Sperm ultrastructure and higher order chromatin structure: Implications for embryogenesis

Traditional concepts about the role the mammalian spermatozoon plays in fertilization and development have centered on the idea that spermatozoa are no more than streamlined carriers of the paternal chromatin to the egg. The central concept considered in this dissertation diverges from this traditional view. Here, it is proposed that the sperm nucleus carries an epigenetic structural organization of DNA that is necessary for embryogenesis to occur. This structural organization is thought to take the form of DNA loop domains that are specifically organized by the underlying substructure of the sperm nucleus—the nuclear matrix.; In the work presented here, the importance of the role that the structural organization of sperm chromatin might play in embryogenesis was investigated in three different ways. First, the DNA loop domain organization of the 5S rRNA gene cluster was analyzed in spermatogenic cells, embryonic stem cells, and differentiated adult somatic cells using fluorescent in situ hybridization (FISH). The results of these analyses indicate that the three dimensional structure of DNA loop domains is specifically regulated during spermatogenesis and early development. Second, ultrastructural analyses of chemically disrupted perinuclear theca material in both mouse and hamster sperm were performed using a high-resolution surface imaging technique (cold field-emission scanning electron microscopy, or FE-SEM) and the results were compared to the rates of ICSI success for similarly treated nuclei. The ICSI experiments were performed with spermatozoa from which the perinuclear theca was entirely or partially removed, and were designed to show that sperm chromatin organized by an intact nuclear matrix is the only sperm component necessary for embryogenesis to occur. The ultrastructural analyses performed in the current work did not confirm that the perinuclear theca is entirely removed using the treatments described for the ICSI experiments, however several novel substructures of the perinuclear theca were characterized that have not been previously described. Lastly, sperm nuclear morphology was investigated at the ultrastructural level using FE-SEM. Specifically, the relationship of chromatin to the sperm nuclear matrix was investigated, as well as the morphology of condensed and decondensed chromatin structures, and isolated nuclear matrices. The data from these novel electron microscopy studies suggest a new model for how condensed sperm chromatin might be arranged with respect to the nuclear matrix.