Genesis of newt sperm axial fiber: cDNA cloning and expression of a 29 kDa protein, a major component of the axial fiber, during spermatogenesis.

Newt sperm has a unique structure: the tail consists of axial fiber, undulating membrane and flagellum. The genesis and chemical composition of the axial fiber remain unknown. The axial fiber consists of about 10 major components, as evidenced by SDS-polyacrylamide gel electrophoresis. In order to clarify the biochemical properties of the components of the axial fiber and study the mechanism of axial fiber formation, we focused our attention on a 29 kDa protein, the major constituent of the axial fiber. Immunofluorescent antibody technique showed that the 29 kDa protein was first expressed in the cytoplasm of early round spermatids but was expressed on fibers in the periphery of the cyst in late round spermatids. Double staining with tubulin antibody and 29 kDa antibody showed that the fibers around the cysts in early round spermatids were flagella alone but those in late round spermatids consisted of flagella and 29 kDa protein. These results indicated that 29 kDa proteins are synthesizsed in the cytoplasm of round spermatids and enter the preformed flagella in late round and elongated spermatids. A cDNA clone for 29 kDA protein was isolated. A database search could not find any homologous clones, indicating that the 29 kDa protein is a new one. Northern blot with the cDNA showed that mRNA for 29 kDa protein was highly expressed in round spermatids but barely in primary spermatocytes, indicating that the mRNA for 29 kDa protein is haploid-expressed.