PARENT SESSION

(T290) THE GTPases Rho, Rac, AND Cdc42 ARE PRESENT IN PORCINE SPERM AND AT LEAST ONE PLAYS A ROLE IN MEDIATING THE ZONA PELLUCIDA INDUCED ACROSOME REACTION.

Ducummon, Carl^{1, 2}, Berger, Trish², ¹ Presbyterian Hospital, Dallas, TX² University of California, Davis, CA

ABSTRACT- Use of the bacterial toxin CNF, which artificially activates Rho family G-proteins, has implicated the Rho GTPases in regulating the acrosome reaction (AR). Rho has been immunolocalized to the acrosomal region of acrosome-intact porcine sperm but is not observed in the acrosomal region of acrosome-reacted sperm. These observations suggest that Rho family G-proteins are located either in the membranes overlaying the acrosome or in the cytosol between the plasma membrane (PM) and the outer acrosomal membrane (OAM), a location consistent with a role in mediating the AR. To further test the role of Rho family G-proteins in the AR, Toxin B, a bacterial toxin that inhibits the activity of Rho, Rac, and Cdc42 was used to treat porcine sperm. Treatment of porcine sperm with Toxin B slightly, but significantly, inhibited the zona pellucida (ZP)-induced AR (11.2 v. 8.6 ± 1%; P<0.015). Additionally, deconvolution microscopy was performed on washed, 6 h capacitated sperm to confirm the cellular localization of the Rho proteins in porcine sperm. The G-proteins were immunolocalized using an anti-Rho(-A,-B,-C), Rac1, or Cdc42 antibody followed by a Texas Red-labeled secondary antibody. Acrosomal contents or the PM were simultaneously labeled with an anti-PM antibody or FITC-PSA to delineate cellular compartments. The sperm were subjected to two- and three-dimensional light microscopy and analyzed computationally to remove out of focus data; these datasets were processed using an iterative, constrained three-dimensional deconvolution algorithm. Deconvolution microscopy of porcine sperm demonstrated that Rho family G-proteins are present in the PM or cytoplasm overlaying the acrosome. Taken together, these data strongly suggest that Rho, Rac, and/or Cdc42 mediate the ZP-induced AR in porcine sperm and are present in the sperm cytoplasm or PM, a localization consistent with the hypothesized role in the acrosome reaction.

KEY WORDS: acrosome reaction, Rho GTPases, sperm