N. S. Kocheshkova, Z. D. Vorobets
The experiments performed on preparations of spermatozoids of men of reproductive age (27–44-year old) studied the ATPase activity (sensitive to inhibited effects of eosine Y) in both normal and oligozoospermia conditions when treating cell suspension with detergents. The methodical approaches for testing the so-called “common” and eosin Y-sensitive ATP-hydrolase activities in spermatozoids were developed. Saponin, the optimal detergent for permeabilisation of their plasma membrane was chosen using laser-correlational spectroscopia method. Saponin perforates effectively the membranes of spermatozoids, decreasing the average hydrodynamical diameter of cells from 10–15??m (the spermatozoids themselves) to 3–8??m (treating cell suspension with 0.05% solution of saponin) and even to 2–3 ?m (treating spermatozoids suspension with 0.5% solution of detergent). A non-specific inhibitor of ATP-hydrolase’s systems, eosin Y, decreases effectively the ATP-hydrolase activity of intact spermatozoids up to 40%. The exact effect of eosin depends on composition of incubation medium. In the model of extracellular conditions (the optimal concentration of detergent is 0.05%), eosin Y-sensitive ATP-hydrolase’s activity of spermatozoids in both normal and oligozoospermia cases is increased by 220–240% (at an average). If enzymatic reaction was performed during intracellular conditions modeling (the optimal concentration of saponin is 0.5%), the increase of eosin Y-sensitive АТРase activity (up to 350–400% in normal conditions, and only to 130–150% in oligozoospermia conditions) was detected. This specificity can be used as easy-to-use clinical test for such pathology of men’s reproductive system. Eosin Y inhibited doze-dependently the common ATPase activity in spermatozoids in both normal and with studied pathology. In both cases, after linearization of curves of catalytic titration of ATPase activity with eosin Y in Hill’s plot the two-phase dependency, of high and low affinities, was found (the average values of imaginary inhibition constant I0,5 are 0.1 and 0.3–0.4 mM correspondingly). In both normal and oligozoospermia conditions, the high-affinity component has a positive cooperativity, while the low-affinity component is characterized by a negative cooperativity. The obtained results may be of both theoretical and practical value for further investigation of membrane mechanisms used in the support of ion homeostasis in men’s spermatozoids and its violation in conditions under different pathological states. Besides, the results can be used as a theoretical basis for improvement of simple and accessible clinical biochemical methods used for testing such a pathology as oligozoospermia.
Key words: spermatozoid, oligozoospermia, АТРase activity, laser-correliation spectrophotometry, saponin, eosin Y.
The original article in Ukrainian is available for download in PDF format.
© The Ukrainian Biochemical Journal