Submission Number: CAR-4-2-11

Abstract Number: 256

THE MONOMERIC G-PROTEIN RhoA UNDERGOES TRANSLOCATION DURING CAPACITATION OF PORCINE SPERM.

Carl Ducummon* ^1,2, Mary Horton ¹ and Trish Berger* ^1,2

Department of Animal Science, University of California, Davis, CA ¹
Cell & Developmental Biology Graduate Group, University of California, Davis, CA ²

Abstract:
The Rho family of GTPases has been implicated in the regulation of exocytosis in secretory cell systems such as mast cells and pancreatic cells. Since the acrosome reaction is an exocytotic event, we hypothesized that Rho family members may be present in sperm and involved in the acrosome reaction. These experiments immunolocalized RhoA in washed sperm (0 h) and after incubation for 4 or 6 h in capacitating (C) and non-capacitating (NC) conditions. Semen from three boars was collected, washed and incubated at 39 C in 5% CO₂ in air in the presence of 5 mg/mL BSA (C) or polyvinyl alcohol (PVA; NC). Aliquots of sperm were evaluated for acrosomal status or fixed and applied to slides for immunostaining. For immunofluorescence, sperm were blocked with 10% normal donkey serum, incubated with a goat anti-RhoA primary antibody, rinsed, and labeled with a Texas Red conjugated donkey anti-goat secondary antibody. Three types of staining were observed. In 0 h sperm, RhoA was seen as two distinct wedge shapes in the equatorial segment frequently connected to form a horseshoe. During capacitation two other staining patterns were observed: 1) a wide band encompassing the equatorial segment or 2) acrosomal cap staining. We believe the band pattern is an intermediate form between the wedge and cap patterns. The percentage of sperm exhibiting the cap staining increased significantly (P<0.05) during capacitation (1.4% vs. 12.0% for 0 h vs. 6 h). Concomitantly the percentage of sperm with wedge staining significantly decreased (96.2% vs. 83.4% for 0 h vs. 6 h; P<0.05). These data indicate that RhoA undergoes a translocation from the equatorial segment to the entire acrosomal region during capacitation suggestive of a role for RhoA in the acrosome reaction. .

Keywords: GTPase, RhoA, Capacitation, Acrosome Reaction, Boar, Sperm

This abstract is being presented at: 2:30 PM in session:
SESSION 13: CAPACITATION AND ACROSOME REACTION / SPERM MOTILITY