Isolation and Characterization of the Fibrous Sheath of Rat Epididymal Spermatozoa1

The fibrous sheath was isolated from epididymal spermatozoa of rat caput epididymidis and cauda epididymidis and its polypeptide composition and subunit substructure determined. Cauda epididymal spermatozoa were extracted sequentially with Triton X-100-dithiothreitol (DDT), potassium thiocyanate-DTT, and urea-DTT; the loss of specific flagellar components as determined by electron microscopy was correlated with the solubilization of specific polypeptide components as determined by SDS-polyacrylamide gel electrophoresis. Triton X-100 treatment solubilized all flagellar membranes but had no effect on the flagellar structural elements. KSCN extraction affected the organization of the 9+2 axoneme, solubilizing the central pair of microtubules, portions of the b-subtubule and disorganizing the remaining axoneme matrix components. KSCN also disorganized the outer dense fiber cortex but did not remove it. SDS gels show that none of the major polypeptide bands present in Triton extracted sperm were removed by KSCN extraction. Brief urea extraction resulted in solubilization of specific outer dense fiber polypeptides; the nonsolubilized portion of the outer dense fiber assumed a very regular corkscrew configuration. Prolonged urea extraction completely solubilized the outer dense fibers and connecting piece, leaving the fibrous sheath as the only intact flagellar organelle. Caput epididymal spermatozoa were treated sequentially with Triton X-100 and sodium dodecyl sulfate (SDS), and the fibrous sheath was recovered by differential centrifugation. SDS-polyacryladmide gel electrophoresis showed that the fibrous sheath of rat sperm is composed predominantly of a single polypeptide with a molecular weight of 80,000 daltons. Study of the substructure of the fibrous sheath, by negative staining of demembranated spermatozoa, show that the ribs are composed of parallel 5–6 nm wide filaments which appear to have a beaded substructure.