The research is designed to elucidate the assembly, structure and functions of the nuclear matrix during mouse spermatogenesis. Nuclear matrices isolated from pachytene spermatocytes lack the peripheral fibrous lamina typically present in somatic cells. Instead, the spermatocyte matrix is composed of a pore-fiber complex, residual nucleoli, interchromatin fibers, XY heterochromatin, and a full complement of synaptonemal complexes (SCs). The spermatid nuclear matrix is similar in appearance, but lacks the XY body and synaptonemal complexes. Although different in morphology, the spermatocyte and spermatid nuclear matrices are comprised of similar proteins, ranging in Mr from 8,000 to 150,000 daltons. However, a temporal study of in vivo synthesis of these proteins has revealed six highly-conserved polypeptides, having Mrs of 33,000-21,000 daltons, that occur only in zygotene and pachytene spermatocyte nuclear matrices. These may be constituents of the SCs. Similarly, spermatic nuclear matrices contain five distinct polypeptides, Mr 51,000-31,000, which may form elements of the perinuclear theca. Both matrices contain at least 12 polypeptides not detectable in somatic nuclear matrices. Current research is directed toward defining the temporal synthesis and morphological assembly of nuclear matrix constituents during meiotic prophase in the mouse. The study will focus on a complete biochemical characterization of matrix constituents, particularly the unique components, by SDS PAGE, NEFGE, and by Western blot analysis using lectin and monoclonal antibody probes. Isolated spermatogonia, primary spermatocytes, and spermatids will be cultured in vitro with [35S]methionine, [3H]fucose and [32P] in an effort to specifically label the unique polypeptides and/or glycoproteins. Moreover, selected monoclonal antibodies will be utilized to identify, characterize and localize individual antigenic constituents with germ cell nuclei, and their matrices, at discrete stages of spermatogenesis. Thus, the research will involve analyzing antigenic determinants by Western immunoblot and localizing these proteins to particular structures by light and ultrastructural immunocytochemistry. Further studies are planned to resolve the molecular interactions of homopolymers and heteropolymers of these protein constituents. Finally, selected polypeptides will be purified by immunoaffinity chromatography, and characterized by amino acid analysis and primary sequencing.
| Thomas, K; Del Mazo, J; Eversole, P et al. (1990) Developmental regulation of expression of the lactate dehydrogenase (LDH) multigene family during mouse spermatogenesis. Development 109:483-93 |
| Thomas, K H; Wilkie, T M; Tomashefsky, P et al. (1989) Differential gene expression during mouse spermatogenesis. Biol Reprod 41:729-39 |
