Adenylate cyclase activity increases concomitantly with the onset of capacitation in heparin-treated bovine spermatozoa

Abstract

This study examined the effect of metal ions Ca2+, Mg2+, and Mn2+ and sonication on ejaculated frozen-thawed bovine sperm adenylate cyclase activity, and whether cAMP levels in sperm changed during capacitation with heparin. The sperm adenylate cyclase was almost insensitive to Ca2+ at concentrations up to 10 mM when cold-shocked homogenized spermatozoa were used. Adenylate cyclase activity, as observed by cAMP formation (pmol/mg protein/min), did not increase significantly in the presence of 5 mM Mg2+. However, a 40-fold stimulation (cAMP 400-800 pmol/mg protein/min) occurred in the presence of 5 mM Mn2+. Although Ca2+ per se had no effect, it acted synergistically with Mg2+ and Mn2+ in stimulating sperm adenylate cyclase. Adenylate cyclase activity was highest in cold-shocked, homogenized spermatozoa. Sonication of cold-shocked spermatozoa resulted in loss of adenylate cyclase activity, and a logarithmic decrease in cAMP production (1,155.5 to 109.7 pmol cAMP) occurred when sonication was increased from 2 x 5 to 2 x 25 s on ice. Cyclic AMP levels in spermatozoa incubated under non-capacitating conditions, both untreated and treated with glucose or heparin plus glucose, remained higher (P < 0.01) compared with those incubated with heparin for the first 4 h of incubation. When spermatozoa were incubated under non-capacitating conditions, cAMP levels increased (P < 0.01), especially during the first hour of incubation, and then declined gradually throughout the incubation. In contrast, cAMP levels of heparin-treated spermatozoa declined gradually for 3 h, at which time they began to rise, peaked at 4 h and then remained fairly stable until 6 h. Glucose antagonized the effect of heparin on adenylate cyclase activity but not for more than 4 h. We conclude that: i) Ca2+ stimulates adenylate cyclase in the presence of Mg2+; ii) homogenization by sonication reduces cyclase activity in frozen-thawed, cold-shocked spermatozoa; iii) adenylate cyclase activity is inhibited by heparin but rises concomitantly with the onset of capacitation (after 4 h) in spermatozoa incubated under capacitating conditions; and iv) glucose, which prevents capacitation by heparin, antagonizes heparin action on adenylate cyclase.