J. Villegas

Bacteria induce expression of apoptosis in human spermatozoa.

An increased number of sperm undergoing apoptosis has been observed during inflammatory processes in the male genital tract, which might be associated with elevated reactive oxygen species (ROS) levels. However, another factor to stimulate apoptosis could be the direct contact with bacteria or its products, even in the absence of ROS. The aim of this study was to investigate whether bacteria can directly initiate apoptosis in human spermatozoa. Human spermatozoa selected by density gradient centrifugation were incubated with polymorphonuclear granulocytes (PMN) isolated from blood and/or E. faecalis, E. coli or S. aureus. As ROS inductor in PMN, phorbol-12-myristate-13-acetate was used. After incubating the cells for 60 min at 37∘C, ROS were determined by chemiluminescence and phosphatidyl serine (PS) externalization was analyzed by flow cytometry with Annexin V-FITC and propidium iodide (PI). The increase in the percentage of spermatozoa Annexin V-FITC-positive/ PI-negative (early event of late apoptosis) was significant after the incubation with PMN plus PMA, PMN plus E. coli and E. coli alone. The percentage of spermatozoa Annexin V-FITC-positive/ PI-positive (apoptosis/necrosis) increased significantly in sperm incubated with E. coli and S. aureus(20.3% ± 3 and 13.6% ± 3.2 compared to sperm alone, 6% ± 0.5). Sperm incubated with PMN-PMA activated showed only a relative increase in apoptosis/necrosis (8.4% ± 1). Our results show that bacteria directly increase the PS externalisation in ejaculated human sperm. This way of inducing apoptosis does not require external ROS and may result from anyone of the molecular mechanisms that account for changes in motility, vitality and DNA integrity, that are characteristics of spermatozoa in male genital tract infection.

Urogenital inflammation: changes of leucocytes and ROS

Summary. The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml−1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase‐positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase‐positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase‐positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase‐positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Integrity of mitochondrial membrane potential reflects human sperm quality.

The aim of this work was to evaluate intracellular reactive oxygen species (ROS) levels, phosphatidylserine (PS) externalisation and mitochondrial membrane potential integrity in the spermatozoa of healthy donors and outpatients who consulted for infertility and to correlate the results with the classic sperm parameters. For the evaluation of intracellular ROS levels, PS externalisation and mitochondrial membrane potential integrity, the fluorescent compounds dihydroethidium, annexin V‐FITC and JC‐1, respectively, were used and analysed by using flow cytometry. Conventional seminal analysis, including motility, viability, morphology, sperm count and volume, was performed according to the WHO criteria. The mitochondrial membrane potential and ROS results showed significant differences between the spermatozoa of individuals with a normal semen analysis and those of the group presenting abnormality in at least one of the sperm parameters. Mitochondrial membrane potential showed a significant and direct correlation with all the sperm parameters analysed. ROS were inversely correlated with motility, viability and morphology. PS externalisation, however, did not show any differences between the two groups, nor was it correlated with the sperm parameters examined. The evaluation of mitochondrial membrane potential integrity is a test that reflects sperm quality, which makes it highly recommendable to be applied as a complement to routine sperm analyses.

Effect of Escherichia coli and its soluble factors on mitochondrial membrane potential, phosphatidylserine translocation, viability, and motility of human spermatozoa

OBJECTIVE To evaluate the effect of Escherichia coli and its soluble factors on the viability and function of human spermatozoa. DESIGN In this prospective study, after removal of seminal plasma, the sperm suspension was incubated in vitro with E. coli or with supernatant from E. coli culture. SETTING Andrology laboratory in a medical research institution. PATIENT(S) Semen was obtained from normozoospermic men. INTERVENTION(S) Semen samples were evaluated to determine the effect of E. coli and its soluble factors on sperm viability, motility, mitochondrial membrane potential (DeltaPsim), phosphatidylserine translocation, and reactive oxygen species generation. MAIN OUTCOME MEASURE(S) To verify the effect of E. coli and its soluble factors on sperm function. RESULT(S) After incubation with E. coli, the percentage of sperm with intact DeltaPsim decreased significantly, as did sperm viability and motility. Reactive oxygen species levels and phosphatidylserine translocation did not increase significantly. After sperm incubation with E. coli supernatant, a significant reduction in DeltaPsim, viability, and motility were also observed. CONCLUSION(S) Escherichia coli and its soluble factors affect sperm function, suggesting that the harmful effects of bacterial infection do not require that the spermatozoon come into direct contact with bacteria.

Canine sperm vitrification with sucrose: effect on sperm function.

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.

Reactive oxygen species induce reversible capacitation in human spermatozoa

Summary. Leucocytospermia has been associated with reduced sperm motility and decreased capacity for sperm–egg interaction. This effect could be mediated through reactive oxygen species (ROS), which, at high concentrations, induce lipid peroxidation and cellular death. The high impact on sperm capacitation reported in other mammalians should be more accurately assessed in the human because premature activation could affect sperm fertilizing capacity. The aim of this study was to evaluate both the effect of ROS on sperm capacitation and the protective role of seminal plasma. Spermatozoa selected by Percoll gradient were incubated with polymorphonuclear (PMN) granulocytes isolated from blood and activated by phorbol‐12‐myristate 13‐acetate (PMA). Different seminal plasma concentrations were added immediately or after 3‐h incubation. Afterwards, ROS production was evaluated by luminescence and sperm capacitation by chlortetracycline stain. In PMN granulocytes and sperm suspensions, the basal ROS production was <32 × 103 relative luminescence units (RLU). After stimulation with PMA, the rate of ROS production by PMN increased to 1287 × 103 RLU. Incubation of sperm with activated PMN resulted in an increase of sperm capacitation (37% versus 19% in the control). Immediate addition of seminal plasma caused a significant reduction in ROS (P < 0.01) and prevented sperm from capacitating. A higher effect in inhibition of sperm capacitation was observed when seminal plasma had been added after 3‐h incubation. The results suggest that human sperm capacitation can prematurely be induced by exogenous ROS and this effect can be reversed by seminal plasma. Thus, human sperm capacitation is another functional parameter that may be affected by nonphysiological ROS production.

Indirect immunofluorescence using monoclonal antibodies for the detection of leukocytospermia: comparison with peroxidase staining.

Summary. The presence of increased number of leukocytes in semen is indicative of inflammation in the male genital tract. Inflammatory processes at this level may lead to marked impairment of sperm function, and finally to a reduction in their fertilizing capability. An immunocytological technique for the detection of seminal leukocytes was evaluated in this study. As part of the standardization technique, different fixation methods were tested to ascertain whether samples could be stored and examined later. It was found that fixation with cold acetone at freezing temperatures retained immunoreactivity until day 11 of storage. All other methods showed a significant loss of immunoreactivity, from as little as day 2 of storage. In 46 specimens with elevated numbers of round cells, number of peroxidase‐positive cells and number and type of leukocytes were evaluated by means of indirect immunofluorescence. Determination of peroxidase‐positive cells to detect leukocytospermia, the standard procedure recommended by the WHO, was compared with the indirect immunofluorescence technique using monoclonal antibodies. While 19 of 46 patients showed high numbers of leukocytes in the ejaculate, as determined by the immunocytological method, only 9 of these were identified to be leukocytospermic, according to the WHO (standard) procedure. This difference was statistically significant (P < 0.01) and indicates that the standard method of detection of seminal leukocytes may be inaccurate.

Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa

STUDY QUESTION Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? SUMMARY ANSWER Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation. WHAT IS KNOWN ALREADY Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied. STUDY DESIGN, SIZE, DURATION Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation. PARTICIPANTS/MATERIALS, SETTING, METHODS To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm. LIMITATIONS, REASONS FOR CAUTION The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. WIDER IMPLICATIONS OF THE FINDINGS We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa. STUDY FUNDING/COMPETING INTERESTS This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.

Mitochondrial membrane potential disruption pattern in human sperm

BACKGROUND Loss of mitochondrial membrane potential (DeltaPsi(m)) in spermatozoa is correlated with high levels of reactive oxygen species in semen, abnormal spermiogram parameters, and low success rates of IVF. In somatic cells, the loss of DeltaPsi(m) is primarily associated with several mechanisms of cell death, mainly the activation of caspases. The impact of mitochondrial dysfunction on sperm function is still not fully elucidated, although disruption of DeltaPsi(m) and activation of caspases are processes thoroughly studied in human ejaculates. Disruption of DeltaPsi(m) in sperm can be externally triggered by the antineoplastic agent betulinic acid (BA). In this study, we determined whether caspase activation is necessary for the BA-induced disruption of DeltaPsi(m) in human sperm. METHODS Viable and highly motile sperm cells were selected through a swim-up process and incubated with 90 microg/ml BA. To elucidate the caspase dependency of BA-triggered disruption of DeltaPsi(m), we used the pan-caspase inhibitor zVAD-fmk and the caspase-3/7 inhibitor DEVD-cho. RESULTS Exposing highly motile sperm to BA caused a specific disruption of DeltaPsi(m) (P < 0.001 versus control) and a corresponding increase in caspase-3/7 activity (P < 0.001 versus control). Pre-incubation of the sperm with zVAD-fmk or DEVD-cho only partially inhibited BA-induced loss of DeltaPsi(m) (P < 0.05 versus control). CONCLUSION We found that caspases directly participate in the loss of DeltaPsi(m) caused by BA in human sperm cells. However, caspase-independent pathways may also be present.

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application

Summary.  α‐Glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of α‐glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of α‐glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean ± SEM): normozoospermia (control): 20.2 ± 1.4 mU ml−1; azoospermia: normal value: 17.6 ± 2.2 mU ml−1, low value: 7.4 ± 1.8 mU ml−1; oligozoospermia: normal value: 22.3 ± 2.5 mU ml−1, low value: 7.3 ± 0.7 mU ml−1; leucocytospermia: increase value: 38.9 ± 3.7 mU ml−1, low value: 11.1 ±1.3 mU ml−1. This study suggests that determination of α‐glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.