PARENT SESSION
SLIDE SESSION 9: SPERMATOGENESIS
Chairs: Deborah O'Brien, John Herr, Cynthia Shirley (Trainee)
Univ Ottawa-UCU Auditorium
1:30 PM-3:30 PM

SAMP32, A NOVEL, TESTIS-SPECIFIC, MEMBRANE-ASSOCIATED PROTEIN, IS LOCALIZED TO THE ACROSOME OF HUMAN SPERMATOZOA.

Hao, Zhonglin^1,2, Wolkowicz, Michael^1,2, Shetty, Jagathpala^1,2, Klotz, Ken^1,2, Coonrod, Scott^1,2, Brolling, Laura^1,2, Sen, Buer^1,2, Westbrook, Vivian^1,2, Flickinger, Charles^1,2, Herr, John^1,2, ^{1 2}

ABSTRACT- Hydrophobic human sperm proteins that partitioned into Triton X-114 were separated by 2-D gel electrophoresis. Four protein spots (pIs from 4.5 to 5.5 and Mr. 32 to 34 kD) contained identical peptides when microsequenced. Cloning of the corresponding cDNA showed that these protein spots were products of a single gene, containing an ORF encoding a predicted protein of 32 kD, pI 4.57(SAMP32). SAMP32 contained a predicted amino terminal signal peptide and a carboxyl terminus transmembrane domain. Three casein kinase II phosphorylation sites were predicted including one that was found phosphorylated on a serine residue by tandem mass spectrometry. Post-translational modification(s) appear to be responsible for the mass and pI differences among the 4 SAMP32 protein spots. Northern blots of 8 human tissues showed a 1.4 Kb transcript that was present only in testis. Testis specificity was further confirmed by RNA dot blotting of 67 human tissues. Immunofluorescence using polyclonal antiserum raised against recSAMP32 stained the equatorial segment and acrosomal cap of human spermatozoa (intense bar and faint cap). Electromicroscopy confirmed the localization of SAMP32 on the equatorial segment and acrosomal membranes leading to the name: Sperm Acrosomal Membrane-associated Protein. A Blast search revealed that SAMP32 consists of 7 exons on chromosome 6q15-16.2. Immunofluorescence staining of enzyme dissociated human testicular cells indicated that SAMP32 expression localized to stages of acrosomal biogenesis in round spermatids. Rat anti-recSAMP32 blocked human sperm binding and fusion to zona free hamster eggs. In summary, SAMP32 is a novel, hydrophobic, testis-specific, serine phosphorylated protein localized to the acrosome. Due to its testis specificity and role in sperm-egg interactions, SAMP32 is a candidate target for contraceptive development.

KEY WORDS: acrosome, phosphorylation, proteomics, transmembrane protein