CHARACTERIZATION OF FIBROUS SHEATH AND MIDPIECE FIBER NETWORK POLYPEPTIDES OF MARSUPIAL SPERMATOZOA WITH A MONOCLONAL-ANTIBODY

In this study cytoskeletal antigens common to brushtail possum and tam mar wallaby spermatozoa were characterised using a monoclonal antibody (PSA-10). Using indirect immunofluorescence, the PSA-10 antibody dete cted antigens predominantly associated with the midpiece and principal piece of mature, permeabilised marsupial spermatozoa. The principal p iece determinant, shared by a variety of other species, was found to a rise in the marsupial testis. Midpiece localisation of the PSA-10 epit ope was detected only in marsupial spermatozoa and shown to arise in t he epididymis. Immunogold labelling demonstrated that the PSA-10 antig ens were predominantly associated with the fibrous sheath and midpiece fibre network of both possum and wallaby spermatozoa. Western blottin g suggested that two major possum and wallaby sperm polypeptides of 15 8 and 182 kDa were associated with the midpiece fibre network, a cytos keletal structure unique to marsupial spermatozoa. A 32 kDa polypeptid e was associated with the principal piece fibre network and/or fibrous sheath. The finding that these marsupial sperm cytoskeletal proteins share a common linear epitope suggests that they share some sequence s imilarity. The midpiece fibre network of marsupial sperm, like the fib rous sheath, has been proposed to have a structural role in providing passive stiffening for the flagellum (Harding et al., 1975, 1979; Olse n, 1975). The PSA-10 monoclonal antibody may provide a tool for compar ative studies of mammalian sperm cytoskeletal proteins, particularly t he marsupial midpiece fibre network. It may also allow the formation o f this unique marsupial cytoskeletal structure, and its fate during th e fertilisation process, to be followed by immunological means. Mol. R eprod. Dev. 50:461-473, 1998. (C) 1998 Wiley-Liss, Inc.