Eosin Y-sensitive ATPase activity in men's spermatozoids as a biochemical test for oligozoospermia

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 microm (the spermatozoids themselves) to 3-8 microm (treating cell suspension with 0.05% solution of saponin) and even to 2-3 microm (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 ATPase 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 I(0,5) 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.     

Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile cells

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).     

System of Ca2+ transport in spermatozoids and biochemical mechanisms of capacitation and acrosomic reaction

The paper systematizes modern ideas regarding the role of macroelements (including calcium ions) in physico-chemical, biochemical spermatozoid processes, that provides the preservation of their biological completeness and fertility. Information concerning ion content and ion distribution in semen of males of different age groups and in the ejaculate with different quality indicators is presented. The basic ion-transport systems that are identified in spermatozoid membranes of males and their role in sperm activation, cell active movement ability, maintenance of cell homeostasis etc. are discussed. General schemes of biochemical mechanisms of capacitation process and acrosomic reaction are proposed.     

Kinetic properties of Na+, K+ activated, Mg2+ -dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis

The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+ -ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+ -ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+ -ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+ -ATPase affinity to ATP. However, Mg2+ -binding center of Na+, K+ -ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+ -ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+ -ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.