Characterization of Fscl cDNA for a Mouse Sperm Fibrous Sheath Component1

The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. We have cloned a cDNA and used it to characterize the expression of mRNA for a mouse sperm fibrous sheath protein. Peptides from a tryptic digest of fibrous sheath proteins were separated by HPLC and a 31 amino acid sequence was obtained from one of the peptides. Through the use of degenerate oligonucleotide polymerase chain reaction (PCR) primers predicted from this sequence, an 80- bp product was amplified from mouse testis first-strand cDNA. This was utilized as a probe to isolate a 2.9-kb cDNA clone from a mouse round spermatid cDNA library. Sequence analysis of the cDNA clone showed that it encodes a protein with an open reading frame of 849 amino acids and includes the original peptide sequence. The predicted protein has a molecular weight of 93 795 and contains 32 cysteine residues and 32 potential phosphorylation sites. It has no significant homology with other known cytoskeletal proteins. Northern blot analysis detected an mRNA of ~3 kb that was abundant in round spermatids of the mouse and in testes from six other mammalian species, but not in twelve somatic tissues from the mouse. In situ hybridization analysis indicated that the mRNA is first detected in step 1–6 spermatids, is most abundant in step 8–12 spermatids, and decreases in amount in step 13–15 spermatids, suggesting that expression of the mRNA occurs in the postmeiotic phase of spermatogenesis. In addition, monoclonal antibody 5A8, which recognizes proteins of the mouse fibrous sheath, was used to isolate cDNAs from a mouse spermatid expression library that were found by PCR analysis to be homologous to the original cDNA clone. These results suggest that the ~3-kb mRNA encodes a major structural component of the mouse fibrous sheath that appears to be a unique cytoskeletal protein of spermatogenic cells. We designate the cDNA for this fibrous sheath component Fscl.