Apoptosis In Human Spermatozoa Research Articles

Melatonin alleviates heat stress-induced oxidative stress and apoptosis in human spermatozoa

Oxidative stress generates a large amount of reactive oxygen species (ROS) and affects sperm quality via damaging sperm DNA and compromising the intracellular homeostasis in human spermatozoa. In assisted reproductive technology (ART), it is substantial to prevent spermatozoa from ROS attack. The pineal hormone melatonin has the natural antioxidant capacity and can scavenge ROS. To the best of our knowledge, however, there are presently no studies investigating if melatonin can protect human spermatozoa from heat-induced oxidative damage. Herein, we induced oxidative stress in human spermatozoa with heat treatment, and determined that melatonin could protect human spermatozoa from heat-induced oxidative stress. We first confirmed that heat stress-induced oxidative stress damaged human spermatozoa by decreasing sperm motility and viability. Furthermore, the pretreatment of human spermatozoa by melatonin was able to alleviate such damage by suppressing sperm mitochondrial ROS generation, increasing mitochondrial membrane potential, reducing the formation of the lipid peroxidation product, 4-HNE, and reducing sperm DNA damage and apoptosis. Collectively, these findings suggest that melatonin is useful as a potential treatment option for male infertility caused by heat-induced oxidative stress.

Impact of silymarin on cadmium-induced apoptosis in human spermatozoa.

Oxidative stress-induced apoptosis in spermatozoa may lead to male infertility. Environmental pollutants and heavy metals such as cadmium cause harmful effects on the reproductive system and sperm parameters through the induction of oxidative stress. Silymarin, as a potent antioxidant, is able to inhibit oxidative stress. This study was performed to investigate the protective effects of silymarin on cadmium-induced toxicity in human spermatozoa. Sperm samples were divided into the following five groups: (a) spermatozoa at 0min, (b) spermatozoa in the control group, (c) spermatozoa treated with cadmium chloride (20μM), (d) spermatozoa treated with silymarin (2μM)+ cadmium chloride (20μM) and (e) spermatozoa treated with silymarin (2μM). Sperm parameters related to apoptosis, such as DNA fragmentation, nucleus diameter, mitochondrial membrane potential (MMP) and expression of caspase-3, were evaluated in all groups. After 180min, spermatozoa treated with cadmium chloride showed a significant decrease in nucleus diameter and MMP but a significant increase in DNA fragmentation; however, caspase-3 expression remained unchanged. At this time point, silymarin in the silymarin + cadmium chloride group could significantly reverse the adverse effects of cadmium chloride on these parameters.Silymarn could partly compensate for the caspase-independent apoptosis in the spermatozoa. Therefore, oxidative stress could be a consequence for cadmium toxicity.

The study of proteins expression as a marker of FAS and FasL apoptosis in human spermatozoa

The study of proteins expression as a marker of FAS and FasL apoptosis in human spermatozoa

Spermicidal and contraceptive potential of desgalactotigonin: a prospective alternative of nonoxynol-9.

Crude decoction of Chenopodium album seed showed spermicidal effect at MIC 2 mg/ml in earlier studies. Systematic isolation, characterization and evaluation revealed that the major metabolite Desgalactotigonin (DGT) is the most effective principle in both in vitro and in vivo studies. The in vitro studies comprises (a) rat and human sperm motility and immobilizing activity by Sander-Cramer assay; (b) sperm membrane integrity was observed by HOS test and electron microscopy; (c) microbial potential was examined in Lactobacillus broth culture, and (d) the hemolytic index was determined by using rat RBCs. The in vivo contraceptive efficacy was evaluated by intra uterine application of DGT in rat. Lipid peroxidation and induction of apoptosis by DGT on human spermatozoa were also studied. The minimum effective concentration (MEC) of DGT that induced instantaneous immobilization in vitro was 24.18 µM for rat and 58.03 µM for human spermatozoa. Microbial study indicated DGT to be friendly to Lactobacillus acidophilus. Implantation was prevented in DGT treated uterine horn while no hindrance occurred in the untreated contra lateral side. At the level of EC50, DGT induced apoptosis in human spermatozoa as determined by increased labeling with Annexin-V and decreased polarization of sperm mitochondria. Desgalactotigonin emerged 80 and 2×104 times more potent than the decoction and Nonoxynol-9 respectively. It possesses mechanism based detrimental action on both human and rat spermatozoa and spares lactobacilli and HeLa cells at MEC which proves its potential as a superior ingredient for the formulation of a contraceptive safer/compatible to vaginal microflora.

Molecular detection of Chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers.

This study was undertaken to determine the prevalence of Chlamydia trachomatis, Mycoplasmas, and Ureaplasmas in semen samples of the male partners of infertile couples and to investigate whether Chlamydia trachomatis could initiate apoptosis in human spermatozoa. A total of 85 males partners of infertile couples undergoing routine semen analysis according to World Health Organization guidelines were included. Specimens were examined for the presence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum and Ureaplasma parvum by Real time PCR (qPCR). Semen specimens were analysed for the appearance of apoptotic markers (sperm DNA fragmentation, activated caspase 3 levels, mitochondrial membrane potential (ΔΨm)) using flow cytometry. C. trachomatis, N. gonorrhoeae, U. urealyticum, M genitalium were detected in semen samples of 13 (15.2%), 5 (5.8%), 5 (5.8%) and 3 (3.5%) male partners of infertile couples, respectively. M. hominis and U. parvum were detected in semen sample of only one patient (1.1%). The semen of infertile men positive for C. trachomatis showed lower mean of semen count and lower rapid progressive motility (category [a]) of spermatozoa compared to uninfected men with statistically significances (p = 0.02 and p = 0.04, respectively). Flow cytometry analyses demonstrated a significant increase of the mean rate of semen with low ΔΨm and caspase 3 activation of infertile men positive for C. trachomatis compared to uninfected men (p = 0.006 and p = 0.001, respectively). DNA fragmentation was also increased in sperm of infertile men positive for C. trachomatis compared to uninfected men but without statistical significances (p = 0.62). Chlamydial infection was associated to loss of ΔΨm and caspase 3activation. Thus, C. trachomatis infection could be incriminated in apoptosis induction of spermatozoa. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.

The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa

The aim of this study was to investigate the relationship between seminal leukocytes, reactive oxygen species (ROS) production in the ejaculate, and markers of apoptosis in human spermatozoa. Semen samples were collected from 60 patients attending fertility clinics at the Reproductive Biology Unit at Tygerberg Academic Hospital and Vincent Pallotti Hospital, Cape Town, South Africa. The concentration of seminal leukocytes was determined and was correlated with ROS production in the ejaculate, the percentage of superoxide (·O2−)- and hydrogen peroxide (H2O2)-positive spermatozoa, glutathione activation in the ejaculate, and with markers of apoptosis in spermatozoa, namely cysteine-dependent aspartate-directed proteases (caspase)-3/7 activation, mitochondrial membrane potential (ΔΨm), and the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive sperm. Significant correlations with the concentration of seminal leukocytes were found for ROS production in the ejaculate, the percentage of ·O2−-positive spermatozoa, and caspase-3/7 activation in the ejaculate. Leukocytospermic samples showed significantly higher ROS production, percentage of ·O2−-positive sperm, GSH activation, and caspase-3/7 activation compared to non-leukocytospermic samples. The percentage of ·O2−-positive sperm was significantly correlated with sperm ΔΨm and caspase-3/7 activation in the ejaculate. Sperm ΔΨm and TUNEL-positive sperm did not correlate with seminal leukocyte concentration. Data demonstrate that high seminal leukocyte concentrations that leads to increased seminal ROS production, and is also associated with caspase activation in the male germ cell and increased mitochondrial ROS production. The latter could possibly be a result of disturbed ΔΨm. The activation of caspase-3/7 could then follow the increased intrinsic superoxide levels due to depleted intrinsic glutathione (GSH). These cellular events might not directly and immediately lead to DNA fragmentation as an endpoint of apoptosis because of topological hindrances.

Implication of apoptosis in sperm cryoinjury

Apoptosis is an ongoing physiological phenomenon that has been documented to play a role in male infertility, if deregulated. Caspase activation, externalization of phosphatidylserine, alteration of mitochondrial membrane potential and DNA fragmentation are markers of apoptosis found in ejaculated human spermatozoa. These markers appear in excess in subfertile men and functionally incompetent spermatozoa. Sperm cryopreservation is a widely used procedure in the context of assisted reproductive techniques. Cryopreservation and thawing is a procedure that inflicts irreversible injury on human spermatozoa. The damage is manifested by a decrease in recovery of viable spermatozoa with optimum fertilization potential. This review describes the implication of apoptosis as one of the possible mechanisms involved in sperm cryoinjury. Evidence shows significant increase in some apoptosis markers following cryopreservation and thawing. On the other hand, the increase in sperm DNA fragmentation following cryopreservation and thawing requires further investigation. Specific technical measures should be applied to minimize the induction of apoptosis in human spermatozoa during cryopreservation and thawing. These include standardization of freezing protocols and cryoprotectant use. Selection of non-apoptotic spermatozoa may also prove to be of benefit.

Assessment of sperm motility, viability and apoptosis in human spermatozoa after hydrogen peroxide exposure

ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2).DesignProspective study.Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence.ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). Male infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). DesignProspective study. Prospective study. Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. Semen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. Exposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage.

Immunomagnetic removal of cryo-damaged human spermatozoa

To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

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 degrees 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.

Assessment of spermatozoal caspases in oxidative stress mediated apoptosis

Reactive oxygen species (ROS) are involved in the pathophysiology of male infertility via a variety of mechanisms that include apoptosis. Oxidative stress is also associated with the activation of effector caspase-3, the main executioner caspase. However, data on ROS dependent caspase activation remain controversial. The aim of our study was to study caspase activation response following exposure to ROS inducers such as hydrogen peroxide (H2O2) hypochlorous acid (HOCl), and superoxide anion (SO•-). Prospective-controlled study. A total of 20 ejaculates from 20 healthy donors were collected after a sexual abstinence of 2 to 3 days. Washed spermatozoa were incubated at 37° with H2O2 (200 μmol/L) or HOCl (10-3 mol/L) for 1 hour or 5 mM of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) for 3 hours to induce SO− generation. Control aliquots were incubated under identical conditions except that ROS inducer was replaced with PBS. Activated caspases- 3 level was measured using carboxyfluorescein derivatives (CaspaTag™, Purchase, NY) by flow cytometry. No significant increase in caspase-3 activation was seen following exposure to the 3 ROS inducers compared with controls: H2O2 (38.8 ± 16.8 vs. 33.3 ± 15, P > 0.05), HOCl (38.66 ±25.24 vs. 38.05 ± 9.85, P > 0.05), and NADPH (45.34 ± 33.34 vs. 39.3 ± 26.51, P > 0.05). Our results indicate that caspase-3 activation may not be the main pathway in oxidative stress pathophysiology. Oxidative stress-induced apoptosis in human spermatozoa may be occurring by other caspase-independent pathways.

Effects of age on DNA double-strand breaks and apoptosis in human sperm

Objective This study was designed to explore the relationship between men's age and DNA damage and apoptosis in human spermatozoa. Design Semen samples were collected from men between the ages of 20 and 57 years. Sperm DNA double-strand breaks were assessed using the neutral microgel electrophoresis (comet) assay, and apoptosis was estimated using the DNA diffusion assay. Setting Academic medical center. Patient(s) Sixty-six men aged 20 to 57 years were recruited from infertility laboratory and general populations and consented to donate a semen sample. Recruitment was determined by time and day of analysis; the only exclusions were for azoospermia, prostatitis, or prior cancer therapy. Intervention(s) None. Main outcome measure(s) DNA damage and apoptosis in human sperm. Result(s) Age correlated with an increasing percentage of sperm with highly damaged DNA (range: 0–83%) and tended to inversely correlate with percentage of apoptotic sperm (range: 0.3%–23%). For example, percentage of sperm with highly damaged DNA, comet extent, DNA break number, and other comet measures was statistically significantly higher in men aged 36–57 years than in those aged 20–35 years, but percentage apoptosis was statistically significantly lower in the older group. Semen analysis showed percentage motility to be significantly higher in younger age groups. Conclusion(s) This study clearly demonstrates an increase in sperm double-stranded DNA breaks with age. Our findings also suggest for the first time an age-related decrease in human sperm apoptosis. These novel findings may indicate deterioration of healthy sperm cell selection process with age.

Porins and lipopolysaccharide induce apoptosis in human spermatozoa.

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.

Measuring apoptosis in human spermatozoa: a biological assay for semen quality?

Objective: [1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures expression of phosphatidylserine on the outer leaflet of the cell membrane, or the TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP [deoxy-uridine triphosphate] nick end labeling) assay, which measures occurrence of DNA strand breaks and [2] to correlate the outcome with routine semen variables and the hypoosmotic swelling (HOS) test. Design: Pilot study and clinical trial. Setting: Large teaching hospital and fertility center. Patient(s): Men whose semen was studied for various reasons. Main Outcome Measure(s): Percentage of apoptotic spermatozoa by two different assays, percentage of necrotic spermatozoa, concentration and motility of spermatozoa, and outcome of the HOS test. Result(s): Apoptosis can be measured in spermatozoa by flow cytometry using the Annexin V assay and the TUNEL assay. Twenty percent of spermatozoa were apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm concentration ( r = −0.389; P<.05) and motility ( r = −0.289; P<.05). A highly significant inverse correlation was seen between DNA double-strand breaks (TUNEL assay) and sperm concentration ( r = −0.629; P<.0001). Conclusion(s): Flow cytometry can easily and reliably detect phosphatidylserine expression on the outer leaflet of the cell membrane and DNA strand breaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spermatozoa are apoptotic, and the concentration of spermatozoa is lower in men with more apoptotic spermatozoa.