Inhibition Of Sperm Motility Research Articles

Localization of MIF-II on mammalian spermatozoa: A study revealing its structure, function and motility inhibitory pathway

Motility of spermatozoa is a crucial factor for determining semen quality. Here we report motility inhibitory factor (MIF-II) from goat epididymal plasma, revealing its structure, function, localization and motility inhibitory pathway. Structural characterization with MALDI revealed novelty of this protein while circular dichroism data confirmed its alpha helical nature. Higher dilutions of MIF-II antibody increased cauda sperm motility and induced immature/immotile caput sperm motility as tested microscopically. Higher number of sperm cells and lower dilutions of antibody induced agglutination in cauda sperm showing surface localization. Indirect immuno-fluorescence showed MIF-II localization throughout the caput sperm surface which relocated more towards acrosomal region with maturation. ELISA assay revealed gradual increase and decrease in concentration of MIF-II in epididymal plasma and plasma membrane respectively from caput to cauda. Signaling cascade that leads to sperm motility inhibition elevates nitric oxide levels through cAMP dependent pathway. MIF-II treatment doesn't alter sperm surface morphology. Expression pattern of MIF-II during epididymal maturation goes hand-in-hand with gaining motility potential as well as dormancy of spermatozoa before ejaculation. Both MIF-II and its antibody inhibit fertilization in-vitro thus expected to open new gateway for future male infertility and contraceptive development research.

Inhibition of sperm motility in male macaques with EP055, a potential non-hormonal male contraceptive.

Men have two practical choices for contraception; the condom which has a high typical use failure rate or vasectomy. New male hormonal and non-hormonal contraceptives are under development that target either the production of sperm (spermatogenesis) or the delivery of sperm. One particular target is the sperm protein EPPIN, which is present on the surface of human spermatozoa. EP055 is a small organic compound that targets EPPIN on the surface of sperm and inhibits motility. EP055 was tested in cynomolgus (Macaca fascicularis) males to determine its plasma half-life after intravenous (i.v.) infusion of a single dose and for binding to its target tissues. Our initial study demonstrated a plasma half-life for EP055 of 10.6 minutes. In a second study examination of macaque testis, epididymis, and plasma after i.v. infusion of a single dose of compound EP055 (63.25 mg/kg) demonstrated that EP055 was detected in testis and epididymis two hours and six hours post-infusion. We initiated a trial in rhesus (Macaca mulatta) males to assess the availability of EP055 in semen and its effect on sperm motility as a measure of the drug's efficacy. Four macaques were infused with a low dose (75–80 mg/kg) followed by a recovery period and a subsequent high dose (125–130 mg/kg) of EP055. After high dose administration, sperm motility fell to approximately 20% of pretreatment levels within 6 hours post-infusion; no normal motility was observed at 30 hours post-infusion. Recovery of sperm motility was obvious by 78 hours post-infusion; with full recovery in all animals by 18 days post-infusion. EP055 has the potential to be a male contraceptive that would provide a reversible, short-lived pharmacological alternative.

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster, Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC50), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa

Metformin is clinically used to treat diabetes. Given its role-impacting metabolism, metformin has been also added to semen cryopreservation media showing specie-dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24hr) and refrigerated (17°C for 10days) boar spermatozoa. Metformin (2hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin-including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ-population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.

In vitro study evaluating the instantaneous treatment of ozonised olive oil on human sperm

Objective: The aim of our study was to evaluate the effects of ozonised olive oil (OOO) on human sperm in vitro.Methods: Human sperm was incubated with OOO for 20 s in vitro. The lowest concentration that completely immobilised all the sperm in 20 s without subsequent recovery of motility was recorded as the minimum effective concentration (MEC). The effects of OOO at MEC on human sperm viability, mitochondrial and acrosomal status, DNA integrity and transmission electron microscopy were observed.Results: Our findings demonstrate that OOO dose-dependently inhibits sperm motility. The MEC of OOO for 100% sperm immobilisation in 20 s was 6 μg/ml. Further experiments showed that sperm ultrastructure, function and DNA integrity were significantly affected after treatment with 6 μg/ml OOO in vitro.Conclusions: OOO has spermicidal potential and may be explored as a promising vaginal contraceptive agent.

A CCR5 antagonist-based HIV entry inhibitor exhibited potent spermicidal activity: Potential application for contraception and prevention of HIV sexual transmission

B07 is a small-molecule CCR5 antagonist-based HIV-1 entry inhibitor that is being developed as an anti-HIV microbicide for preventing sexual transmission of HIV. Here we evaluated its spermicidal and contraceptive potential, including sperm motility, plasma membrane integrity, and contraceptive efficacy tested in rabbits. We found that B07 inhibited sperm motility and movement patterns in a concentration- and time-dependent manner. Within 30 min, B07 induced sperm immobilization with the minimum 100% effective concentration and median effective concentration of 640.0 and 64.4 μg/mL, respectively. The hypo-osmotic swelling test showed that plasma membranes of B07-treated sperms exhibited slight disruption, as verified by electron micrographs. In both B07 gel and N-9 gel groups, not a single implantation site or embryo was observed based on the contraceptive efficacy test in rabbits, indicating that B07 could effectively block the potential of sperm to reach and/or fertilize oocytes. The safety profile of B07 in vivo was evaluated by use of an optimized rabbit vaginal irritation test. While the pathological scores of the N-9 gel group was 14.67 ± 1.21, those of the blank control and B07 gel groups were 2.17 ± 0.76 and 4.00 ± 0.89, respectively, which were within the clinically acceptable range (<8). The proportion of inflammatory cells and CD45+ cells in the cervicovaginal lavages of the B07 gel group showed no significant change compared to those of the control group. Therefore, our results confirmed that B07 exhibited significant spermicidal and contraceptive effects, suggesting its potential for development as a microbicidal spermicide for contraception and prevention of HIV sexual transmission.

A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO-cGMP pathway.

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 μM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility.

Determining the relationship between bull sperm kinematic subpopulations and fluorescence groups using an integrated sperm quality analysis technique.

The aim of the present study was to determine whether there is an association between the kinematic sperm subpopulations and fluorescent groups in bulls using a new fluorescent staining method that allows classification of spermatozoa into groups depending on their acrosomal and membrane integrity, as well as functional status, without inhibiting sperm motility. Cryopreserved semen samples from 10 Holstein bulls were used in the study. A multiparametric analysis of results obtained by the ISAS 3Fun kit (Proiser) was performed. The different fluorescent groups were detected and their motility characteristics evaluated using ISAS software. Clustering procedures using the kinematic data resulted in the classification of spermatozoa into three kinematic sperm subpopulations. The distribution of kinematic sperm subpopulations was different between the fluorescent sperm groups (P<0.001), although the correlation between them was low (r=0.113; P<0.01).

PH controls spermatozoa motility in the Pacific oyster (Crassostrea gigas).

ABSTRACTInvestigating the roles of chemical factors stimulating and inhibiting sperm motility is required to understand the mechanisms of spermatozoa movement. In this study, we described the composition of the seminal fluid (osmotic pressure, pH, and ions) and investigated the roles of these factors and salinity in initiating spermatozoa movement in the Pacific oyster, Crassostrea gigas. The acidic pH of the gonad (5.82±0.22) maintained sperm in the quiescent stage and initiation of flagellar movement was triggered by a sudden increase of spermatozoa external pH (pHe) when released in seawater (SW). At pH 6.4, percentage of motile spermatozoa was three times higher when they were activated in SW containing 30 mM NH4Cl, which alkalinizes internal pH (pHi) of spermatozoa, compared to NH4Cl-free SW, revealing the role of pHi in triggering sperm movement. Percentage of motile spermatozoa activated in Na+-free artificial seawater (ASW) was highly reduced compared to ASW, suggesting that change of pHi triggering sperm motility was mediated by a Na+/H+ exchanger. Motility and swimming speed were highest in salinities between 33.8 and 42.7‰ (within a range of 0 to 50 ‰), and pH values above 7.5 (within a range of 4.5 to 9.5).

Proteinaceous sperm motility inhibitory factor from the female Indian garden lizard Calotes versicolor.

Female sperm storage is an intriguing adaptation exhibited by a wide array of both vertebrates and invertebrates. The mechanisms underlying female sperm storage have remained elusive. Using the Indian garden lizard Calotes versicolor as a model organism, we investigated the role of low and high molecular weight factors in this phenomenon. Previously, we demonstrated three distinct phases of the reproductive cycle in this animal with live, motile spermatozoa recovered from the uterovaginal region during the reproductive phase. In the present study, we analysed the uterovaginal contents using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified an abundant protein band corresponding to ~55 kDa regardless of the phase of the reproductive cycle. Analysis of the purified protein by liquid chromatography-tandem mass spectrometry suggested a unique protein without any homology to the National Center for Biotechnology Information database. Exogenous addition of this protein to washed spermatozoa derived from the epididymis reversibly inhibited sperm motility in a concentration- and time-dependent manner, suggesting it plays a key role in sperm storage. These studies are likely to offer new avenues to unravel the secrets of female sperm storage seen across the animal taxa and may have novel applications not only in reproductive biology, but also in general cell storage and preserving endangered animal species.

AB025. Semen liquefaction molecular pathways

BackgroundHuman semen is the jelly-like substance mainly containing Semenogelin 1 (Sg1) and fibronectin (Fn) with the characteristics of coagulation and liquefaction in a short time. In our previous study, we have identified that Eppin could interact with Sg. Eppin75–133 C-terminal fragment bind the Sg164–283 fragment containing the only cysteine in human Sg1 (Cys-239). Besides that, during semen liquefaction, physiologically prostate specific antigen (PSA) hydrolyzes some region of Sg1 which inhibits sperm motility. Therefore, complex interaction among Eppin, Sg and PSA plays a major role in regulating semen liquefaction process. The aim of this study is to investigate the molecular pathways during semen liquefaction.MethodsMolecular cloning was used to recombination in vitro 6-His-Eppin protein with N- and C-terminal fragments. Protein in seminal vesicle fluid was transferred to Immobilon-P Polyvinylidene Difluoride membrane by western blot analysis, followed by incubation with 6-His-Eppin protein, Eppin283–423 C-terminal fragment and Eppin73–288 N-terminal fragment at 4 °C overnight respectively in order to find the protein which can be bind to 6-HisEppin protein. 2-D electrophoresis was used to identification of Eppin binding partners. After that, anti-His was used to visualize using enhanced chemiluminescence and mass spectrometry to identify the sequence of protein.ResultsWe found that the protein specifically binding to Eppin through Far-western immunoblot analysis demonstrated only the N-terminal of recombinant epididymal protease inhibitor (N-rEppin) and rEppin can binds to reduce seminal plasma protein, while MS identified that Fn can specifically bind to Eppin. Our study was the first evidence that some protein existed in seminal vesicle fluid does bind to Eppin, regulating the semen coagulation and liquefaction.ConclusionsTwo molecular pathways occurred in semen liquefaction. Eppin C- and N-terminal fragment interacted with Sg and Fn. Eppin N-terminal has a binding site of fibronectin, which is an important protein for semen coagulation. EpiPen regulated the process of semen coagulation and liquefaction through its N- and C-terminal bound to fibronectin and Sg respectively, influencing sperm capacitation. What’s more, the C- and N-terminal fragment of Eppin self-formed double ring type molecular structure respectively and closely bound to the core structure named β—sheet by four-disulfide, which make the combination of Wap and Kunitze type inhibitor external surface just in the opposite two terminals of the transection of molecular structure. This structure contributes to interact with other proteins in increasing its roles. During human ejaculation, spermatozoa pass through the ampulla of the vas deferens and then move into the proximal extension of the seminal vesicle and finally enter into the ejaculatory duct. At this juncture spermatozoa are first mixed with copious secretion from the seminal vesicles. Thereafter the spermatozoa and seminal fluid is mixed with prostatic secretions when they enter into the prostatic urethra. It can be imagined that after spermatozoa enter into the ejaculatory ducts their surface Eppin would be saturated by binding with Sg and Fn. This process inhibits human sperm capacitation, making the initial ejaculated spermatozoa be in an immotile state. Purified plasma Fn, added at various concentrations to a preparation of live spermatozoa, was found to inhibit sperm motility in a dose dependent manner. During semen liquefaction, physiologically PSA hydrolyzes Sg and Fn to increase sperm motility. Therefore, seminal liquefaction is a process increasing the capacitation of sperm progressive motility and fertilization. Fibronectin could affect the process of sperm coagulation and liquefaction through specificity combined with Eppin, involving in sperm capacitation and fertilization. Our findings revealed novel molecular pathways of semen liquefaction.

Decline of a giant salamander assessed with historical records, environmental DNA and multi‐scale habitat data

Abstract Freshwater species are declining rapidly but more complete data are needed for determining the extent and cause(s) of population declines and extirpations. Integrating newer survey techniques, freely available data, and traditional field work may allow for more effective assessment of population decline. We used detailed historical species records and environmental DNA (eDNA) survey methods to identify changes in population distribution of a long‐lived, imperiled stream salamander, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis: Cryptobranchidae). We used logistic regression with Bayesian inference to test whether selected environmental variables may be good predictors of hellbender population persistence and extirpation. Hellbenders persisted in only 42% of the 24 historical record sites. The best fit model indicated electrical conductivity (EC) was the strongest predictor of hellbender population persistence (EC &lt; 278 μS/cm) and extirpation. Conductivity was strongly negatively correlated with canopy cover within the total watershed (r = −0.83, n = 21, p &lt; 0.001) and riparian buffer of the watershed (r = −0.77, n = 21, p &lt; 0.001). Electrical conductivity tends to increase following deforestation, and may inhibit sperm motility and thus limit recruitment of hellbenders and other aquatic vertebrate species with external fertilisation. By integrating historical data, eDNA, field data, and freely available high resolution remote sensing data, our study design allowed for rapid assessment of predictors of and changes in hellbender distribution over a relatively broad geographic area. This cost‐ and time‐effective approach may be used for evaluating other rare aquatic species.

Ochrona przed stresem oksydacyjnym w męskim układzie płciowym

Reactive oxygen species (ROS) play in the male reproductive system important physiological functions in cell signaling, spermatogenesis and sperm maturation in epididymis. The influence of various factors, e.g. environmental, could be the reason for oxidative stress. This can lead to the exposure of cells to the toxic effects of many oxidants such as O•⁻₂, O₃, H₂O₂, •OH. The source of ROS may be spermatozoa, which also, due to the composition of the cytoplasmic membrane lipids and great amount of mitochondria are particularly susceptible to oxidative damage. Oxidative stress can damage their membranes, DNA, inhibit sperm motility and reduce their fertilization ability. This dual effect of ROS confirms the unique role of antioxidant enzymes (such as SOD, CAT and GPX) and non-enzymatic (e.g. GSH, vitamins A, E, C, transferrin) responsible for maintaining adequate levels of ROS in the male reproductive system.

Effects of cadmium on protein tyrosine phosphorylation of mouse spermatozoa and the protective role of EGTA

In this study, we explored the effects of cadmium (Cd) on mouse sperm motility parame-ters, protein tyrosine phosphorylation and the location of tyrosine-phosphorylated targets using computer-assisted sperm analysis (CASA), western blot (WB) and immunofluorescence technique coupled to sperm in vitro culture method, respectively. The results showed sperm motility was inhibi-ted by Cd in a dose-dependent manner and when Cd increased to 1.0 μmol·L-1, sperm motility was inhibited significantly (P<0.05). Simultaneously, protein tyrosine phosphorylation was enhanced by Cd and in particular, the tyrosine phosphorylation of ～55 kDa proteins was greatly promoted when Cd concentrations were greater or equal to 1.0 μmol·L-1 (P<0.05). Importantly, these tyrosine-phosphorylated proteins were mainly localized in the middle piece of mouse sperm. However, when sperm was incubated with 30 μmol·L-1 ethylene glycol tetraacetic acid (EGTA) and 10 μmol·L-1 Cd concurrently, both the tyrosine phosphorylation of ～55 kDa proteins and sperm motility were not changed obviously (P>0.05). These results suggested that Cd may inhibit sperm motility by inducing the tyrosine phosphorylation of ～55 kDa proteins in the middle piece and EGTA could chelate Cd ions to relieve its toxicity. This study demonstrated that Cd induced the tyrosine phosphorylation of a specific subset of proteins and thus decreased sperm motility. Interes-tingly, EGTA acted as an inhibitor to block Cd from entering the sperm, which provided a novel research method for revealing the molecular mechanisms of reproductive toxicity caused by Cd.

In Vitro Effects of Selected Trichothecenes on the Rabbit Spermatozoa Motility Behavior – A Comparative Study

Summary This study was designed to describe and compare the time- and dose-dependent in vitro effects of selected trichothecenes (deoxynivalenol-DON, zearalenone-ZEA and T-2 toxin) on the motility behavior of rabbit spermatozoa. The rabbit semen was diluted in PBS supplemented with different concentrations (1, 5, 10, 50, 100 μmol/L) of DON, ZEA or T-2 while the Control carried no mycotoxin. At culture times of 0h, 2h, 4h and 8h, the spermatozoa motility was assessed using the computer-aided sperm analysis (CASA) with the help of the IDENT stain and fluorescent illumination. The motility assessment revealed different behavior patterns, specific and unique to each of the studied mycotoxins. DON exhibited the ability to temporarily increase the sperm motility, followed by its rapid decline at later stages of the experiment (P&lt;0.001). ZEA proved to act as a highly toxic substance on the spermatozoa, causing a rapid decline of the motion and resulting in a fast and complete sperm motility inhibition (P&lt;0.001). Lastly, T-2 revealed to be highly detrimental to the sperm activity even at small concentrations (P&lt;0.001). Our data suggest that further experiments are needed due to the lack of evidence emphasizing the toxinogenic effects of trichothecenes on male reproductive capacity.

Microbiological profile of cervix of females attending in-vitro fertilization clinic of a tertiary care hospital, North India

ObjectivesBacterial infections of the female reproductive tract have long been recognized as having an association with infertility. Microorganisms in the female genital tract might themselves or by producing some extracellular metabolites inhibit sperm motility leading to infertility. So, microbiological screening of female genital tract is needed before in vitro fertilization (IVF) procedures. The aim of the present study was to look for the microbiological profile of cervical samples of females attending IVF clinic for infertility. Materials and methodsThe study was conducted in the Department of Microbiology, Bacteriology Division, Maulana Azad Medical College, New Delhi. Cervical swabs from the females attending the IVF clinics for infertility from January 2015 to June 2015 were screened for the bacterial isolates. The isolates were identified by standard microbiological procedures. ResultsTotal 296 samples were analyzed. Majority was from females belonging to 31–40 years age group (56.4%) followed by 21–30 years (37.1%). Most of the samples (54.3%) were sterile, while normal vaginal flora and environmental contaminants were obtained from 34.7% of the samples. Various isolates obtained were, Micrococcus spp. (12.5%), diphtheroids (9%), Non enterococcal group D streptococcus (6.7%), Staphylococcus aureus (0.3%), Coagulase negative staphylococcus (2%), Enterococcus spp. (1%), Bacillus spp. (3%), Escherichia coli (5.7%), Klebsiella spp. (2%), Acinetobacter spp. (1.3%) and Candida spp. (2.3%). ConclusionGram negative organisms in the female reproductive tract might be a cause of female infertility. There is still a need for practising proper procedure while collecting endocervical samples from females attending infertility clinic.

Role of potassium and pH on the initiation of sperm motility in the European eel

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pHi in the quiescent stage was also induced. Intracellular pH (pHi) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pHi (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0mm=H000; H2 O2 50mm=H050; H2 O2 100mm=H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p<0.05) for NS (59%±3.3) than BSS (36.9%±5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p<0.05) for BSS (21.6%±5.0) than NS sperm (14.6%±1.2). The presence of H2 O2 increased DNA damage in NS (H000 =4.1%±0.9; H050=9.3%±0.7; and H100=10.9%±2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p<0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5±4.8 vs 21.1±3.9 and BSS: 33.3±8.1 vs 8.9±1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells.