Role of the major ecto-phosphoprotein in sperm flagellar motility using a cell electroporation method

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.