal effects of gene expression soon after fertilization and during early embryogenesis. These studies will define the regulatory roles that differential sperm chromatin packaging play at fertilization and in early embryogenesis, will elucidate mechanisms of nuclear-mitochondrial genome communication during spermatogenesis, and will help determine how "isozyme switching" for cytochrome c, an essential nuclear encoded mitochondrial sperm protein is regulated. The cytochrome c studies will also define a general mechanism for gene shutdown in highly differentiated mammalian cells. Overall, the studies outlined in this proposal seek to identify epigenetic and nuclear regulated processes essential for normal mammalian development. A better understanding of these events is needed to explain how perturbations lead to birth defects. In addition, a more detailed comprehension of the molecular events of the male gamete before, at, and following fertilization will provide necessary insight to aid in the treatment of the infertile and lead to novel approaches for fertility control.