Regulation of sperm motility bycAMP and calcium/calmodulin
Paupard, Marie-Christine Nelly
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In this study we: (1) characterize the non flagellar regulatory subunit of cAMP-dependent protein kinase; (2) determine whether multiple 56,000 cAMP-dependent phosphoproteins exist in the cytosolic or detergent extract of bovine sperm; and (3) determine with which compartment of the cell the initiator protein is associated.;We have established that the sperm cytosolic and membrane associated RII is a conventional RII. The subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 dalton, respectively. Using either immobilized cAMP or immobilized monoclonal anti-RII antibodies, we were able to remove quantitatively RII as well as all of the 56,000 dalton cAMP-dependent phosphoproteins from cytosol and detergent extracts of sperm. Since it is known that phospho-RII cannot initiate motion, the motility initiator protein cannot be the 56,000 dalton phosphoprotein. We have also shown that it is possible to initiate motion in demembranated sperm, devoid of all cytosolic and membrane components, by the addition of cAMP/Triton/ATP. These results demonstrate that the initiation of sperm motion requires the cAMP-dependent phosphorylation of flagellar, rather than cytosolic or membrane associated proteins. We have also demonstrated that the cAMP-dependent phosphorylation of the initiator motility protein can be achieved by the endogenous flagellar-associated cAMP-dependent protein kinase. Once motion has been initiated, the addition of protein kinase inhibitor does not inhibit motion suggesting that cAMP-dependent phosphorylation is only a prerequisite for initiation. Another important result was the requirement of a "detergent-like" molecule in order to initiate motion.;The requirement of cAMP for the initiation of sperm motion is contrasted by the inhibitory and wave form modulating effects of calcium. The effect of micromolar levels of calcium are consistent with a role for calmodulin in these processes. In order to elucidate the role played by calmodulin in motility, it is necessary to characterize the calmodulin regulated enzymes or proteins associated with the flagellum. We have identified a major 67,000 dalton calcium-dependent calmodulin binding protein present on the flagella of rat and bovine sperm as well as on Paramecium cilia. Partial purification of the calmodulin binding protein was achieved. The isoelectric point of the 67,000 dalton calmodulin binding protein from rat sperm flagellum was determined to be 6.0.;In order to see if the 67,000 dalton calmodulin protein found on rat sperm flagellum and on Paramecium cilia was calcineurin we used peptide mapping procedures on the partially purified protein. The digestion profiles of the proteins are different. We looked for calcineurin transcripts in adult testes from mouse spermatogenesis mutants. The message was present only in somatic cells, none was found in germ cells. (Abstract shortened with permission of author.).