Submission Number: SRI-4-32-6

Abstract Number: 252

GLYCOGEN SYNTHASE KINASE - 3 PHOSPHORYLATION EVIDENCE FOR A ROLE FOR THE PHOSPHOINOSITIDE 3- KINASE SIGNALING PATHWAY IN SPERM MOTILITY REGULATION.

Shannan Jack ¹, Riddish Acharya ¹, Douglas Kline ¹, Gary E Olson* ² and Srinivasan Vijayaraghavan* ¹

Kent State University, Kent, OH ¹
Vanderbilt University, Nashville, TN ²

Abstract:
The long-term goal of our work is to understand biochemical mechanisms underlying regulation of sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55 kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the 55 kDa protein, measured by immunoreactivity to phosphotyrosine antibody 4G10, was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility resulted in a corresponding decrease or increase in tyrosine phosphorylation of the protein [Biol Reprod (1997) 56:1450)]. Protein purification showed that this motility associated protein is glycogen synthase kinase-3 (GSK-3) [Biol Reprod (in Press)]. This report describes studies on the mechanisms by which GSK-3 is phosphorylated in proportion to motility. Phosphorylation-specific antibodies show that, in addition to tyrosine phosphorylation, GSK-3 is also serine phosphorylated. Initiation and stimulation of motility of bovine epididymal sperm by cAMP, calyculin A, and okadaic acid lead to a dramatic increase in GSK-3 serine phosphorylation. Concentration and time-course measurements with these compounds show that motility stimulation is closely associated with GSK-3 serine phosphorylation. A similar increase in serine phosphorylation also parallels capacitation of mouse epididymal sperm. Enzyme activity measurements show that serine phosphorylation inactivates GSK-3. We next showed that the enzymes, cAkt and phosphoinositide 3-kinase (PI3-kinase), the upstream signaling enzymes involved in GSK-3 phosphorylation, are both present in sperm. Fluorescence immunocytochemistry showed that the enzymes GSK-3 and PI-3 kinase are present in the head and tail regions of sperm. Our work has unraveled a novel signaling system involving GSK-3 phosphorylation in the regulation of sperm motility and fertility. (Supported by funds from Kent State University).

Keywords: sperm motility, GSK-3, tyrosine phosphorylation, serine phosphorylation, PI-3 kinase

This abstract is being presented at: 1:30 PM in session:
SESSION 13: CAPACITATION AND ACROSOME REACTION / SPERM MOTILITY