PARENT SESSION
Capacitation & Acrosome Reaction

CYTOTOXIC NECROTIZING FACTOR, A BACTERIAL ACTIVATOR OF THE MONOMERIC G-PROTEIN RhoA, ENHANCES SPONTANEOUS ACROSOME REACTIONS IN PORCINE SPERM.

Ducummon, Carl¹, Berger, Trish¹, ¹

ABSTRACT- The acrosome reaction is an exocytotic event induced physiologically by the zona pellucida. Other cell types, such as mast cells, also undergo induced exocytosis. Activation of the monomeric G-protein Rho enhances induced mast cell exocytosis. Cytotoxic Necrotizing Factor (CNF) is a bacterially derived toxin that is cell membrane permeable and artificially activates Rho proteins. We have previously shown that the monomeric G-protein RhoA is present in porcine sperm and that it undergoes translocation from a wedge/horseshoe pattern in the equatorial segment of ejaculated sperm to a full cap staining pattern during capacitation. To further study the role of RhoA in the porcine sperm acrosome reaction, we added CNF to the capacitation medium to determine if CNF activation of RhoA would further stimulate the zona pellucida-induced acrosome reaction. Briefly, sperm were capacitated in Tris-buffered medium supplemented with 5 mg BSA/mL for 6 h either in the presence or absence of CNF. When indicated, heat solubilized porcine zona pellucida was added after 5.5 hours. An aliquot of each sperm sample was stained with FITC-conjugated Pisum sativum agglutinin and scored for acrosomal status. Cells were also fixed, permeabilized, and indirect immunofluorescence performed using a polyclonal RhoA antibody. Sperm were scored for wedge/horseshoe, band, or cap staining pattern and correlated with acrosomal status. The percentage of sperm undergoing the acrosome reaction after 6 h in the presence of CNF was 1.9 times greater than in its absence (P < 0.05). Surprisingly, addition of zona pellucida to CNF-treated sperm did not have an additive effect on the percentage of sperm undergoing the acrosome reaction (1.95 times greater than 6 h samples in the absence of CNF or zona pellucida). Spontaneous acrosome reactions after 4 h capacitation in the presence or absence of CNF were similar to spontaneous acrosome reactions after 6 h capacitation in the absence of CNF (1-1.1 times the 6 h values.) We conclude that RhoA may play a role in signal transduction during the acrosome reaction in porcine sperm.

KEY WORDS: Sus scrofa, acrosome reaction, G-protein, signal transduction