The sperm centrosome is intimately associated with organization and morphogenesis of the spermatocyte, particularly with regard to establishment of extreme cell polarity in mature sperm. In addition, the sperm centrosome is delivered to the oocyte at the time of fertilization where it plays a fundamental role in the organization of the cytoskeleton of the zygote and early embryo through the formation of the microtubule-based sperm aster and the first mitotic spindle. We have recently established that centrin, a 21 kDa calcium-binding protein found in centrosome-associated fibers, is a prominent component of the sperm centrosome. Previous studies in other systems demonstrate a role for centrin-based fibers in centrosome/mitotic spindle pole positioning, segregation, and reorientation. Based on these observations, we present the specific hypothesis that sperm centrin is an essential component of the sperm connecting piece which functions in centrosome dynamics during sperm morphogenesis, and in zygotes and early embryos during spindle assembly and function. The work outlined in this proposal is directed toward achieving a greater understanding of centrosome dynamics in the male reproductive cell during spermatocyte differentiation and following fertilization of the oocyte. For a thorough understanding of the contribution by sperm to the zygotic centrosome it is important to determine the components of the sperm connecting piece that are essential to the process of spermatocyte differentiation, and sperm aster and centrosome formation and function. What proteins are carried by the sperm connecting piece to the egg? Are they essential for centrosome function? What is their relationship to preexisting pools of centrosomal components present in the oocyte? Can their function be perturbed by specific antibodies and/or drug treatments? Our laboratory has developed immunological, biochemical, and molecular tools which will allow us to readily probe the role of a recently discovered and novel calcium-binding cytoskeletal protein, centrin, in these processes.
The specific aims of the proposed studies include; 1) characterization of molecular and biochemical properties of sperm centrin through the purification, peptide and epitope mapping, partial peptide sequencing of centrin from sperm, and the molecular cloning of sperm (testicular) centrin. 2) To define the precise structural organization of centrin in developing spermatids, mature sperm, and in fertilized eggs and early embryos by immunofluorescence and immunoelectron microscopy using polyclonal and monoclonal antibodies raised against centrin. And 3), to determine if i centrin plays an essential role in sperm aster formation and centrosome behavior in early fertilized eggs. Finally, the proposed studies are likely to shed light on the nature of several human infertility disease states that involve centrosomal and spermatid malformations including 'decapitated' sperm.
| Wolfrum, U; Salisbury, J L (1998) Expression of centrin isoforms in the mammalian retina. Exp Cell Res 242:10-7 |
| Uzawa, M; Grams, J; Madden, B et al. (1995) Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation. Dev Biol 171:51-9 |
| Errabolu, R; Sanders, M A; Salisbury, J L (1994) Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles. J Cell Sci 107 ( Pt 1):9-16 |
