M. R. C. Vassallo

Dynamics of the Global Tyrosine Phosphorylation During Capacitation and Acquisition of the Ability to Fuse with Oocytes in Human Spermatozoa

Abstract 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.

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.

Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli.

Unidentified soluble factors secreted by E. coli, a frequently isolated microorganism in genitourinary infections, have been reported to inhibit mitochondrial membrane potential (ΔΨm), motility and vitality of human spermatozoa. Here we explore the mechanisms involved in the adverse impact of E. coli on sperm motility, focusing mainly on sperm mitochondrial function and possible membrane damage induced by mitochondrial-generated reactive oxygen species (ROS). Furthermore, as lactobacilli, which dominate the vaginal ecosystem of healthy women, have been shown to exert anti-oxidant protective effects on spermatozoa, we also evaluated whether soluble products from these microorganisms could protect spermatozoa against the effects of E. coli. We assessed motility (by computer-aided semen analysis), ΔΨm (with JC-1 dye by flow cytometry), mitochondrial ROS generation (with MitoSOX red dye by flow cytometry) and membrane lipid-peroxidation (with the fluorophore BODIPY C11 by flow cytometry) of sperm suspensions exposed to E. coli in the presence and in the absence of a combination of 3 selected strains of lactobacilli (L. brevis, L. salivarius, L. plantarum). A Transwell system was used to avoid direct contact between spermatozoa and microorganisms. Soluble products of E. coli induced ΔΨm loss, mitochondrial generation of ROS and membrane lipid-peroxidation, resulting in motility loss. Soluble factors of lactobacilli prevented membrane lipid-peroxidation of E. coli-exposed spermatozoa, thus preserving their motility. In conclusion, sperm motility loss by soluble products of E. coli reflects a mitochondrial dysfunction-related membrane lipid-peroxidation. Lactobacilli could protect spermatozoa in the presence of vaginal disorders, by preventing ROS-induced membrane damage.

Effect of vaginal probiotic lactobacilli on in vitro–induced sperm lipid peroxidation and its impact on sperm motility and viability

A combination of three selected strains of lactobacilli (Lactobacillus brevis [CD2], L. salivarius [FV2], and L. plantarum [FV9]), whose effectiveness in treating bacterial vaginosis in the form of vaginal tablets has been reported recently, prevented sperm lipid peroxidation that was induced in vitro by a ferrous ion promoter, thus preserving sperm motility and viability. This finding suggests the potential of vaginal probiotic lactobacilli for protecting human spermatozoa from radical oxygen species in the presence of vaginal disorders, thereby improving the fertilization potential of the female host.

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.

Low testosterone and non-alcoholic fatty liver disease: Evidence for their independent association in men with chronic spinal cord injury

Objective: Non-alcoholic fatty liver disease (NAFLD) has been claimed as a liver phenotype of metabolic syndrome, which in turn is associated with male hypogonadism. We assessed whether an independent association between NAFLD and androgen deficiency could be revealed in men with chronic spinal cord injury (SCI), who exhibit a high prevalence of biochemical androgen deficiency and a combination of risk factors for metabolic syndrome. Design: Fifty-five consecutive men with chronic SCI admitted to a rehabilitation program underwent clinical/biochemical evaluations and liver ultrasonography. Results: NAFLD was diagnosed in 27 patients (49.1%). Men with NAFLD were older and exhibited significantly higher body mass index, Homeostatic model assessment of insulin resistance, triglycerides and gamma-glutamyl transpeptidase values, lower total and free testosterone levels and they were engaged in a significantly poorer weekly leisure time physical activity (LTPA). At the multiple logistic regression analysis, only total and free testosterone levels exhibited a significant independent association with NAFLD. The risk of having NAFLD increased indeed of 1% for each decrement of 1 ng/dL of total testosterone and of 3% for each decrement of 1 pg/mL of free testosterone, after adjustment for confounders. In men with total testosterone < 300 ng/dL (36.4%) the prevalence of NAFLD reached 85%: they had a risk of having NAFLD significantly higher (∼12-fold) than those with total testosterone ≥ 300 ng/dL, after adjustment for confounders. Conclusion: The evidence of an independent association between NAFLD and low testosterone is strongly reinforced by its demonstration in men with chronic SCI, in spite of the many confounders peculiar to this population.

Association between 25(OH)-vitamin D and testosterone levels: Evidence from men with chronic spinal cord injury

Objective: As an independent linear association between 25-hydroxyvitamin D (25(OH)D) and testosterone levels is controversial, this study aimed to explore this topic in men with chronic spinal cord injury (SCI), who exhibit a high prevalence of both androgen and vitamin D deficiency. Design: Forty-nine men with chronic SCI consecutively admitted to a rehabilitation program underwent clinical/biochemical evaluations. Results: Deficiency of 25(OH)D (<20 ng/mL) was found in 36 patients (73.5%). They exhibited significantly lower total testosterone and free testosterone levels, higher parathyroid hormone (PTH) and HOMA-IR, a poorer functional independence degree, and were engaged in poorer weekly leisure time physical activity (LTPA). Significant correlates of 25(OH)D levels were: total testosterone, free testosterone, PTH, functional independence degree and weekly LTPA. At the linear regression models, lower 25(OH)D levels were associated with both lower total and free testosterone after adjustment for age, smoking, alcohol consumption, comorbidities and HOMA-IR. However, after full adjustment, also including functional independence degree, BMI and LTPA, only the association of lower 25(OH)D with lower free testosterone was still significant. Conclusion: In men with SCI, 25(OH)D correlates with total and free testosterone and exhibits an independent linear association with free testosterone. Regardless of this independent link, hypovitaminosis D and androgen deficiency are markers of poor health, sharing common risk factors to take into account in the rehabilitative approach to patients 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.

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.