Measurement of adenylate cyclase activity in ram spermatozoa

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Abstract
Semen was collected from 3 rams by electroejaculation and pooled. The following techniques were used to disrupt spermatozoa before assay for adenylate cyclase activity: freeze-thawing, cold shock, passage through a French pressure cell, homogenization and sonication. Freeze-thawing resulted in the greatest increase in adenylate cyclase activity, followed in order by cold-shock, passage through the French press and sonication. Homogenizing had no effect. Similarly, phosphodiesterase activity was highest after freeze-thawing, while cold-shocked cells showed a smaller increase in activity than did untreated cells. Examination of stained spermatozoa revealed various degrees of damage. Assay of untreated and freeze-thawed spermatozoa in the presence of 20 mM-MnCl2 showed a greater than 20-fold stimulation of adenylate cyclase activity, but both in the presence and absence of Mn2+ there was a 5-fold difference between the activities of untreated and freeze-thawed cells. The effect of freeze-thawing apparently increased the premeability of cells to ATP, rather than producing a real increase in the activity of adenylate cyclase. A real increase in enzyme activity should either be additional to the increase produced by Mn2+, or should overlap this increase.