PARENT SESSION
FERTILIZATION

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(227) ASSOCIATION OF PDE4A WITH AKAP110 IN BOVINE SPERMATOZOA.

Bajpai, Malini¹, Fiedler, Sarah¹, Vijayaraghavan, Srinivasan², Livera, Gabriel³, Conti, Marco³, Carr, Daniel¹, ¹ Oregon Health and Sciences University and VA Medical Center, Portland, OR² Kent State University, Kent, OH³ Stanford University, Stanford, CA.

ABSTRACT- The cAMP/PKA (Protein Kinase A) signaling pathway plays an important role in the regulation of sperm motility and other sperm functions. Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and thus play an important role in controlling the concentration of cAMP in spermatozoa. Inhibitors of PDE are potent stimulators of motility. Spermatozoa are terminally differentiated cells with highly specialized compartments. The signal transduction enzymes in sperm need to be correctly targeted to ensure proper functionality. PKA is directed to specific subcellular locations through its interaction with A-Kinase Anchoring Proteins (AKAPs). The location of PDEs is also tightly controlled and recent reports have provided evidence that PDE4D binds to AKAP450 and mAKAP in Sertoli cells and cardiomyocytes, respectively. In spermatozoa there is evidence of many isoforms of PDE's, which could be localized in different sub-cellular locations. The main goal for this study was to determine if PDEs and AKAPs interact in spermatozoa. Using immunofluorescent labeling techniques, PDE4A was localized in the principal piece of the tail similar to AKAP110. In-vitro pull-down assays were then performed using recombinant AKAP110-GST and PDE4A protein. PDE4A bound to AKAP110-GST but not to the GST control. Immunoprecipitation of the co-transfected AKAP110 and PDE4A into COS cells with anti-AKAP110 antibody followed by Western blotting detected an increase in the PDE4A band compared to IgG control. PDE assays performed on the immunoprecipitates from COS cells further support this interaction, showing higher activity in cells co-transfected with the AKAP110 and PDE4A and immunoprecipitated with AKAP110 compared to IgG control. In conclusion, these data suggest that PDE4A is associated with AKAP110 in the principal piece of the sperm tail where they are in a prime position to regulate sperm motility. This work was supported by NIH HD36408 and VA Merit Grant (DWC) and HD31544 (MC).

KEY WORDS: PDE, cAMP, sperm, AKAP110