Semen leukocytes and oxidative-dependent DNA damage of spermatozoa in male partners of subfertile couples with no symptoms of genital tract infection.

The influence of seminal leukocytes on generation of oxidative damage to sperm DNA was here investigated on male partners of subfertile couples asymptomatic for a genital tract infection. The study included 111 ejaculates from men attending the Andrology Centre at University of L'Aquila. Semen leukocytes subset included round cells expressing pan-leukocyte CD45 antigen, monocyte/macrophage lineage antigen CD14, and activated macrophages HLA-DR antigen. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression identified spermatozoa with DNA oxidative adducts while terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labeling (TUNEL) assay detected spermatozoa with DNA fragmentation. Flow cytometry and immunocytochemistry was used for determinations. Main outcome measure was the association of semen leukocyte subpopulations with spermatozoa showing oxidative-related DNA damage and with routine semen parameters. Leukocyte subpopulations were strictly correlated (p < 0.0001), but no association was found between the concentration of leukocytes, semen parameters, the percentage of TUNEL-positive and of 8-OHdG-positive spermatozoa. The percentage of 8-OHdG-positive spermatozoa was positively correlated with the percentage of TUNEL-positive spermatozoa (r = 0.48; p < 0.0001) and negatively correlated with sperm concentration (r = -0.44; p < 0.0001). Sperm concentration and the percentage of TUNEL-positive spermatozoa independently contributed (ß = -0.25, p = 0.008; ß = 0.23, p = 0.05, respectively) to the variation in percentage of 8-OHdG-positive spermatozoa after adjusting for age, abstinence time, and smoking. In conclusion, oxidative-dependent DNA damage in spermatozoa was associated to poor semen quality but not to different leukocyte subpopulations in ejaculates of men asymptomatic for a genital tract infection.

Correlates of low testosterone in men with chronic spinal cord injury.

Although high rates of serum testosterone deficiency have been reported in men with spinal cord injury (SCI), its determinants and attributes are not yet established. The aim of this study was to recognize, among putative determinants and attributes of androgen deficiency, those significantly associated to low testosterone after adjustment for confounders recognizable in men with chronic SCI. A biochemical androgen deficiency (total testosterone <300 ng/dL) was exhibited by 18 of 51 patients (35.3%). Significant correlates of testosterone levels were as follows: weekly leisure time physical activity (LTPA) explored by the LTPA Questionnaire for people with SCI, body mass index (BMI), homeostatic model assessment of insulin resistance (HOMA-IR), triglycerides and sexual symptoms, explored by the aging males' symptom (AMS) questionnaire. At the multiple linear regression analysis, among putative determinants of low testosterone, only weekly LTPA and BMI exhibited a significant association with testosterone levels, explaining 54.2 and 9.0% of testosterone variability respectively. At the linear regression models, among various putative attributes of androgen deficiency, only lower sexual desire and, at a lesser extent, higher HOMA-IR, exhibited significant associations with lower testosterone levels, after adjustment for BMI, age, comorbidities and weekly LTPA. In conclusion, poor LTPA, high BMI and low sexual desire are independent predictors of low testosterone in men with chronic SCI. This is relevant to clinical practice, as all these features are routinely assessed in rehabilitation settings for SCI. As poor LTPA and high BMI are modifiable life-style related risk factors, prospective studies could clarify whether life-style modification could increase the level of testosterone and improve the low sexual desire, relevant clinical attribute of low testosterone in men with SCI.

Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa.

Gram-negative bacteria frequently involved in urogenital tract infections release the endotoxin lipopolysaccharide (LPS); its receptor, toll-like receptor-4 (TLR4), has been recently identified in human spermatozoa, and its direct activation has been suggested in mediating adverse effects of LPS on human spermatozoa. However, the underlying signal transduction remains to be clarified. In other cell types, LPS induces the generation of endocannabinoids, which are involved in mediating endotoxin effects. In human spermatozoa, which exhibit a completely functional endocannabinoid system, the activation of cannabinoid receptor-1 (CB1) inhibited sperm mitochondrial membrane potential (ΔΨm). In this study, we tested the hypothesis of a contribution of CB1 activation by sperm-generated endocannabinoids in the adverse effects exerted by LPS on human spermatozoa. The exposure of motile sperm suspensions to E. coli LPS produced a significant decrease in sperm ΔΨm, assessed at flow cytometry with JC-1, similar to that induced by Metanandamide (Met-AEA), a non-hydrolyzable analogue of the endocannabinoid AEA. The LPS-induced inhibition of ΔΨm was prevented by the selective CB1 cannabinoid receptor antagonist, SR141716. However, the inhibition of ΔΨm induced by either LPS or Met-AEA did not affect sperm motility. Consistent with this finding, the CB1-mediated inhibition of ΔΨm was neither associated to mitochondrial generation of reactive oxygen species as evaluated by flow cytometry with MytoSox Red nor to apoptosis pathway activation as evaluated with cytoflorimetric assay for activated caspase-9 and caspase-3. Any oxidative genomic damage was also ruled out with the cytoflorimetric quantification of the oxidized base adduct 8-hydroxy-2'-deoxyguanosine. In conclusion, E. coli LPS inhibited sperm ΔΨm through the activation of CB1, but this effect was not accompanied to the activation of mitochondrial dysfunction-related apoptotic/oxidative mechanisms, which could affect sperm motility and genomic integrity.

Involvement of mitochondrial dysfunction in the adverse effect exerted by seminal plasma from men with spinal cord injury on sperm motility.

The aetiology of severe asthenozoospermia in men with spinal cord injury includes an adverse impact of seminal plasma (SP) on sperm motility. In this study we investigated the effect exerted by SP from men with SCI on donor sperm mitochondrial activity and its reflection on motility. Donor spermatozoa were exposed (1 h) to SP from 22 ejaculates of men with SCI. Only SP from samples exhibiting both a low fructose level and an inhibitory effect on mitochondrial membrane potential (ΔΨm), assessed at flow cytometry with JC-1, affected donor sperm motility when evaluated 1 h after co-incubation. This effect was reverted by washing from SP and sperm re-suspension in medium containing glucose, in spite of persistently depressed ΔΨm. In the same samples, sperm motility and vitality dramatically decreased when evaluated 6 h after washing and re-suspension in the glucose-containing medium. Seminal plasmas which induced a disruption of ΔΨm, also enhanced a mitochondrial ROS generation, as assessed by MitoSOX red. The enhanced mitochondrial ROS generation was associated with a late induction of sperm membrane lipid peroxidation, as assessed by BODIPY C11 , when evaluated at 6 h, but not at 1 h, after washing from SP. Furthermore, activation of caspase-9 and caspase-3 accompanied the loss of ΔΨm. In conclusion, a double energetic blockage (glycolysis and mitochondrial respiration) can represent a metabolic determinant of the early adverse effect exerted by SP from men with SCI on sperm motility. Mitochondrial dysfunction-related oxidative/apoptotic mechanisms can account for later consequences on sperm motility/vitality.

Energetic metabolism and human sperm motility: impact of CB1 receptor activation.

It has been reported that the endocannabinoid anandamide (AEA) exerts an adverse effect on human sperm motility, which has been ascribed to inhibition of mitochondrial activity. This seems to be at variance with evidence suggesting a major role of glycolysis in supplying ATP for sperm motility; furthermore, the role of AEA-binding receptors in mediating mitochondrial inhibition has not yet been explored. In this study, human sperm exposure to Met-AEA (methanandamide, nonhydrolyzable analog of AEA) in the micromolar range significantly decreased mitochondrial transmembrane potential (ΔΨm), similarly to rotenone, mitochondrial complex I inhibitor. The effect of Met-AEA (1 µm) was prevented by SR141716, CB(1) cannabinoid receptor antagonist, but not by SR144528, CB(2) antagonist, nor by iodoresiniferatoxin, vanilloid receptor antagonist. The effect of Met-AEA did not involve activation of caspase-9 or caspase-3 and was reverted by washing. In the presence of glucose, sperm exposure either to Met-AEA up to 1 µm or to rotenone for up to 18 h did not affect sperm motility. At higher doses Met-AEA produced a CB(1)-independent poisoning of spermatozoa, reducing their viability. Under glycolysis blockage, 1 µm Met-AEA, similarly to rotenone, dramatically abolished sperm motility, an effect that was prevented by SR1 and reverted by washing. In conclusion, CB(1) activation induced a nonapoptotic decrease of ΔΨm, the detrimental reflection on sperm motility of which could be revealed only under glycolysis blockage, unless very high doses of Met-AEA, producing CB(1)-independent sperm toxicity, were used. The effects of CB(1) activation reported here contribute to elucidate the relationship between energetic metabolism and human sperm motility.

Beta-chemokine receptor CCR5 in human spermatozoa and its relationship with seminal parameters.

BACKGROUND: Chemokine receptor CCR5, the main HIV-1 coreceptor, is present in the human spermatozoa. This study aimed to investigate (i) whether the percentage of CCR5-positive spermatozoa varies under conditions associated with changes in the membrane architecture, such as capacitation and fixation/permeabilization procedures; (ii) whether there is any relationship between individual variability in sperm CCR5 expression and semen parameters. METHODS AND RESULTS: In cytometric analysis, the percentage of CCR5-positive unfixed spermatozoa varied from approximately 10 to approximately 60%, and it significantly decreased after 5 h capacitation. The percentage of CCR5-positive spermatozoa was increased to more than 90% following fixation and permeabilization, suggesting the existence of large intracellular pools of the receptor. Immunocytochemistry showed positive staining in the anterior region of the sperm head. In ejaculates from male partner of 102 infertile couples, the CCR5 expression rate significantly correlated with sperm count, total sperm number and forward motility, but not with sperm morphology. In stepwise analysis, only forward motility entered into the model; however, this explained only approximately 8% of the variability in CCR5 expression. Interquartile analysis showed significant differences between the first and fourth quartiles of CCR5 expression for all semen parameters, except morphology. CONCLUSIONS: The percentage of CCR5-positive spermatozoa may vary under conditions associated with changes in membrane architecture and spermatozoa showed large intracellular pools of CCR5. A lower expression of CCR5 in asthenozoospermia seems to be suggested; however, it would only partially contribute to the inter-individual variability in the CCR5 expression. A genetic basis can be hypothesized to explain the variability.

Characterization of the endocannabinoid system in human spermatozoa and involvement of transient receptor potential vanilloid 1 receptor in their fertilizing ability.

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Dynamics of the global tyrosine phosphorylation during capacitation and acquisition of the ability to fuse with oocytes in human spermatozoa.

Phosphorylation of tyrosine residues in cellular proteins represents a major event during sperm capacitaton, but its relationship with the acquisition of sperm-fertilizing ability is still unclear. In this study we explored the relationship between the kinetics of the global tyrosine phosphorylation, monitored with a flow cytometric assay, and the acquisition of the human sperm ability to fuse with oocytes, evaluated with the progesterone-enhanced hamster egg penetration test. Sperm tyrosine phosphorylation appeared to be an early event in the capacitation process, with a 3.6-fold mean increase within 1 h of capacitation, but at this time sperm-oocyte fusion was extremely poor compared with that observed at 5 h of capacitation. Capacitation in calcium-free medium produced a 2-fold mean increase in tyrosine phosphorylation compared with that seen in complete capacitation medium both at 1 h and 5 h of capacitation, whereas sperm-oocyte fusion significantly increased only at 1 h, remaining unchanged at 5 h of capacitation. The cAMP analog, N,2-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP), prevented the inhibitory effect of seminal plasma on tyrosine phosphorylation but not on sperm-oocyte fusion. In conclusion, these results suggest that the acquisition of sperm-fertilizing ability is always associated with an increase of the global tyrosine phosphorylation, but tyrosine phosphorylation does not necessarily reflect the acquisition of the sperm-fertilizing ability. Flow cytometry assay, a reliable technique to quickly quantify the global levels of the human sperm tyrosine phosphorylation, could be useful for a further elucidation of the biological meaning of this process, with the perspective of its clinical use as a measure of the sperm-fertilizing potential.

RANTES and human sperm fertilizing ability: effect on acrosome reaction and sperm/oocyte fusion.

Beta-chemokine, regulated on activation and normally T-cell expressed and presumably secreted (RANTES), is present in both the male and female genital tract fluids where its levels increase in diseases related to infertility, such as endometriosis and male genital tract infections. beta-Chemokine receptors (CCR3 and CCR5) are expressed on freshly ejaculated human sperm cells and a sperm chemoattractant effect for RANTES has been reported. No information exists on other possible roles of RANTES on sperm functions involved in the fertilization process. In the present study, the exposure of sperm suspensions to high concentrations of the chemokine, comparable to those observed in inflammatory diseases, significantly decreased the stimulatory effect exerted by progesterone on sperm/oocyte fusion, evaluated by means of the hamster egg penetration test. Accordingly, a large proportion of spermatozoa preincubated under capacitating conditions with high concentrations of RANTES underwent a premature acrosome reaction (AR) that prevented subsequent progesterone-induced AR. Finally, sperm samples exposed to the same high levels of chemokine showed a significant increase in the intracellular levels of cAMP, which is involved in capacitation and AR dynamics. These results indicate a negative interference of high levels of RANTES on the sperm fertilizing ability, thereby suggesting a potential contribution of this chemokine to subfertility associated with endometriosis and genital tract inflammatory diseases.