Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa

AbstractDefective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64 –65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55–68 kDa, consistent with the original spot used f or immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with an  anti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with an ti-ubiquitin...
Source: Cell and Tissue Research - Category: Cytology Source Type: research