Isolation and characterization of rat sperm tail outer dense fibres and comparison with rabbit and human spermatozoa using a polyclonal antiserum

Rat outer dense fibres were isolated from cauda epididymal spermatozoa usin g mechanical and chemical dissection methods. Sperm tail isolation procedur es were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated oute r dense fibres revealed at least nine Coomassie brilliant blue stained band s, and 12 silver staining bands. The most abundant proteins were a large ba nd between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolat ed proteins showed similar compositions, being abundant in aspartic and glu tamic acid, serine, glycine and leucine. However, the content of cysteine a nd proline was highly variable among the isolated proteins. Immunofluoresce nce microscopy demonstrated that a polyclonal antiserum to isolated rat out er dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum als o showed crossreactivity in the perforatorium of rat sperm heads and the ac rosome and equatorial segment of rabbit sperm heads. These data indicate th at it is technically possible to isolate proteins from the outer dense fibr es that will enable further studies of the amino acid sequences of sperm ta il proteins.