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.