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7BIOLOGICAL SCIENCES
IONS CONCENTRATION AND ITS RATIOS IN TISSUES AND SECRETIONS OF GENITALS OF
MALES AND FEMALES
Maksymyuk A.,
D Sci., Professor, Department of Laboratory and Clinical Diagnostics, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Maksymyuk V.,
PhD, Leading researcher of the Institute of Agriculture of Carpathian Region NAAN of Ukraine, Lviv reg., Ukraine
Sedilo H., D Sci., Professor, Adviser of the Institute of Agriculture of Carpathian Region NAAN of Ukraine, Lviv reg., Ukraine
Melnyk O.,
PhD, Assistant Professor, Department of Microbiology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Vorobets M.,
PhD, Assistant Professor, Department of Urology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Vorobets Z.,
D Sci., Professor, Department of Medical Biology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Abstract
It is established the ability of breeders (bulls), regardless of breed, age (2-9 years) and season of the year constantly secrete semen with low (17-33 mM), medium (36-43 mM) and high (60-66 mM) K+ concentration limits. The constancy of different levels of concentration is related to the quality parameters of sperm, namely: low and high values reduce their motility and viability. Harmful effect of extreme conditions of sperm cryopres-ervation technologies stages significantly changes the initial levels of ion concentration. In sperm of freshly obtained, diluted, equilibrated and deconserved sperm, the dynamics of Ca2+ parameters is: 1,2-1,5-1,7-0,9 mM respectively; K+ - 15-14-14-4 mM; Na+ - 19-15-15-41 mM.
The imbalance of homeostasis of the components of the binary system "sperm - uterine-vaginal mucus" formed by the harmful effect of endogenous factors leads to low fertilization of egg-cells by spermatozoons of freshly obtained and deconserved sperm.
The possibility of objective monitoring and forecasting the levels of fertility of sperm by certain parameters of the ratios of the concentration of one- (Ca2+: Ca2+, K+:K+, Na+:Na+) and different (Na+: Ca2+, K+: Ca2+, Na+:K+) pairs of alkali metal ions is established.
The developed methods of research of the "reaction-response" of the components of open and closed systems such as "environment - cell (substance)" can be used for in-depth study of physical and chemical, transmembrane processes that occur due to the harmful effects of exogenous and endogenous factors on isolated and non-isolated cells of humans and animals.
Keywords: spermatozoa, genitals, ions concentration, Ca2+,Mg2+-ATPase.
Introduction. Clarifying and detailing certain components of sperm protection against the harmful effects of endogenous factors still remains an urgent problem of the biology and medicine to cryopreserva-tion of cells [1, 14]. In this regard, modern researchers should focus on the fact that have already developed a large number of effective multicomponent diluents of sperm, which significantly increase the volume of ejaculate and create the best possible conditions for sperm survival under the harmful effects of sperm cryopreser-vation technologies extreme conditions (SCT). The basic components of the protective media recommended for production are mainly glycerin, yolk, mono- (glucose, galactose, xylose, rhamnose), di- (lac-
tose, sucrose, maltose) and polycarbohydrates (raffi-nose) and conditionally indifferent to the functional state of spermiums organic substances (dimetil-sulfox-ide, polyvinylpyrrolidone, ethyleneglycol etc.). However, the total result of their protective effect ensures the preservation of only 30-35% of functionally complete cells. In addition, destructive changes in the acro-some and cytoplasmic membrane of deconserved sperm, which have almost halved relative sperm motility and fertilization, do not allow them to come into effective contact with the egg.
In this regard, we believe that developed and recommended to researchers of various kinds methods for determining the dynamics of the parameters of mass,
content, concentration of components (organic and inorganic substances, Ca2+, K+, Na+) biological systems such as "environment - cell (substance)" under the action of exo- (semen) and endogenous factors (extracts of tissues and secretions) and the proposed techniques and methods for assessing the features of their "response to the action" of certain factors will help minimize their harmfulness [11-14].
Materials and Methods. Sperm was obtained from bulls (n = 68) of different breeds (Holstein, British-Frisian, German, local, Simmental, Red Danish, Limousine, Aberdeen-Angus), of different ages (2-9 years) in different seasons of the year (winter, spring, summer, autumn). According to parameters of K+ concentration, ejaculates were divided into three separate gradation levels: (HKK), medium (CKK), high (BKK).
It was found that 13 bulls, or 19% of the selection, secrete semen with a low concentration of K+. The CKK group consisted of 40 animals, which is 59%; BKK -15 animals, or 22 %.
Results. Because the course of the processes of formation (gametogenesis), the formation of viability,
Secrets of uterine and vaginal mucus have other indices. Thus, if the levels of Ca2+ concentration of tissues of organs and mucus of cows coincide (6 mM), K+
- are 1,1 times less (55 vs. 48 mM), then Na+ - 4,9 times higher (700 vs. 142 mM). Of particular note is the very significant difference in the concentration of Na+ in sperm and mucus. Its average value in semen is 8.6 times lower (700 vs. 81 mM).
The determined difference in the concentration of Ca2+, K+, Na+ is even more clearly emphasized by the ratio of the concentrations of different pairs of ions, namely: if the average Na+:Ca2+ pair in semen is 2.2 times smaller than in the tissues of the genitals, K+:Ca2+
- in 2,0, then Na+:K+ only 1.5 times.
A significant difference in the indicators is also characteristic of the ratio of the concentration of ions in the tissues of the genitals and the secretion of uterine-vaginal mucus, which is released during 1-3 days of heat. Thus, if the ratio of the concentration of Na+:Ca2+ and Na+:K+ pair of mucus is 4.9 and 5.0 times higher than the tissues, then K+:Ca2+ pair is 1.1 times smaller.
fertilization of gametes (spermatozoons, egg-cells) and the synthesis of secretions (sperm plasma, uterine-vaginal mucus) provides a tissue environment (epithelial, connective, trophic, parenchymal, muscular) of the genital systems of males and females, the purpose of this of research is reduced to a comparative analysis of the response of components of closed and open systems such as "environment - cell (substance)" to the harmful effects of endogenous factors. The results, which illustrate the indicators of table 1, are represented by the limits (min - max) of the average parameters (M) of the concentration and its relations between different ion pairs (Na+:Ca2+, K+:Ca2+, Na+:K+).
The obtained indicators indicate that the level of concentration of Ca2+ in the semen and tissues of the genitals of bulls is almost the same (8 vs. 7 mM). In this regard, it should be emphasized that the limits of its deviations in semen are quite narrow (7-9 mM), and in tissues very wide (3-11 mM). In addition, the average concentration of K+ (36 vs. 80 mM) and Na+ (81 vs. 209 mM) in semen, respectively 2.2 and 2.6 times is lower than in tissues.
In this regard, it should be noted that with a slight difference in the average parameters of the concentration of Ca2+ (8 vs. 6 mM) and K+ (80 vs. 55 mM) in the tissues of males and females and 1.5 times greater Na+ in bulls (209 mM) than in cows (142 mM), the ratios of its pairs of different names are almost the same (26-10-3: 1 vs. 24-9-3: 1). To this should be added that the high concentration of Na+ (500-900 mM) of mucus secretions is opposite to the ratio of its concentration in semen, namely: if the ratio of Na+:Ca2+ pair of mucus ions in 9.8, K+:Ca2+ - in 1,6, then Na+:K+ - -7.5 times larger than in sperm.
Some aspects of the reaction of the components of the biological system type "environment - cell (substance)" to the action of exogenous and endogenous factors allow us to state that the researchers can use the difference in the composition of the optimal composition of new media, which will increase the percentage of complete spermatozoons in deconserved sperm doses. The results of biochemical studies confirm the indicators obtained in determining the degree of polymorphism of spermatozoons and the destruction of
Table 1
Levels of ions concentration and its ratios in tissues and secretions _ of genitals of males and females (M)_
Sexual organs Stat. indi-cator Bulls (semen) Cows (uterine-vaginal mucus)
Concentration, mM
Ca2+ K+ Na+ Ca2+ K+ Na+
Tissues M 8 80 209 6 55 142
Lim 7-9 60-100 162-256 4-8 19-91 68-215
Secrets M 7 36 81 6 48 700
lim 3-11 25-46 64-97 3-9 34-61 500-900
Ratio, IC:1
Pairs of ions Na+:Ca2+ K+:Ca2+ Na+:K+ Na+:Ca2+ K+:Ca2+ Na+:K+
Tissues M 26:1 10:1 3:1 24:1 9:1 3:1
Lim 23-28:1 9-11:1 3:1 17-27:1 5-11:1 4-2:1
Secrets M 12:1 5:1 2:1 117:1 8:1 15:1
Lim 21-9:1 8-4:1 3-2:1 167-100:1 11-7:1 15:1
their acrosome and cytoplasmic membrane at each stage of SCT [12, 15].
However, the published information on the results of research aimed at studying the features of the imbalance in the concentration of Ca2+, Na+, K+ in the sperm of offsprings is very ambiguous and contradictory, which requires a detailed analysis of its changes in systems such as "environment - cell (substance)". According to1, the concentration of Ca2+, Na+, K+ in semen are different. Their limit is 6-11, 39-126 and 38-106 mM respectively. The concentration of Ca2+ in sperm plasma and spermatozoons is the same. A high concentration of K+ was found in the ejaculates of individual broodstock, the limit of which is 65-106 mM. Therefore, the ratio of Na+:K+ for sperm with a low concentration of K+ is 1:1; with high - <1:1. The concentration ratios of Na+:Ca2+ and K+:Ca2+ are similar, which is 7-11 and 6-10: 1, respectively.
However, the presence of a high concentration of K+ is denied by40, 64, which prove that K+ is higher in spermatozoons, and Na+ - in sperm plasma. The concentration of these ions is characterized by an inverse negative and a positive correlation. Therefore, taking into account the results and conclusions of the above studies, (bulls secrete semen with individually constant levels of low (HKK = 17-24), medium (CKK = 36-43) and high (BKK = 60-67 mM) concentration of K+) we state that the given concentration limits are constantly the same in broodstocks of different breeds and ages. They remain the same during the mating season, but are the cause of high (group CKK) and low (groups HKK i BKK) fertility of spermatozoons in native and cryo-preserved sperm.
During the reproductive period (2-10 years), the constancy of the ions concentration in sperm plasma and spermatozoons is associated with hypo- and hyper-function of the glandular parenchyma of sperm- and plasmagenic tissues of the testes and adnexa. The change in the ratios of different-named ion pairs of sperm plasma is ± 2-13, spermatozoons - ± 1-8, and between plasma and spermatozoons - ± 6-47. The change in the ratio of eponymous pairs in the system "sperm plasma - spermatozoons" is insignificant - only ± 2:1 [11, 13]. Ejaculates of the BKK group also have lower concentration parameters of linoleic, linolenic and arachidonic FA [10], but their concentration in the HKK group is higher than in the CKK group. This suggests that it may be the cause of low resistance of the acrosome and cytoplasmic membrane of spermatozoons to the harmful effects of physical and chemical properties of the components of mono- and multicomponent AP and extreme conditions of SCT stages [14, 19]. In such circumstances, the barrier, buffer, compensatory role of ratios of the concentration of free and bound ions of the tissues of the genitals of males and females is aimed at coordinating the protective, trophic, generative, transport functions, which promotes passive and/or active movement of spermatozoons.
The variability of the levels of indicators of different and eponymous pairs in closed and open systems such as "environment - cell" is associated with the pro-
cesses that provide the intensity of sperm and plasmagenesis in the tissues of the glandular parenchyma of the generative organs. The osmotic pressure created by the difference in concentration transports spermatozoons and sperm plasma through the male genitalia. Physicochemical properties of ions also contribute to the penetration of spermatozoons into the egg-cell [2, 3, 18].
In the pathological state, the ratio of the concentration of the same-named pairs Ca2+:Ca2+, K+:K+ and Na+:Na+ are virtually unchanged, but of different names K+:Ca2+<Na+:K+<Na+:Ca2+. Determined relative to the norm, their difference is 8; 23; 25 % [11, 14].
This may mean that the products of inflammatory processes secreted by the tissues of the adnexa change the concentration of pro- (IL-6, IL-8) and anti-inflammatory (IL-10) interleukins in the sperm plasma. Therefore, very wide limits of concentration of pro- and anti-inflammatory interleukins in sperm plasma and certain parameters of high activity of transport ATPases of perforated spermatozoons' membranes can be used to diagnose the degree of inflammatory processes in the generative tissues of the reproductive system and the level of reproductive capacity of germ cells.
Indicators of enzyme activity correlate with 2 times lower than normal concentration and motility of spermatozoons and 3 times more of their abnormal forms in isolated ejaculates. It is possible that this feature is associated with the functional state of the reproductive organs, which affects the level of ion concentration in semen [13].
Because Ca2+/Mg2+-ATPase regulates the active transport of ions from cells into the environment, their low concentration in spermatozoons and high concentration in plasma are associated with passive permeability of Ca2+ across the membrane. It is known3, 60, that increasing the concentration of Ca2+ stimulates the "exit" of K+ from cells. But keep in mind that traffic stimulation has a limit. Ca2+ excess stimulates and inhibits the "input" of K+ and the "output" of Na+. Therefore, their movement can be adjusted by changing the ratio of concentrations Ca2+:Ca2+.
The movement of ions across the membrane also depends on the interaction of Ca2+ with the components of the internal structure of the membranes. Such interactions include the binding of the phosphorus ester modification of phospholamban Ca2+ with calmodulin-dependent protein kinase and the "protein-protein" binding of Ca2+/Mg2+-ATPhase to calmodulin [6, 23, 24].
An additional driving force that initiates the "exit" of ions from cells is the concentration gradient of Na+. Its parameters change the speed of Ca2+ movement and facilitate the antiportic motion of ions in the "environment - cell" system. Under these circumstances, the stoichiometry of ion transports 2 Na+ per 1 Ca2+. If the increase in concentration in the cell is carried out by passive diffusion, the decrease is due to active Mg2+-dependent transport. This leads to the conclusion that its transport is regulated by Ca2+:Ca2+ ratio [8, 24]. An important role of regulators of metabolic processes, which preserve the structure and function of
spermatozoons, belongs to enzymes20. They form electro-chemical and osmotic gradients of concentration of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations, initiate reorganization of the cytoplasmic membrane domain structure, move amino acid residues from hydrophilic to hydrophobic environment [18, 20]. Their protein and lipid fractions indicate that the active transport of ions provides corporate interaction of ATPases protomeres in membranes. Inactivation of interactions occurs under the action of phospholipases. The process causes the degradation of the lipid environment, which is determined by the nature of enzymes and membrane functions. Their corrective role depends on the magnitude of the charge and steric effects. The sensitivity of enzymes is manifested only after their significant inactivation [6, 8, 23].
The activity of Na+/K+-ATPase also depends on the Na+:K+ ratio of concentrations. At a ratio of 6: 1, the activity of this enzyme is 2 times higher than 1:1. This action can be activated by ensuring the effective binding of ions to the active center of the enzyme, ie - to reduce its affinity for the action of endogenous calcium [6, 8].
The physiological significance of these interactions is that under normal environmental conditions, the movement of structural domains is optimal. But during the changes that occur in testicular pathology, spermatozoons accumulate hydrogen ions and products of oxidative processes. Their mitochondria have an increased concentration of Ca2+ and a reduced intensity of ATP synthesis. The activity of proteolytic enzymes increases in sperm plasma [5, 6, 23, 24]. The analysis of the obtained results indicates that the detected limits of high, medium, low concentration of K+ and limits of low and high activity of ATPases open new possibilities for studying the peculiarities of changes in ion homeostasis, development of effective methods of correction and regulation of biochemical processes in open systems such as "environment - cell" under the harmful effects of extreme conditions of the SCT stages.
Analysis of the work results indicates that the dynamics of the concentration of Ca2+, K+, Na+ open system type of the "environment - cell" type and features of the "response" of spermatozoons to exogenous factors can be used to study the relevant parts of the protective mechanism influence of cryoprotectors on a condition of structure and functions of spermatozoons.
In this regard, the data obtained indicate that the harmful effect of the SCT stages on spermatozoons is associated with an imbalance of ion homeostasis in the system "environment (sperm plasma, SF components) - spermatozoons". After equilibration of undiluted sperm, the changes are insignificant, but if deconserva-tion is performed without 2.8% sodium citrate solution, the changes are significant. The effect of SCT stages on spermatozoons is differentiated with respect to ions, namely: the concentration of Ca2+ and K+ in cells becomes lower, Na+ - higher.
The conditions of the stages of dilution and equilibration of sperm in monocomponent SF are the reason that the concentration of Na+ in cells decreases. However, after deconservation of granules, its level in spermatozoons is significantly higher. It should also be
noted that the absolute and relative changes in ion ho-meostasis depend on the action of extreme conditions of the SCT stages. After equalization of undiluted sperm, they are insignificant. Their limit is 1-4%, but after deconservation of granules - 9-10 times higher. If the deconservation of the granules is carried out without 2.8% sodium citrate solution, the imbalance of the concentration is K+<Ca2+<Na+, and if in citrate, then -Ca2+<K+<Na+.
After dilution of sperm by monocomponent SFs, changes in Ca2+, K+, Na+ homeostasis are 30-40, after equilibration - 5-12, after deconservation - 50-300% of its initial level in undiluted sperm. Under the action of the factors of the first stage of SCT changes in mul-ticomponent SF are 1-3 times smaller, for the second -almost the same, for the third (conservation - deconser-vation) - 2 times less, which is respectively 10-30, 2-19, 50-150 %.
In all cases, the indicators of displaced Ca2+<K+<Na+. From 50 to 80% of their content is involved in metabolic processes. The result of ion exchange in open systems such as "environment - cell" is a significant change in the homeostasis of their different pairs in spermatozoons and environment. The limit of ion concentration ratios in cells is: K+:Ca2+ - 2-6:1, Na+:K+ - 2-24:1, Na+:Ca2+ - 6-73:1, but between medium and spermatozoons - 7-53:1, 6-38:1, 28-100:1, which is 3-4 times more. Therefore, changes in the ratios of Na+:Na+<Ca2+:Ca2+<K+:K+.
The adaptive response of spermatozoons of undiluted and diluted sperm to the harmful effect of extreme conditions of SCT stages after dilution of sperm by monocomponent SF is expressed by the symportal movement of Ca2+, K+, Na+ from cells; multicomponent SF - antiportal movement of Ca2+ in cells relative to K+ and Na+ out of them. After the action of the factors of the equilibration stage there is a slight symportal transfer of Ca2+, K+, Na+ from the cells into the environment. Whereas deconservation conditions without sodium citrate initiate the symportal transfer of ions into cells, and their deconservation in solution - antiportal Na+ in spermatozoons relative to the "yield" of K+ and Ca2+ out of them. The adaptive response of spermatozoons to the harmful effects of SCT stages, expressed by symportal and/or antiportal methods of ion transfer, is associated with changes in the initial state of cell structure and function.
The results of long-term cryobiological studies, which were aimed at developing ways to minimize the harmful effects of SCT, show that their introduction into production allows to store in deconserved sperm the maximum possible number of full-fledged spermatozoons that can come into effective contact with the egg-cell [9, 14, 17, 22].
The current knowledge level about the peculiarities of metabolic processes during the life cycle of spermatozoons does not provide a positive solution to this problem. A possible way to solve it may be a natural phenomenon, or a state of "physiological conservation" of vital processes of spermatozoons in the female genitals. Factors inhibiting the functions are the secretions of the uterine-vaginal glands, which are called "sperm-preserving" [4]. These secretions have 4 times
higher concentration of K+ than sperm plasma [8, 25]. However, in this case it should be noted that researchers will have to use substances, the physical and chemical nature of the properties of which are still not studied. The optimal composition of the SF should preserve the full structure and functions of spermatozoons, and the methods and means of its composition - to reproduce the primary osmotic balance, or homeostasis of ions of inorganic and molecules of organic compounds. It is achieved by combinations of aqueous solutions of organic (glutamates, citrates, acetates) and inorganic (salts of potassium, sodium, magnesium, carbohydrates, impermeable to the cell cryoprotectants) substances.
Acid-base balance is created by inorganic and organic buffer systems, ionic strength - by alkaline metals, energy supply - by carbohydrates and monosodium glutamate. The change in the equilibrium state of ions is a significant cause of irreversibility of metabolic processes, which reduces the viability of spermatozoons. Their undesirable consequence is the induction of lipid peroxidation, which inhibits the active and stimulates the passive transport of Ca2+, changes the value of the transmembrane concentration gradient. The result of the above series of processes is low membrane permeability and accumulated high electric potential (-75 mV), which causes passive transmembrane movement and increased concentration of ions in spermatozoons. This situation suggests that the change in the structure of annular lipids may also be the cause of deep disintegration of the membrane matrix of spermatozoons. Significant quantitative losses and modification of the ex-ostructure of the acrosome and cytoplasmic membrane are inactivated by enzymes that initiate denaturation and the formation of an inactive form of protein [7, 16, 20, 21, 23].
This means that the concentration limits of lino-leic, linolenic and arachidonic acids and their ability to switch from solid to liquid temperatures at low (-5-49 ° C) can be used to enhance the natural stability of sperm membranes. In addition, reactive oxygen species and free radicals of unsaturated FA have the ability to regulate such physiological processes as: transduction of formed signals, acrosomal reaction, capacitation of spermatozoons and their penetration into the egg-cell.
Carbohydrates, due to the formation of a stable hydrogen bond between the hydroxyl groups and polar groups of phospholipids of the acrosome and cytoplas-mic membrane, enhance its natural resistance to the harmful effects of SCT stages [5, 17]. Monocarbohy-drates (glucose), due to their high penetration abilities have less protective effect than carbohydrates (lactose). This statement is consistent with the results of our research [17, 21]. Under the action of low and ultra-low temperatures, their protective effect is due to low eutec-tic and high viscosity. The degree of protective effect on the cell is as follows: glucose> lactose> sucrose> maltose> trehalose> raffinose.
Until recently, this role was assigned to the lipids of chicken egg yolk. However, it has already been established that purified phosphatidylcholine also has a positive effect. The results of our research coincide with this point of view. Sperm plasma and
spermatozoons of bull ejaculates contain both high and low concentrations of saturated and unsaturated, ester-ified and non-esterified FA, which has both protective and harmful effects on the exostructure of spermatozoons under the harmful effects of the SCT stages.
The accumulated experience of experimental research indicates that in systems such as "environment -cell" to correct the parameters of osmotic pressure, buffer capacity, homeostasis of ions of salts of macro-and micronutrients, it is better to use solutions of organic salts. Their physicochemical properties play the role of a buffer component [25]. They normalize pH, change sperm motility, are donors of K+, Mg2+ i Zn2+. This position is confirmed by changes in the concentration of Ca2+, K+, Na+ in spermatozoons of decon-servated sperm. Cryoprotectants such as glycerin and DMSO must be included in the SF. Despite the harmful effects on the cell, no effective replacement has yet been found for them. Recently, amides and diols have been used to prevent the formation of water crystals in sperm. The diffusion of disubstituted molecules provides their 20% permeability to the cell [4, 17].
Summarizing the results of research and discussed aspects of the problem of protection of the structure and functions of spermatozoons under the harmful effects of SCT, it should be assured that the established ability of offspring to produce sperm with constant, individually low, medium and high levels of K+ concentration and observed phenomenon of high and low spermatozoons' membranes stability in extreme freezing conditions (-196 ° C) can be useful for finding tools that minimize their harmful effects.
The results and analysis of the presented work expand the understanding of the possibilities of objective monitoring and forecasting the level of sperm fertility by certain parameters of the ratio of the concentration of identical and different pairs of ions in samples of native and cryopreserved sperm
CONCLUSIONS
The features of the response of systems such as "environment - sperm (substance)" to the harmful effects of endogenous factors indicate that some details of its complex mechanism can be used to create a new generation of protective environments. The fact of individually set constant levels of K+, concentration deserves special attention, the indicators of which are related to sperm motility and fertilizing ability and anti-portic movement of alkali metal ions while maintaining and restoring their viability under extreme, harmful effects of SCT stages.
The barrier, buffer and compensatory role of different levels of concentration and ratios of Ca2+, K+, Na+ is aimed at coordinating the protective, trophic, generative and transport functions of the reproductive organs of males (bulls) and females (cows), what quite possible causes active movement of spermatozoons in channels of their reproductive system. The possible presence of such a connection is indicated by the revealed significant difference between the concentration limits of Na+ (64-97mM) of sperm and uterine-vaginal mucus (500-900 mM).
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