Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa

Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca: fascicularis) spermatozoa; inst ead, both have an absolute requirement for exogenous stimulation with caffe ine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting cri teria for automated analysis of macaque sperm exhibiting hyperactivated mot ility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteris tics were assessed by computer-assisted motion analysis. Tyrosine phosphory lation of sperm tail proteins was determined by immunocytochemistry with PY -20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) greater than or equal to 150 mu m/sec; amplitude of lateral head disp lacement (ALH) greater than or equal to 8.0 mu m, and linearity (LIN) less than or equal to 60%, Using these criteria, caffeine and dbcAMP significant ly stimulated HA (61 +/- 8%) compared to control conditions (12 +/- 2%), p < 0.01, with a concomitant increase in PY-20 labeling (88 +/- 12%) vs. cont rol (13 +/- 2%), p < 0.01. PY-20 labeling significantly correlated with HA (r = 0.75, p < 0.01) and with some motion characteristics used for HA sorti ng including ALH (r = 0.86, p = 0.0013) and LIN (r = -0.88, p < 0.001) but not VCL (r = 0.21). Treatment with genistein (10 mu M) had no effect on HA or PY-20 immunocytochemistry in the absence of caffeine and dbcAMP, but the tyrosine kinase inhibitor significantly decreased caffeine- and dbcAMP-sti mulated HA and PY-20 labeling in a dose-dependent manner (p < 0.01). These results demonstrate that tyrosine phosphorylation of sperm tail proteins is an integral signaling pathway modulating some but not all of the motion ch aracteristics associated with cAMP- and caffeine-stimulated HA in cynomolgu s monkey spermatozoa.