Molecular cloning and developmental expression of the major fibrous sheathprotein (FS 75) of rat sperm

The fibrous sheath (FS) is a cytoskeletal structure that encases the axonem e in the principal piece of the spermatozoon tail. In the rat, it is compos ed of several proteins, of which a 75-kDa polypeptide (FS 75), as estimated by PAGE, is the most prominent. The objectives of this study were to clone and sequence this protein and to characterize its transcriptional and tran slational origins during spermatogenesis. Initially, we isolated two overla pping cDNA segments that encoded a large part of the FS 75 protein but lack ed the initiation codon for translation. Both clones were obtained by scree ning a rat testicular phagemid cDNA library with an anti-FS 75 polyclonal a ntibody. An upstream portion of the FS 75 mRNA containing the initiation co don was obtained by polymerase chain reaction with a pair of specific prime rs. Accounting for the overlap in all segments, 2786 nt of an approximately 3-kb FS 75 mRNA was obtained. The amino acid sequence of the longest possi ble open reading frame of the rat FS 75 was found to be similar to two prev iously cloned variants of the major FS polypeptide of mouse spermatozoa. Se quence analysis of the rat FS cDNA revealed anchoring kinase A protein doma ins and several kinase phosphorylation sites, supporting the idea that this protein plays a crucial role in the motility of spermatozoa. The presence of a potential N-myristoylation site suggests that this protein may covalen tly bind to the inner leaflet of the plasma membrane (PM), which in turn ma y explain the close association of the FS and PM from early development. De velopmental northern blot analysis and in situ hybridization revealed that the FS 75 mRNA is haploid expressed, with an abundant level of mRNA in late round spermatids. Maximum levels of the FS 75 polypeptide, as determined b y immunocytochemistry, correlated with a rapid decline in corresponding mRN A levels in step 14-16 spermatids. Since transcription termination occurs s everal steps earlier, the bulk of FS 75 mRNA appears to be translationally regulated.