Presence of cyclic nucleotide phosphodiesterases PDE1A, existing as a stable complex with calmodulin, and PDE3A in human spermatozoa.
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. Calmodulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities 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 segment of the sperm head as well as on the mid and principal pieces of the flagellum, and that PDE3A is found on the postacrosomal 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 + calmodulin. Results obtained with electrophoresis in native conditions indicated that calmodulin is tightly bound to PDE1A. Incubation with EGTA + EDTA, trifluoperazine, or urea did not dissociate the PDE1A-calmodulin complex. These results suggest that PDE1A is permanently activated in human spermatozoa.
Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.
Linda
L
Lefièvre
Eve
E
de Lamirande
Claude
C
Gagnon
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000328:Adult; D002147:Calmodulin / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D020105:Milrinone / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000201:enzymology* / Q000378:metabolism
D002147:Calmodulin; D010726:Phosphodiesterase Inhibitors; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517265:PDE1A protein, human; C517287:PDE3A protein, human; D020105:Milrinone; D015056:1-Methyl-3-isobutylxanthine