Evidence that 68-Kilodalton and 54–51-Kilodalton Polypeptides are Components of the Human Sperm Fibrous Sheath1

Abstract

This study characterizes a common antigen recognized by two monoclonal antibodies (mAbs) that immunoreact with the principal piece of the human sperm flagellum. By means of immunofluorescence microscopy, mAbs S69 (IgM) and S70 (IgG1) (WHO Task-force nomenclature) were observed to bind to the principal piece of methanol- or detergent-permeabilized human spermatozoa, but did not react with live swimming spermatozoa as assessed by immunofluorescence microscopy. Faint immunofluorescence was also seen on the connecting piece in approximately 40% of the spermatozoa. Immunoreactivity in both regions was resistant to sequential extraction with Triton X-100, sodium thiocyanate, and urea. Pre-embedding electron microscopic immunogold labeling of ejaculated spermatozoa with mAb S69 showed gold particles located on the fibrous sheath. Immunoreactive peptides of 68, 53, and 45 kDa were recognized by both S69 and S70 mAbs on immunoblots of nonreduced human sperm extracts, while a 68-kDa band and a strongly immunoreactive triplet from 54 to 51 kDa were recognized in reduced sperm extracts. Human fibrous sheaths were isolated by differential solubilization and centrifugation and characterized by transmission electron microscopy. The 68-kDa and 54-51-kDa bands were enriched and found to be major polypeptides in the isolated fibrous sheath fraction. These results suggest that the S69/S70 antigen, which we term SP (sperm protein) (68 kDa, 54-51 kDa), is a component of the human fibrous sheath.

Immunochemical Characterization of a Human Sperm Fibrous Sheath Protein, Its Developmental Expression Pattern, and Morphogenetic Relationships with Actin

“…The GDA-J/F3 MAb (Jassim et al 1990) and the FS K 32 MAb (Sakai et al 1986;Koyama et al 1984;Jones et al 1983) recognize proteins of the human FS but are not reactive in Western blotting. MAbs S69 and S70 recognize human 68-kD and 54-51-kD major FS polypeptides (Beecher et al 1993) and AJ-FS1 multiple bands ranging between 97 and 28 kD (Jassim and Chen 1994). Unlike MAb 4F7, AJ-FS1 (Jassim and Chen 1994) and S69 (Beecher et al 1993;Homyk and Herr 1992) bind only to the outer side of the FS.…”

“…MAbs S69 and S70 recognize human 68-kD and 54-51-kD major FS polypeptides (Beecher et al 1993) and AJ-FS1 multiple bands ranging between 97 and 28 kD (Jassim and Chen 1994). Unlike MAb 4F7, AJ-FS1 (Jassim and Chen 1994) and S69 (Beecher et al 1993;Homyk and Herr 1992) bind only to the outer side of the FS. The RT97 anti-neurofilament antibody recognizes a single 97-kD peptide of the human FS, showing that the human FS shares an epitope common to anti-200-kD neurofilament (Jassim et al , 1992Jassim 1991).…”

“…Both human (Kim et al 1995;Jassim 1991Jassim ,1994Jassim and Chen 1994;Beecher et al 1993) and rodent (Brito et al 1989;Fenderson et al 1988;Oko 1988;O'Brien and Bellvé 1980;Olson et al 1976) FS contain many polypeptides ranging from M r 11 ϫ 10 3 to M r 120 ϫ 10 3 . Crossreactions of anti-rat FS antibody with the outer dense fibers reveals common antigenic determinants between these flagellar cytoskeletal structures (Tres and Kierszenbaum 1996;Oko 1988).…”