Presence of Cyclic Nucleotide Phosphodiesterases PDE1A, Existing as a Stable Complex with Calmodulin, and PDE3A in Human Spermatozoa1

Mammalian sperm motility, capacitation, and the acrosome reaction are regulated by signal transduction systems involving cAMP as a second messenger. Levels of cAMP are controlled by two key enzymes, adenylyl cyclase and phosphodiesterases (PDEs), the latter being involved in cAMP degradation. Calmod-ulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) ac-tivities were previously identified in spermatozoa via the use of specific inhibitors. Here we report that human sperm PDEs are associated with the plasma membrane (50%–60%) as well as with the particulate fraction (30%–50%) and have more affinity for cAMP than cGMP. Immunocytochemical data indicated that PDE1A, a variant of PDE1, is localized on the equatorial seg-ment of the sperm head as well as on the mid and principal pieces of the flagellum, and that PDE3A is found on the postac-rosomal segment of the sperm head. Immunoblotting confirmed the presence of PDE1A and PDE3A isoforms in spermatozoa. Milrinone, a PDE3 inhibitor, increased intracellular levels of cAMP by about 15 % but did not affect sperm functions, possibly because PDE3 represents only a small proportion of the sperm total PDE activity (10 % and 25 % in Triton X-100 soluble and particulate fractions, respectively). PDE1A activity in whole sperm extract or after partial purification by anion-exchange chromatography was not stimulated by calcium 1 calmodulin. Results obtained with electrophoresis in native conditions indi-cated that calmodulin is tightly bound to PDE1A. Incubation with EGTA 1 EDTA, trifluoperazine, or urea did not dissociate the PDE1A-calmodulin complex. These results suggest that PDE1A is permanently activated in human spermatozoa. cyclic adenosine monophosphate, gamete biology, phosphodi-esterases, sper