Herr J C, Thomas D, Bush L A, Coonrod S, Khole V, Howards S S, Flickinger C J
Department of Cell Biology, The Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, Virginia 22908, USA.
Biol Reprod. 1999 Aug;61(2):428-35. doi: 10.1095/biolreprod61.2.428.
A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22-25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.
