J. Villegas - Academia.edu (original) (raw)
Papers by J. Villegas
Free Radical Biology and Medicine
Asian journal of andrology, Jan 22, 2018
Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosi... more Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT...
Cryobiology, Dec 13, 2017
Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic o... more Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incuba...
Systems biology in reproductive medicine, Jan 3, 2016
Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it ca... more Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite an...
Human reproduction (Oxford, England), 2015
Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxyge... more Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? 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. 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. 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. To evaluate the MPT in sperm cells, the calcein-AM and cobalt chlori...
Fertility and Sterility, 2015
To determine the effect on human sperm of Escherichia coli strains separated on the basis of thei... more To determine the effect on human sperm of Escherichia coli strains separated on the basis of their ability to produce hemolysis. Experimental study. University-based laboratory. Semen samples from healthy donors. Five million sperm, selected via the swim-up method, were incubated with 3 E. coli concentrations to obtain ratios of sperm to E. coli of 1:2, 1:16, and 1:128. The E. coli strains were: a hemolytic isolated strain (H), a nonhemolytic American Type Culture Collection strain (NH-ATCC), and a nonhemolytic isolated strain (NH-I). Aliquots of human sperm were used to measure progressive motility using computer-aided sperm analysis, mitochondrial membrane potential (ΔΨm) with a JC-1 (5,5&amp;amp;amp;amp;amp;amp;amp;#39;,6,6&amp;amp;amp;amp;amp;amp;amp;#39; tetrachloro-1,1&amp;amp;amp;amp;amp;amp;amp;#39;,3,3&amp;amp;amp;amp;amp;amp;amp;#39;-tetraethylbenzamidazolocarbocyanin iodide) and propidium iodide stain, and intracellular reactive oxygen species (iROS) with a dihydroethidium (DHE) stain. Sperm ΔΨm and iROS were measured by flow cytometry. Sperm vitality was considered the mean of propidium iodide-negative and DHE-negative cells. Sperm incubated with the H strain in a 1:2 sperm to bacteria ratio demonstrated a significant decrease in motility and ΔΨm, and an increase of iROS. The NH-ATCC strain decreased sperm motility and ΔΨm, but in a ratio of sperm to bacteria of 1:128; it increased iROS at a ratio of 1:16. The NH-I strain did not affect the analyzed sperm functions, even at a 1:128 sperm to bacteria ratio. Results show a greater pathogenic effect on human sperm of E. coli strains with, versus without, hemolytic capacity.
Theriogenology, 2014
High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are ... more High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.
Human Reproduction, 2009
Loss of mitochondrial membrane potential (DC m) in spermatozoa is correlated with high levels of ... more Loss of mitochondrial membrane potential (DC 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 DC 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 DC m and activation of caspases are processes thoroughly studied in human ejaculates. Disruption of DC 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 DC m in human sperm. methods: Viable and highly motile sperm cells were selected through a swim-up process and incubated with 90 mg/ml BA. To elucidate the caspase dependency of BA-triggered disruption of DC 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 DC 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 DC m (P , 0.05 versus control). conclusion: We found that caspases directly participate in the loss of DC m caused by BA in human sperm cells. However, caspaseindependent pathways may also be present.
Fertility and Sterility, 2010
Objective: To evaluate the effect of Escherichia coli and its soluble factors on the viability an... more 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 (DJm), 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 DJm 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 DJm, 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. (Fertil Steril Ò 2010;94:619-23. Ó2010 by American Society for Reproductive Medicine.
Fertility and Sterility, 2010
Objective: To verify whether chemotaxis is in part an oxidative process mediated by reactive oxyg... more Objective: To verify whether chemotaxis is in part an oxidative process mediated by reactive oxygen species (ROS). Design: In this prospective study, after removal of seminal plasma, the sperm suspension received no treatment (control), ROS formation by stimulation with phorbol 12-myristate 13-acetate (PMA), antioxidant treatment (with catalase), or PMA stimulus in the presence of catalase. At time zero and after 3 hours of incubation, the percentage of capacitated and oriented spermatozoa and the ROS levels were determined. Setting: Andrology laboratory in a medical research institution. Patient(s): Normal semen was obtained from eight men. Intervention(s): The semen samples were evaluated to determine the effect of ROS production by stimulation with PMA and/or antioxidant treatment (with catalase) on the percentage of capacitated and oriented spermatozoa. Main Outcome Measure(s): The sperm capacitation, chemotaxis and reactive oxygen species were assessed before and after PMA and/or antioxidant treatment. Result(s): Prolonged exposure to high quantities of ROS decrease the sperm chemotactic response, probably because of oxidative damage of the cell. However, this effect may be reduced by the addition of antioxidants like catalase. Conclusion(s): Similar to capacitation, chemotaxis seems to depend on the production of ROS, but in the latter process there may be a critical level of ROS necessary for chemotaxis to occur.
Fertility and Sterility, 2005
Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis i... more Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis in mature human spermatozoa Human spermatozoa incubated with polymorphonuclear leukocytes and Escherichia coli showed a significant increase in the annexin V binding (PϽ.05). This increase was similar to that obtained when spermatozoa were incubated with polymorphonuclear leukocytes activated by phorbol-12-myristate-13-acetate.
Andrologia, 2011
The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome r... more 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.
Andrologia, 2004
The acrosome reaction (AR), a modified exocytotic process, is prerequisite for successful mammali... more The acrosome reaction (AR), a modified exocytotic process, is prerequisite for successful mammalian fertilization. The protein component that is responsible for the AR-inducing activity of human follicular fluid, has been found to be immunologically identical with corticosteroid-binding globulin (CBG), which is well characterized and serves as a transport protein for progesterone and cortisol in the plasma. Our findings have shown that the CBG-like protein is expressed by endothelial cells of the Fallopian tube depending on the hormonal cycle. In the culture medium of human epithelial tubal cells, the CBG-like protein was detected by Western blot analysis. The protein was also found in biologically active form in human tubular fluid. Our investigations strongly indicate that human Fallopian tube cells actively express and secrete a CBG-like progesterone-binding protein, which might play a role in the in vivo modulation of human sperm AR.
Andrologia, 2003
The presence of excess leucocytes in the semen has been associated with male infertility. Accordi... more The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than lo6 leucocytes ml-' 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 = 0.02 19) 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 uitro 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
Andrologia, 2009
Acrosome reaction-corticosteroid-binding globulin-cumulus oophorus-fallopian tubes-progesteronebi... more Acrosome reaction-corticosteroid-binding globulin-cumulus oophorus-fallopian tubes-progesteronebinding protein Summary. The acrosome reaction (AR)inducing effect of follicular cells, like that of the cumulus oophorus and granulosa cells, has been described previously. In addition to the well known steroid secreting activity of cumulus cells, the results obtained here demonstrate the secretion of a corticosteroid-binding globulin (CBG)-like protein. An AR-inducing effect was shown with the culture medium of human cumulus oophorus. This effect could be eliminated by treating the sample with monoclonal antibodies against CBG. Moreover, Western blotting after SDS-PAGE of the culture medium strongly indicates that human cumulus cells actively express and secrete a CBGlike protein. This might give an indication as to the origin of the acrosome reaction-inducing substance found in follicular fluid. Furthermore, AR-inducing activity and the elimination of this activity by antibodies against CBG was shown for oviductal fluid. With immunohistochemical techniques the CBG-like protein was localized in the epithelial lining of the fallopian tubes, giving possible evidence for the involvement of this molecule in fallopian tube function.
Andrologia, 2002
The presence of increased number of leukocytes in semen is indicative of inflammation in the male... more 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.
Andrologia, 2011
In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertil... more In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.
Free Radical Biology and Medicine
Asian journal of andrology, Jan 22, 2018
Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosi... more Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT...
Cryobiology, Dec 13, 2017
Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic o... more Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incuba...
Systems biology in reproductive medicine, Jan 3, 2016
Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it ca... more Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite an...
Human reproduction (Oxford, England), 2015
Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxyge... more Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? 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. 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. 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. To evaluate the MPT in sperm cells, the calcein-AM and cobalt chlori...
Fertility and Sterility, 2015
To determine the effect on human sperm of Escherichia coli strains separated on the basis of thei... more To determine the effect on human sperm of Escherichia coli strains separated on the basis of their ability to produce hemolysis. Experimental study. University-based laboratory. Semen samples from healthy donors. Five million sperm, selected via the swim-up method, were incubated with 3 E. coli concentrations to obtain ratios of sperm to E. coli of 1:2, 1:16, and 1:128. The E. coli strains were: a hemolytic isolated strain (H), a nonhemolytic American Type Culture Collection strain (NH-ATCC), and a nonhemolytic isolated strain (NH-I). Aliquots of human sperm were used to measure progressive motility using computer-aided sperm analysis, mitochondrial membrane potential (ΔΨm) with a JC-1 (5,5&amp;amp;amp;amp;amp;amp;amp;#39;,6,6&amp;amp;amp;amp;amp;amp;amp;#39; tetrachloro-1,1&amp;amp;amp;amp;amp;amp;amp;#39;,3,3&amp;amp;amp;amp;amp;amp;amp;#39;-tetraethylbenzamidazolocarbocyanin iodide) and propidium iodide stain, and intracellular reactive oxygen species (iROS) with a dihydroethidium (DHE) stain. Sperm ΔΨm and iROS were measured by flow cytometry. Sperm vitality was considered the mean of propidium iodide-negative and DHE-negative cells. Sperm incubated with the H strain in a 1:2 sperm to bacteria ratio demonstrated a significant decrease in motility and ΔΨm, and an increase of iROS. The NH-ATCC strain decreased sperm motility and ΔΨm, but in a ratio of sperm to bacteria of 1:128; it increased iROS at a ratio of 1:16. The NH-I strain did not affect the analyzed sperm functions, even at a 1:128 sperm to bacteria ratio. Results show a greater pathogenic effect on human sperm of E. coli strains with, versus without, hemolytic capacity.
Theriogenology, 2014
High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are ... more High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.
Human Reproduction, 2009
Loss of mitochondrial membrane potential (DC m) in spermatozoa is correlated with high levels of ... more Loss of mitochondrial membrane potential (DC 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 DC 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 DC m and activation of caspases are processes thoroughly studied in human ejaculates. Disruption of DC 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 DC m in human sperm. methods: Viable and highly motile sperm cells were selected through a swim-up process and incubated with 90 mg/ml BA. To elucidate the caspase dependency of BA-triggered disruption of DC 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 DC 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 DC m (P , 0.05 versus control). conclusion: We found that caspases directly participate in the loss of DC m caused by BA in human sperm cells. However, caspaseindependent pathways may also be present.
Fertility and Sterility, 2010
Objective: To evaluate the effect of Escherichia coli and its soluble factors on the viability an... more 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 (DJm), 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 DJm 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 DJm, 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. (Fertil Steril Ò 2010;94:619-23. Ó2010 by American Society for Reproductive Medicine.
Fertility and Sterility, 2010
Objective: To verify whether chemotaxis is in part an oxidative process mediated by reactive oxyg... more Objective: To verify whether chemotaxis is in part an oxidative process mediated by reactive oxygen species (ROS). Design: In this prospective study, after removal of seminal plasma, the sperm suspension received no treatment (control), ROS formation by stimulation with phorbol 12-myristate 13-acetate (PMA), antioxidant treatment (with catalase), or PMA stimulus in the presence of catalase. At time zero and after 3 hours of incubation, the percentage of capacitated and oriented spermatozoa and the ROS levels were determined. Setting: Andrology laboratory in a medical research institution. Patient(s): Normal semen was obtained from eight men. Intervention(s): The semen samples were evaluated to determine the effect of ROS production by stimulation with PMA and/or antioxidant treatment (with catalase) on the percentage of capacitated and oriented spermatozoa. Main Outcome Measure(s): The sperm capacitation, chemotaxis and reactive oxygen species were assessed before and after PMA and/or antioxidant treatment. Result(s): Prolonged exposure to high quantities of ROS decrease the sperm chemotactic response, probably because of oxidative damage of the cell. However, this effect may be reduced by the addition of antioxidants like catalase. Conclusion(s): Similar to capacitation, chemotaxis seems to depend on the production of ROS, but in the latter process there may be a critical level of ROS necessary for chemotaxis to occur.
Fertility and Sterility, 2005
Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis i... more Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis in mature human spermatozoa Human spermatozoa incubated with polymorphonuclear leukocytes and Escherichia coli showed a significant increase in the annexin V binding (PϽ.05). This increase was similar to that obtained when spermatozoa were incubated with polymorphonuclear leukocytes activated by phorbol-12-myristate-13-acetate.
Andrologia, 2011
The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome r... more 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.
Andrologia, 2004
The acrosome reaction (AR), a modified exocytotic process, is prerequisite for successful mammali... more The acrosome reaction (AR), a modified exocytotic process, is prerequisite for successful mammalian fertilization. The protein component that is responsible for the AR-inducing activity of human follicular fluid, has been found to be immunologically identical with corticosteroid-binding globulin (CBG), which is well characterized and serves as a transport protein for progesterone and cortisol in the plasma. Our findings have shown that the CBG-like protein is expressed by endothelial cells of the Fallopian tube depending on the hormonal cycle. In the culture medium of human epithelial tubal cells, the CBG-like protein was detected by Western blot analysis. The protein was also found in biologically active form in human tubular fluid. Our investigations strongly indicate that human Fallopian tube cells actively express and secrete a CBG-like progesterone-binding protein, which might play a role in the in vivo modulation of human sperm AR.
Andrologia, 2003
The presence of excess leucocytes in the semen has been associated with male infertility. Accordi... more The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than lo6 leucocytes ml-' 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 = 0.02 19) 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 uitro 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
Andrologia, 2009
Acrosome reaction-corticosteroid-binding globulin-cumulus oophorus-fallopian tubes-progesteronebi... more Acrosome reaction-corticosteroid-binding globulin-cumulus oophorus-fallopian tubes-progesteronebinding protein Summary. The acrosome reaction (AR)inducing effect of follicular cells, like that of the cumulus oophorus and granulosa cells, has been described previously. In addition to the well known steroid secreting activity of cumulus cells, the results obtained here demonstrate the secretion of a corticosteroid-binding globulin (CBG)-like protein. An AR-inducing effect was shown with the culture medium of human cumulus oophorus. This effect could be eliminated by treating the sample with monoclonal antibodies against CBG. Moreover, Western blotting after SDS-PAGE of the culture medium strongly indicates that human cumulus cells actively express and secrete a CBGlike protein. This might give an indication as to the origin of the acrosome reaction-inducing substance found in follicular fluid. Furthermore, AR-inducing activity and the elimination of this activity by antibodies against CBG was shown for oviductal fluid. With immunohistochemical techniques the CBG-like protein was localized in the epithelial lining of the fallopian tubes, giving possible evidence for the involvement of this molecule in fallopian tube function.
Andrologia, 2002
The presence of increased number of leukocytes in semen is indicative of inflammation in the male... more 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.
Andrologia, 2011
In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertil... more In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.