Loss Of Sperm Motility Research Articles

Time-dependent effect of ground water fluoride on motility, abnormality and antioxidant status of spermatozoa: An in vitro study.

The present study was undertaken to investigate the toxic effect of ground water fluoride (F) on motility, abnormality, and antioxidant status of spermatozoa. Treatment of ground water F with epididymal sperm suspension caused complete loss of sperm motility and decrease in the activities of superoxide dismutase (SOD) and catalase (CAT) and increase in the concentration of malondialdehyde (MDA) and abnormality of spermatozoa at 15 and 30 min time intervals. Further, incubation of spermatozoa with ground water F for 5, 10, and 15 min time intervals significantly reduced the sperm motility and activities of SOD and CAT and increased the concentration of MDA and abnormality of spermatozoa. The study revealed that F-induced effect on sperm motility and antioxidant status is time dependent. Increase in oxidative stress and concomitant decrease in motility of spermatozoa in ground water F clearly indicates that F-induced oxidative stress affected the sperm motility. The present study for the first time demonstrated the toxic effect of ground water F on spermatozoa at shorter duration of exposure, which affects the capability of spermatozoa in fertilization.

Interaction of different Chlamydiae species with bovine spermatozoa

BackgroundInteraction of spermatozoa and Chlamydiae spp. might contribute to reduced fertility in cattle. To proof this hypothesis, bovine semen was incubated with viable or heat inactivated Chlamydia (C.) abortus or psittaci (Multiplicity of infection = 1) and sperm motility was monitored with a computer-assisted sperm analyzer over 24 h. Additionally, the interaction with the spermatozoa was further investigated by means of light and transmission electron microscopy (TEM).ResultsOnly viable Chlamydiae of both species decreased sperm motility and this only after about 9 h. Taking binding rates into account, the loss of sperm motility after about 9 h could likely be a consequence of Chlamydiae attachment to the spermatozoa. About two thirds of the Chlamydiae elementary bodies were bound to the front third of the sperm, the acrosomal region. No inclusions of Chlamydiae in spermatozoa were observed in TEM after 2 h co-incubation.ConclusionsAs initial motility was not affected following co-incubation of viable Chlamydiae and bovine sperm, it seems likely that sperm could serve as a carrier/vehicle for Chlamydiae facilitating cervical passage of Chlamydiae spp. in cattle. Additionally, our results suggest that spermatozoa carrying Chlamydiae may have no initial disadvantage in reaching the oviduct, but are immotile at the time of ovulation what might have an impact on fertilization capacities of the individual sperm. Consequently, high concentrations of the investigated Chlamydiae in the seminal plasma or female genital tract might play a role in reduced fertility in cattle.

Influence of cryopreservation on the CATSPER2 and TEKT2 expression levels and protein levels in human spermatozoa.

This study designed to assess the expression level of CATSPER2 and TEKT2 and to evaluate the levels of CatSper2 and Tektin2 proteins in human spermatozoa before and after cryopreservation. One hundred and twenty semen samples were included in this study. All the samples were subjected to qPCR and Western blot analysis. The results showed a significant reduction in the expression levels of CATSPER2 and TEKT2 in the cryopreserved compared to the fresh samples (P = 0.0039 and P = 0.0166, respectively), and the results showed down-regulation in the expression level of CATSPER2 and TEKT2 genes between the study groups. Moreover, the protein levels of the CatSper2 and Tektin2 were lower in cryopreserved samples compared to fresh samples (P = 0.0001). In conclusion, the reduction in the proteins level and expression level of the CATSPER2 and TEKT2 in cryopreserved samples could be used as an indicator of sperm motility loss.

Buck Semen Does Not Easily Succumb to Oxidative Stress

Semen processing and manipulation generally result in loss of sperm motility and sperm velocity due in part to oxidative stress. In this study we investigated the vulnerability of South African indigenous unimproved buck semen to oxidative stress induced by an oxidative stress inducing agent, namely, hydrogen peroxide (H2O2). Semen ejaculates were collected from four superior South African indigenous unimproved bucks in a total of ten collections and then each duplicate was treated with different concentrations of H2O2 in presence or absence of Dithiothreitol (DTT). Sperm motility and velocities were determined using the computer aided sperm class analyser (CASA). SYBR-14 and propidium iodide (PI) Live/Dead assay kit was used to determine cell viability and Yo-Pro-1 plus PI Apoptosis kit was used to determine apoptosis. Statistical analysis was performed on the data using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL). South African indigenous unimproved buck raw semen motility was between 97% with 98% viability and 0% apoptotic cells. Comparisons of the untreated controls at 0 and 3 hrs incubations revealed that after 3 hrs there was overall a decrease in the number viable cells with the majority of remaining cells exhibiting circular movements accompanied by high progressive (PM) and rapid (RAP) motilities. In treated South African indigenous unimproved buck semen, H2O2 marginally increased total motility (TM) with few apoptotic sperm cells while retaining high viability. Also, H2O2 increased straight line distances travelled of more than 4 fold as compared to untreated controls with no circularly moving cells. Moreover, inclusion of DTT, an antioxidant, had minimal effects on TM, RAP, curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN) and wobble (WOB) but positively affected PM, average path velocities (VAP), apoptosis and viability. Our Pearson’s correlation data revealed that only straightness (STR) was highly positively affected by H2O2. Overall, the South African indigenous unimproved buck semen resisted deterioration in TM, RAP, VCL, VAP, VSL, LIN, WOB, viability and apoptosis under oxidative stress conditions. These data suggest that the South African indigenous unimproved buck semen does not easily succumb to oxidative stress.

Dose and time-dependent effects of sodium fluoride on sperm motility: An in vitro study.

The present study was undertaken to investigate the toxic effects of sodium fluoride (NaF) on sperm motility and abnormality. Treatment of epididymal sperm suspensions with different doses of NaF (0.1, 1, 10, and 100 mg/ml) significantly reduced sperm motility at 15- and 30-min time intervals. Further, incubation at the same concentrations of NaF for 5-, 10-, and 15-min time intervals decreased sperm motility in a dose- and time-dependent manner. The present investigation revealed for the first time the adverse effect of lower doses of NaF (0.1 mg/ml) on sperm motility. Further, higher doses of NaF (10 and 100 mg/ml) caused a complete loss of sperm motility with a concomitant increase in sperm abnormality, which reflected the toxic effect of NaF on spermatozoa and its capability in fertilization.

Effects of different extenders and additives on liquid storage of Awassi ram semen

The aim of this study was to evaluate the effects of different extenders and additives on ram semen during liquid storage and to demonstrate the role of oxidative stress parameters on this process. In the present study, ejaculates taken by artificial vagina twice a week from 4 rams during the breeding season were used. They were mixed and used if motility and viability were above 70% and there was a 95% intact acrosome. The semen specimens were diluted by Tris-citrate-glucose (TRIS), Tris-TES (TEST), HEPES-buffered Tyrode lactate (TL-HEPES), and Dulbecco?s phosphate-buffered saline (PBS) extenders supplemented with different additives [centrifuged egg yolk, Equex-STM, bovine serum albumin (BSA), and ethylenediaminetetraacetic acid (EDTA)]. The specimens were stored at 4 °C for up to 96 h and evaluated for motility, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), and oxidative stress parameters. At the end of the 96-h storage, the highest sperm motility was 64.2 ± 3.7% (P < 0.05) and significant loss of sperm motility and membrane integrity were not detected in extender TEST-3, but the MMP rate significantly declined. Acrosome integrity was not affected by storage time or extender types. BSA and EDTA decreased lipid peroxidation (LPO) and total oxidant status (TOS), but did not positively affect motility or membrane integrity. As a result, TRIS, TEST, and TL-HEPES-3 were observed to provide better protection for ram semen during liquid storage at 4 °C than other extenders. The role of oxidative stress and MMP are considerable in liquid storage.

Sperm characteristics of wild and captive lebranche mullet Mugil liza (Valenciennes, 1836), subjected to sperm activation in different pH and salinity conditions

In this article we describe basic aspects of the sperm biology of lebranche mullet (Mugil liza) in the wild and in captivity, in particular assessing the effects of salinity (0, 10, 20, 30, 35, 40, 50 and 60 g L−1) and pH (6, 7, 8, 9 and 10) on sperm motility. Our results indicate that the highest percentage of motility was recorded with salinity 34.6 g L−1 (95 ± 10%) and the longest motility time was obtained with a salinity of 34.8 g L−1 (189 ± 15 s). Variations in the salinity between 30 and 35 g L−1 did not produce any significant alterations in sperm motility; however salinities of 20 and 50 g L−1 produced a significant loss of sperm motility. The highest percentage of motility was obtained at pH 8.5 (93 ± 12%), and the longest motility period at pH 8.7 (218 ± 13 s), while pH lower than or equal to 7 and equal to 10 both produced a significant loss in sperm motility. A positive correlation was found between pH/salinity and the motility percentage (R2 = 0.94 and R2 = 0.97) and motility time (R2 = 0.86 and R2 = 0.98). In seminal and morphometric parameters, statistically significant differences were observed in semen volume, sperm density, plasma membrane integrity and sperm morphometry between the groups studied, showing that the characteristics of the fish have a direct influence on sperm quality. The information generated in this research will be useful for developing biotechnology tools for the effective management of Mugil liza gametes.

Low concentrations of glyphosate-based herbicide cause complete loss of sperm motility of yellowtail tetra fish Astyanax lacustris.

Environmental relevant concentrations of glyphosate-based herbicide as 50 µg l-1 , 300 µg l-1 and 1800 µg l-1 can affect sperm quality of yellowtail tetra fish Astyanax lacustris. Viability of sperm cells was impaired at 300 µg l-1 , a concentration that is within legal limits in U.S.A. waterbodies, while motility was impaired at 50 µg l-1 , which is the more stringent limit set in Brazilian law. Therefore, environment protection agencies must review regulations of glyphosate-based herbicides on water bodies.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

Changes in Gene Expression and Metabolism in the Testes of the Rat following Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating changes to almost all aspects of a patient's life. In addition to a permanent loss of sensory and motor function, males also will frequently exhibit a profound loss of fertility through poorly understood mechanisms. We demonstrate that SCI causes measureable pathology in the testis both acutely (24 h) and chronically up to 1.5 years post-injury, leading to loss in sperm motility and viability. SCI has been shown in humans and rats to induce leukocytospermia, with the presence of inflammatory cytokines, anti-sperm antibodies, and reactive oxygen species found within the ejaculate. Using messenger RNA and metabolomic assessments, we describe molecular and cellular changes that occur within the testis of adult rats over an acute to chronic time period. From 24 h, 72 h, 28 days, and 90 days post-SCI, the testis reveal a distinct time course of pathological events. The testis show an acute drop in normal sexual organ processes, including testosterone production, and establishment of a pro-inflammatory environment. This is followed by a subacute initiation of an innate immune response and loss of cell cycle regulation, possibly due to apoptosis within the seminiferous tubules. At 1.5 years post-SCI, there is a chronic low level immune response as evidenced by an elevation in T cells. These data suggest that SCI elicits a wide range of pathological processes within the testes, the actions of which are not restricted to the acute phase of injury but rather extend chronically, potentially through the lifetime of the subject. The multiplicity of these pathological events suggest a single therapeutic intervention is unlikely to be successful.

The control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail.

Male infertility due to abnormal spermatozoa has been reported in both animals and humans, but its pathogenic causes, including genetic abnormalities, remain largely unknown. On the other hand, contraceptive options for men are limited, and a specific, reversible and safe method of male contraception has been a long-standing quest in medicine. Some progress has recently been made in exploring the effects of spermatid-specifical genetic factors in controlling male fertility. A comprehensive search of PubMed for articles and reviews published in English before July 2016 was carried out using the search terms ‘spermiogenesis failure', ‘globozoospermia', ‘spermatid-specific', ‘acrosome', ‘infertile', ‘manchette', ‘sperm connecting piece', ‘sperm annulus', ‘sperm ADAMs', ‘flagellar abnormalities', ‘sperm motility loss', ‘sperm ion exchanger' and ‘contraceptive targets'. Importantly, we have opted to focus on articles regarding spermatid-specific factors. Genetic studies to define the structure and physiology of sperm have shown that spermatozoa appear to be one of the most promising contraceptive targets. Here we summarize how these spermatid-specific factors regulate spermiogenesis and categorize them according to their localization and function from spermatid head to tail (e.g., acrosome, manchette, head-tail conjunction, annulus, principal piece of tail). In addition, we emphatically introduce small-molecule contraceptives, such as BRDT and PPP3CC/PPP3R2, which are currently being developed to target spermatogenic-specific proteins. We suggest that blocking the differentiation of haploid germ cells, which rarely affects early spermatogenic cell types and the testicular microenvironment, is a better choice than spermatogenic-specific proteins. The studies described here provide valuable information regarding the genetic and molecular defects causing male mouse infertility to improve our understanding of the importance of spermatid-specific factors in controlling fertility. Although a male contraceptive ‘pill' is still many years away, research into the production of new small-molecule contraceptives targeting spermatid-specific proteins is the right avenue.

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm.

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.

Resveratrol prevents oxidative damage and loss of sperm motility induced by long-term treatment with valproic acid in Wistar rats

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400mgkg−1) by gavage for 28days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10mgkg−1) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment.

Utility of different sugar extenders for cryopreservation and post-thaw storage of sperm from Salmonidae species

The effect of replacement of glucose in glucose-methanol extender (GM) with either sucrose and trehalose on the sperm motility characteristics of cryopreserved semen and the post-thaw storage of salmon, brown trout, brook trout, sex-reversed female rainbow trout and whitefish was investigated. Semen samples were diluted 1:5 or 1:9 (for sex-reversed females) in 9% methanol containing 0.18M glucose, sucrose or trehalose. After thawing (40°C, 5s), sperm was stored for 0, 30, 60, 120 and 240min at 4°C and assessed for sperm motility. The type of sugar had no effect on the percentage of sperm motility of brown trout (65–73%), brook trout (51–58%) or sex-reversed female rainbow trout (38–40%). Glucose and trehalose secured the highest post-thaw sperm motility after thawing (0min) in salmon (64±7%) and whitefish (52±8%), respectively. The longest period (determined as the last time point when the percentage of motility did not significantly change) was recorded for cryopreserved semen of brook trout (60–120min). The shortest was recorded for whitefish semen (30min). Our results suggest that glucose, sucrose or trehalose can be used interchangeably for the cryopreservation of brown trout, brook trout and sex-reversed semen. However, the best results in terms of post-thaw sperm motility were achieved for salmon with glucose and sucrose and for whitefish with trehalose and sucrose. Cryopreserved sperm can be stored for prolonged periods of at least 30min without a loss in sperm motility, which can facilitate more organised hatchery work. Statement of relevanceThe results of this study confirmed the high level of utility of cryopreservation employing a 0.18M glucose and 9% methanol extender for freezing, among Salmonidae species. We have demonstrated for the first time the utility of this method for freezing Atlantic salmon semen. Furthermore it was shown that disaccharides are more efficient than glucose for preserving of whitefish post-thaw motility.

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

Oxidative Status of Boar Semen during Storage

The intensification of the use of artificial insemination in the swine industry has increased the interest to develop proper conditions to store semen better for longer periods of time without affecting its fertility. Trolox is a vitamin E analog with antioxidant property to prevent oxidative stress. Therefore, the objective of this study was to evaluate the Trolox antioxidant capacity on boar semen during storage period (86 h) at 15°C. For that, semen samples from 5 different boars were collected and diluted with a Long-Term (LT) extender with and without the addition of Trolox in a concentration of 50 µM. The sperm motility, oxidative stress and antioxidant protection were measured in those samples during an 84 h storage time. Our results indicated that Trolox was able to minimize the loss of sperm motility after 24 h of storage. The storage time was positively correlated with TBARS production. However in the presence of Trolox, after 12 h, TBARS content were significantly lower than those of sperm cells stored with LT diluent only. The concentrations of Non-Protein SH groups (NPSH), after 36 h of storage, were lower in sperm cells stored only with LT diluent when compared to the group supplemented with Trolox. Catalase and Superoxide Dismutase activities were decreased after 48 h storage at 15°C in both groups (with or without Trolox). Spontaneous spermatozoa ROS formation was high in viable swine sperm during the LT storage period in samples without Trolox. In conclusion, LT diluent with the addition of antioxidant as Trolox can reduce the oxidative stress in sperm cells, which could be used to protect spermatozoa against oxidative damage, preventing motility losses in swine semen.

In situ polyethylene sebacate particulate carriers as an alternative to Freund’s adjuvant for delivery of a contraceptive peptide vaccine—A feasibility study

The present study evaluates the feasibility of particulate carriers of a biodegradable polymer polyethylene sebacate (PES) as an alternative to Freund’s adjuvant in the design of a peptide vaccine formulation. The vaccine formulation comprised of PES and the antigen KLH conjugated 80kDa HSA peptide-1 dissolved in N-methyl-2-pyrrolidone (NMP)/NMP-water as solvent. The antigen revealed good stability and the formulations were readily syringeable. Intradermal injection of the formulations resulted in the formation of PES particulates in situ at the site of injection. The NMP formulations revealed larger particulates which elicited no immunogenic response when injected in rabbits. On the other hand the NMP-water formulation revealed formation of microparticles which were significantly smaller in size, in combination with a small fraction of nanoparticles. It elicited an antibody titer up to 1:3200 in rabbits following intradermal injection. Western blot confirmed generation of antibodies specific to the peptide. Contraceptive efficacy was confirmed by loss of sperm motility and head-to-head agglutination of sperms in the treatment group. Unlike the severe reactions observed with administration of Freund’s adjuvant, only mild hypersensitivity reaction was observed with the PES formulations. The mild reaction coupled with the contraceptive efficacy observed suggested PES particulates as a viable alternative to Freund’s adjuvant.

The immobilization of human spermatozoa by STAT3 inhibitory compound V results from an excessive intracellular amount of reactive oxygen species.

We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca(2+) concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA, acrosomal integrity by FITC conjugated Pisum sativum agglutinin (PSA-FITC) labeling, intracellular pH, and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N-acetyl-L-cysteine (NAC), prevented Stattic V-induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.

Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca(2+)]c in sperm to down-regulate them, and thus prevent elevated levels of NO, known to induce asthenozoospermia via oxidative stress. Our studies point to the potential underlying cause of infertility in PMCA4's absence, and suggest that inactivating mutations of PMCA4 could lead to asthenozoospermia and human infertility. Screening for these mutations may serve both diagnostic and therapeutic purposes.

Sperm motility under exposure of hydrogen dioxide

The paper contains research data on the effect of low concentrations of hydrogen dioxide on human sperm motility and specific enzyme activity of sperms of glyceraldehyde-3-phosphate dehydrogenase. It is shown that incubation of sperms with hydrogen dioxide in a low concentration leads to a change and motility in sperm and activity of sperm enzyme. Intensity of observed effect depended on the concentration of hydrogen dioxide: active mobility increased by 17–19 % and the total mobility – 11 %. Motility changes in sperms were accompanied by increased activity of glyceraldehyde-3-phosphate dehydrogenase by 24 %, in normozoospermia response was higher than in pathozoospermia and also depended on the concentration of hydrogen dioxide. The use of sperm analyzer enabled revealing changes in the diapason of different speeds of the active fraction of sperm, which have been observed in the first 15 min of incubation with hydrogen dioxide. A possible mechanism of action of the detected effect is discussed. Reactive oxygen species easily oxidize enzyme for glyceraldehyde-3-phosphate dehydrogenase of sperms, which leads to a loss of sperm motility, for example, in varicocele. Initially low enzyme activity in varicocele (pathozoospermia) may be associated with the suppression of sperm antioxidant defense. Addition of low concentrations of hydrogen dioxide into sperm samples leads to an increase in the concentration of reduced glutathione in a cell. Increase of sperm motility in this case can serve as an indicator of normal operation of the cellular antioxidant defense system. Obtained experimental results provide a background for their introduction into clinical practice in the program of assisted reproductive technologies.