PARENT SESSION
SLIDE SESSION 3: REGULATION OF SPERM FUNCTION
Chairs: Gary Killian, Marc-Andre Sirard, TanYa Gwathmey (Trainee)
Arts Hall 026
2:30 PM-4:30 PM
21
SYNAPTOTAGMIN VIII MAY FUNCTION IN MAMMALIAN SPERM ACROSOME EXOCYTOSIS.
Hutt, Darren1,2, Cardullo, Richard5, Baltz, Jay2,3,4, Ngsee, John2,4, 1 2 5 3 4
ABSTRACT- Sperm cells possess a single secretory granule—the acrosome—which undergoes exocytosis when receptors on the sperm surface bind to ligands in the egg extracellular matrix. The binding event is transduced into exocytosis via a rise in intracellular Ca++, and thus the acrosome reaction resembles stimulated secretion in neurons. Sperm have recently been found to contain several proteins of the SNARE complex, which is proposed to mediate Ca++-stimulated exocytosis in neurons. One family of SNARE proteins, the synaptotagmins (Syt), of which 12 isoforms are currently known, are thought to function as Ca++ sensors. Santos et al. (Dev. Biol. 223: 54-69, 2000) have recently used an antibody designed to cross-react with all Syt isoforms to show that mammalian sperm contain Syt, and identified it as the widely-expressed SytI. We, however, were unable to detect SytI in spermatocyte, spermatid, or testis cDNA libraries using PCR, but instead found SytVIII. We raised an antiserum against a SytVIII-specific peptide, which recognizes SytVIII but does not cross-react with other Syt isoforms (including SytI). The SytVIII antiserum labels a single prominent band on western blots of whole mouse sperm. SytVIII was also found to be restricted to sperm membrane fractions enriched in acrosomal markers. Fluorescence immunohistochemical localization on intact fixed mouse sperm showed that SytVIII is localized to the acrosomal cresent. Acrosome-reacted sperm lose SytVIII labelling, and western blots confirm that the amount of SytVIII decreases stoichiometrically with acrosome reaction. Furthermore, SytVIII appears to exist in a large complex in mouse sperm as demonstrated by sucrose gradient centrifugation. Thus, SytVIII is present in mammalian sperm where it may function as a Ca++ sensor in the acrosome reaction. (Supported by NSERC grant OGP0203132; cDNA libraries a gift of Dr. J. McCarrey).
KEY WORDS: acrosome, exocytosis, synaptotagmin, sperm