Ecto-Cyclic AMP Independent Protein Kinase-A Potent Regulator of L-type Calcium Channel and Forward Motility of Goat (Capra indicus) Epididymal Spermatozoa

Abstract

Objective: Caprine (Capra indicus) spermatozoa possess a novel ecto-cAMP independent protein kinase (ecto-CIK) on their membrane surface. This enzyme showed remarkable alteration through the maturation process of spermatozoa in epididymis. We investigated the role of CIK in the regulation of forward motility of epididymal spermatozoa by controlling the intracellular level of [Ca 2+ ].Method: Cauda epididymal mature sperm cells were treated with CIK antibody and maximum inhibition of enzyme activity (85%) was observed at 120 minutes of exposure. To analyze the calcium uptake mechanisms cells were exposed to 45 Ca 2+ after treatment with CIK antibody and pretreated with different calcium channel regulators. The intracellular [Ca 2+ ] i signal was determined fluorometrically by using fura 2-AM. The computerized spectrophotometric assay method was used to measure the percentage of forward motility.Result: It was shown that the uptake of calcium through the L-type voltage-dependent Ca 2+ channels of the plasma membrane is regulated by CIK. Pretreatment of sperm cells with varapamil (20 μM), nifedipine (20 μM) significantly inhibited the increased intracellular [Ca 2+ ] i induced by the CIK antibody. Whereas calmodulin antagonists trifluoperazine and w 13 (N-(4-Aminobutyl)-5-chloro-2 naphthalenesulfonamide hydrochloride and sodium azide, a potent mitochondrial inhibitor, showed no effect on this calcium entry. The treatment with ca ionophore A123187 of the CIK antibody treated cells showed channel inhibitor insensitive increase of calcium uptake. It was observed that verapamil (20 μM) has significant role in decreasing (~50%) forward motility of CIK-antibody treated spermatozoa. Conclusion:We concluded that CIK is an important regulator of forward motility in epididymal spermatozoa and the regulation may be activated partly by the intracellular [Ca 2+ ] i level the extent of which is functionally imparted by the voltage gated L type calcium channel.