Kinetic properties of na+,K+-АТРase of spermatozoa from fertile and infertile men under effect of calix[4]arene c-107

the calix[4]arene c-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) effects on the kinetic properties of Na+,k+-atPase in spermatozoa of fertile (normozoospermia) and infertility men (oligozoospermia, and asthenozoospermia) were studied. It was shown that in spermatozoa of healthy men calix[4]arene С-107 inhibited Na+,k+-atPase activity and decreased the maximum reaction rate of ATP hydrolase reaction without affecting the coefficient of (half-) activation by ATP and Hill coefficient nh. In оligoand asthenozoospermic samples of spermatozoa almost a 2-fold decrease of cooperativity coefficient nh of atPase inhibition with calyx[4]aren c-107 was observed. In normozoospermic samples of spermatozoa the kMgcl2 for Na +,k+-atPase was decreased at calix[4]arene c-107 high concentrations (≥50 nM) in the incubation medium in contrast to oligozoospermic samples of spermatozoa where KMgcl2 was increased only at high calix[4]arene c-107 concentration (100 nM). the increase of the kMgcl2 in the entire range of investigated calix[4]arene concentrations and the decrease of cooperativity coefficient nh of Mgcl2 activating effect were detected in asthenozoospermic samples of Na +,k+-atPase.

C alix [4]arenas attracted more and more attention in recent years. They are macrocyclic oligomers formed by cyclic condensation of n-substituted phenols and formaldehyde. Calix [4]arenas are low molecular, low-toxic supramolecular compounds capable of altering the activity of iontransporting systems [1,2]. Due to their ability to form supramolecular complexes with biologically important molecules and ions they affect biochemical processes. Calix [4]arene matrices have low toxi city and sufficiency conformational mobility in solution which provides a great advantage for their application in biomedical studies as enzyme effectors, receptor simulators, organic polymer stabilizers, DNA microarray platform, etc [3][4][5]. Therefore calix [4]arenas are considered as promising molecular platforms for the creation of physiologically active compounds [6].
Approximately 50% of the infertility cases are related to male factor. The most common ab-normal parameters are low motility (asthenozoospermia), low sperm count (oligozoospermia), or a combination of these abnormalities (oligoasthenozoospermia) [7,8]. From the standpoint of modern biomembranolo gy, many diseases are associated with changes in the structure and function of biomembranes. Membrane binding proteins, inclu ding the integral ATP-dependent transport enzyme, play an important role in proper functioning of the membranes. Therefore, it is necessary to further the study of properties of membrane-bound ion transport systems in the normal and at pathological states. Na + ,K + -ATPase (Na + ,K + -activated, Mg 2+ -dependent АТP-hydrolase, ЕС 3.6.1.37) is an electrogenic Ca 2+ -independent transport system which reacts quickly to changes in intracellular and extracellular concentration of Na + and K + ions and maintains the transmembrane gradient of these ions and resting membrane potential needed for normal cell functioning [9]. The activity of many other biochemi cal processes depends on the activity of this enzyme. Specifically, Na + ,K + -ATPase transmits signals to the nucleus of the cell by protein-protein interac tions with sensor proteins [10]. Na + ,K + -ATPase is present in male germ cells and differentiated spermatozoa [11]. A unique α4 subunit was identified in the structure of Na + ,K + -ATPase of spermatozoa [12]. In addition to α4 subunit, α1 Na,K-ATPase isoform, which is ubiquitously present in all tissues, is also expressed in spermatozoa [11]. Na + ,K + -ATPase plays a crucial role in sperm physiolo gy [13]. Many studies showed a significant relation between Na + ,K + -ATPase activity and sperm motility [9,14]. Obviously, the search for effective inhibitors and activators of Na + ,K + -ATPase is important from both fundamental and practical point of view. Specifically, such studies may be useful for the develop ment of potential pharmacological agents for the modulation of sperm motility.
donors and semen sample preparation. Human semen was obtained from 10 healthy volunteers and 14 pathozoospermic men undergoing routine semen analysis for couple infertility at Lviv Regional Clinical Hospital (Ukraine). Approval for the study was taken from the ethics committee of Danylo Halytsky Lviv National Medical University (Ethical Committee Approval, protocol No 6 from March 29, 2017). Terms of sample selection meet the requirements of the principles of Convention of Europe Council on human rights, Helsinki Declaration on protection of human rights and biomedicine and the laws of Ukraine. All patients and healthy donors were matched by age and gave written informed consent to participate in research. Exclusion criteria: subjects currently on any medication or antioxidant supplementation were not included. In addition, subjects with infertility over 10 years, azoospermia, testicular varicocele, genital infection, chronic illness and serious systemic diseases, smokers and alcoholic men were excluded from the study because of their well-known high seminal reactive oxygen species levels and decreased antioxidant activity which may affect calcium level. Samples were obtained by masturbation after 3-4 days sexual abstinence and processed immediately upon liquefaction. The classical semen parameters of spermatozoa concentration, motility, and morphology were examined according to World Health Organization criteria (2010).
Biochemical study. Biochemical studies were carried out in the Department of Medical Biology of Danylo Halytsky Lviv National Medical University. Sperm cells were washed from semen plasma by 3 times centrifugation at 3000 xg for 10 min in media which contained (mM): 120 NaCl, 30 KCl, 30 Hepes (pH 7.4). The protein concentration in the samples was determined by Lowry method using a kit to determine its concentration (Simko Ltd). Determination of ATPases activities was carried out in permeabilized spermatozoa. The detergent saponin in a final concentration of 0.5% was added to sperm suspension for permeabilization of sperm membranes. enzymatic studies. The total Na + ,K + -ATPase activity was assayed with the following incubation medium (mM): 120 NaCl, 30 KCl, 5 МgС1 2 , 3 АТР, 1 ЕGTA, 0.01 thapsigargin (specific inhibitor of SERCA), 1 NaN 3 (specific inhibitor of mitochondrial Fig. 1

. Structural formulas of calix[4]arene c-107
ATPase), 20 Hepes-Tris (рН 7.4; at 37 °C) [16]. The protein concentration did not exceed 50 mg/ml. The reaction was started by addition of an aliquot of permeabilized sperm cells. After a 5 min incubation, 1 ml of a stop solution containing (mM) 1.5 M sodium acetate, 3.7% formaldehyde, 14% ethanol, 5% trichloroacetic acid (рН 4.3) acid was added. Pi was determined by the Fiske-Subbarow method using the assay kit Simko Ltd (Ukraine). Ouabain-sensitive Na + ,K + -ATPase activity was determined as the difference of ATP hydrolysis in the absence and presence of 1 mM ouabain, which is known to completely block the Na + ,K + -ATPase. Activity was expressed as nmoles of P i released /mg of protein per min. The effect of calix [4]arene C-107 at different concentrations (10-100 nM) on Na + ,K + -ATPase was studied using the standard incubation medium (as described above) to which the solution of calix [4]arene was added.
kinetic calculations. For studying of the calix [4]arene effect on enzyme activity, the kinetic parameters were calculated using linearized Hill plots where a is specific enzyme activity, a max is maximum specific enzymatic activity, k is coefficient of (half-)activation by ATP (k ATP ) or MgCl 2 (k Mg2+ ), S is the concentration of the substrate or ion activator in the incubation medium, n H is Hill coefficient. The maximum reaction rate of ATP hydrolysis (V max ) reaction was calculated in the Hanes-Woolf plot {[S]/V as a function of [S]}. The typical value of the meansquare deviation of the approximation coefficient was 0.85-0.99.
Statistical analysis. Data are expressed as means ± standard error of the numbers of determinations. Differences between paired sets of fluorimetric experiments were analysed using paired Student's t-tests in Microsoft Excel. Differences were considered significant at P < 0.05 as the minimum significance level.
Reagents. The following reagents were used in the present study: ATP, ouabain, thapsigargin, EGTA (Sigma, USA), saponin (from Quillaja Saponaria Molina pract.; Acros organics, Belgium). Other reagents of domestic production were of reagent grade or laboratory grade.

results and discussion
In our previous study [17], control samples (fertile men with normozoospermia) showed activity of Na + ,K + -ATPase about 46.3 ± 4.2 nmol P i /min per mg protein. In patients with oligo-, astheno-and oligoasthenozoospermia the Na + ,K + -ATPase activity was 22.4 ± 3.5, 23.6 ± 2.6 and 21.8 ± 3.8 nmol P i /min per mg protein correspondingly, that was two fold lower vs. control group. In previous experiments with calix [4]arenas, it was shown that C-107 calixarenphosphonic acid in 100 µM concentration inhibited sperm Na + ,K + -ATPase more effectively than ouabain [15]. Therefore, for further kinetic interpretation of calix [4]arene C-107 effect on enzymatic activity of Na + ,K + -ATPase of spermatozoa we studied its effects on the concentration dependence of ATPase activity on ATP and Mg 2+ concentration. As substrate concentration in incubation medium increases (under constant level of cofactor) the Na + ,K + -ATPase activity of sperm cells from normo- (Fig. 2, a) and pathozoospermic men increases (Fig. 2, b, c). Similar concentration dependences were also obtained for oligoasthenozoospermic samples (results are not presented in the paper). The highest enzyme activity was observed under mM ATP in the incubation medium. Presence of calix [4]arene C-107 in a concentration range from 10 to 100 nM in the incubation medium leads to inhibition of sperm Na + ,K + -ATPase activity in both normo-and pathozoospermic men. The maximum Na + ,K + -ATPase activity of sperm cells decreases as the calixaren concentration increases.
The coefficient of (half-)activation by ATP (k ATP ) and Hill coefficient (n H ) were calculated using Hill's plot for all studied groups (Fig. 3).
As can be seen from Fig. 3,a the calix [4]arene C-107 did not affect coefficient of (half-)activation by ATP k ATP and Hill coefficient n H for Na + ,K + -ATPase in spermatozoa of normozoospermic men. The values of Hill coefficient n H were higher than 1 with and without calix [4]arene C-107 in the incubation medium which indicates positive cooperation between enzyme and ATP. Since calix [4]arene C-107 inhibi ted sperm Na + ,K + -ATPase activity with respect to control [15] and did not change coefficient of (half-)activation by ATP and enzyme co-operation to ATP (values of K ATP and n H were unchanged) we assume that enzyme activity can be decreased due to the decrease in the maximum reaction rate (V max ). Therefore, next, we calculated V max catalyzed by Na + ,K + -ATPase Hanes-Woolf plot. As can be seen from Fig. 4 calix [4]arene C-107 effect on maximum reaction rate V max in a dose-dependent manner. Similar dependences of V max of Na + ,K + -ATPase were obtained for sperm cells from oligozoospermic and asthenozoospermic men.
Our results are in perfect agreement with studies carried out on uterus myocyte plasma membrane Na + ,K + -ATPase [18]. It was shown that calix [4]arene C-107 inhibited enzyme without affection of kinetic parameters (k ATP , n H ) of reaction velocity dependence on substrate concentration.
For oligo-and asthenozoospermic samples the Hill coefficient n H was almost twice lower in the presen ce of 100 nM calix [4]arene C-107 in the incubation medium compared to control. These values of Hill coefficient (n H > 1) indicate the positive cooperation of Na + ,K + -ATPase to ATP in pathospermic samples obtained from infertile men. Changes in values of n H might indicate subunit changes of enzyme structure under increasing concentration of calix [4]arene C-107 in the incubation medium. The maximum reaction rate (V max ) of Na + ,K + -ATPase significantly decreases as calix [4]arene C-107 increases.
It is known that besides ATP, magnesium is required for the functioning of Na + ,K + -ATPase. The Mg 2+ ions, which act as a cofactor, form a chelating complex Mg•АТР, which is a substrate of enzymatic reaction. Also, Mg 2+ ions bind to the regulatory center of Na + ,K + -ATPase [19]. The Na + ,K + -ATPase activi ty of sperm cells increases as MgCl 2 concentration increases in the range from 0.1 to 10 mM under constant concentrations of ATP (3 mM) in the incubation medium (Fig. 5, a, control). The dependences of enzyme activity on MgCl 2 concentration were similar to dependence without calix [4]arene C-107, howe ver, the plateau level of Na + ,K + -ATPase activi ty decreases as the calix [4]arene concentration increases. Similar concentration dependences were obtained for oligozoospermic (Fig. 5, b), asthenozoospermic (Fig. 5, c)  Calix [4]arene C-107 effect on the coefficient of (half-)activation by MgCl 2 of Na + ,K + -ATPase in spermatozoa of normo-and pathozoospermic men was different. Specifically, in normozoospermic samples of spermatozoa, the coefficient of (half-)activation by MgCl 2 k MgCl2 decreases at high concentrations of calix [4]arene C-107 (≥50 nM) in the incubation medium (Fig. 6). Contrary to this in oligozoospermic samples of spermatozoa, k MgCl2 increases only at high concentration (100 nM) of calix [4]arene C-107. For sperm Na + ,K + -ATPase from asthenozoospermic men, an increase in calix [4]arene C-107 concentration leads to an increase in k MgCl2 in the entire range of investigated concentrations of calix [4]arene. Previously it was shown complex two-phase nature of k MgCl2 dependence on the concentration on calix [4]arene C-107 for uterus myocyte plasma membrane Na + ,K + -ATPase [18]. The Hill cooperativity coefficient n H of activation by MgCl 2 decreases in the presence of calix [4]arene C-107. This may indicate changes in the subunit composition of the enzyme as a result of calix [4]arene C-107 effect on it.
The obtained results indicate that highly effective inhibitory effect of calix [4]arene C-107 on Na + ,K + -ATPase in normozoospermic samples has non-competitive character and is associated with a decrease in the maximum reaction rate (Fig. 7). In oligo-and asthenozoospermic men inhibitory effect of calix [4]arene C-107 causes an increase in k MgCl2 for MgCl 2 .