CHARACTERIZATION OF FSC1 CDNA FOR A MOUSE SPERM FIBROUS SHEATH COMPONENT

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 s heath protein. Peptides from a tryptic digest of fibrous sheath protei ns were separated by HPLC and a 31 amino acid sequence was obtained fr om 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. Th is 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 sho wed that it encodes a protein with an open reading frame of 849 amino acids and includes the original peptide sequence. The predicted protei n has a molecular weight of 93 795 and contains 32 cysteine residues a nd 32 potential phosphorylation sites. It has no significant homology with other known cytoskeletal proteins. Northern blot analysis detecte d an mRNA of similar to 3 kb that was abundant in round spermatids of the mouse and in testes from six other mammalian species, but not in t welve somatic tissues from the mouse. In situ hybridization analysis i ndicated that the mRNA is first detected in step I-G spermatids, is mo st abundant in step 8-12 spermatids, and decreases in amount in step 1 3-15 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 f ound by PCR analysis to be homologous to the original cDNA clone. Thes e results suggest that the similar to 3-kb mRNA encodes a major struct ural component of the mouse fibrous sheath that appears to be a unique cytoskeletal protein of spermatogenic cells. We designate the cDNA fo r this fibrous sheath component Fsc1.