pyrimidinones and Infertility--Male

M540 bodies are membrane-surrounded round bodies occurring in semen of sub-fertile men. They appear variable in size and density, virtually devoided of chromatinic material and especially frequent in oligoasthenoteratozoospermic patients. Up to now, data collected by our group suggest that they may be apoptotic bodies that somehow escaped from testicular/epididymal phagocytosis. Indeed, they promptly stain with Merocyanine 540, a probe detecting the changes occurring in the membrane of apoptotic cells. In addition, they exhibit many of the apoptotic markers occurring in testicular apoptosis, including caspases activity, Fas receptor, p53 and DNA fragmentation (the latter detected by TUNEL assay). Due to the similarity in size and density between head sperm and M540 bodies, traditional protocols of sample preparation fail to yield sperm population completely free of M540 bodies, except for swim-up selection. In flow cytometry, it is possible to distinguish sperm from bodies by labelling samples with nuclear probes, because the latter fail to stain M540 bodies. Occurrence of M540 bodies in semen has been revealed only recently, at least in quantitative studies, and ,hence, many flow cytometric studies have not accounted for them. We summarise two studies (one on sperm ubiquitination and another on sperm DNA fragmentation) in which flow cytometric analyses were conducted both including and excluding M540 bodies from the sperm population. We found that in both cases M540 bodies largely affected the results. The study on sperm ubiquitination reveals that the direct correlation between sperm ubiquitination and good semen parameters is unmasked only after exclusion of M540 bodies from the analysis. The study on sperm DNA fragmentation shows that the amount of DNA damage in sub-fertile patients is more dramatic than expected from past investigations that included M540 bodies in the analysis.

Topics: Apoptosis; Biomarkers; Cell Separation; DNA Fragmentation; Flow Cytometry; Fluorescent Dyes; Humans; Infertility, Male; Male; Pyrimidinones; Semen; Spermatozoa; Staining and Labeling; Ubiquitin

To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa.. Case-control, prospective study.. Academic centers.. Fertile and subfertile subjects.. Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies.. Presence of M540 apoptotic bodies.. Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups.. This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

Topics: Adult; Annexin A5; Apoptosis; Biomarkers; Case-Control Studies; Cross-Sectional Studies; DNA Fragmentation; Ejaculation; Flow Cytometry; Fluorescent Dyes; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Prospective Studies; Pyrimidinones; Spermatozoa

Topics: Apoptosis; Flow Cytometry; Humans; Infertility, Male; Male; Pyrimidinones; Spermatozoa

Topics: Apoptosis; Flow Cytometry; Humans; Infertility, Male; Male; Pyrimidinones; Spermatozoa

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Topics: Adult; Bacteroides Infections; Benzimidazoles; Carbocyanines; Cell Membrane; Cell Survival; Escherichia coli Infections; Humans; Infertility, Male; Male; Mitochondria; Pyrimidinones; Spermatozoa; Staphylococcal Infections; Staphylococcus haemolyticus

The packaging of heterochromatin during spermatogenesis has been correlated with the expression of residual apoptotic bodies (which stain with merocyanine A) that will impact on sperm function in the fertilization process; as well as the joint expression of the transmembrane translocation phosphatidyl serine and oligonucleosomes.. To evaluate the expression of bodies stained with merocyanine in the functional processes of sperm and their level of agreement with apoptotic Annexin V and TUNEL biomarkers.. We performed a prospective, cross, including 11,000 cells belonging to semen samples from infertile men, were evaluated according to WHO criteria (1999), bounded by the lines of Tygerberg. The biomolecular transformation processing of the membrane and the expression of oligonucleosomes in the terminal cascade of apoptosis were quantified by cytometry flow, using an argon lasser as a reading source of 480 nm, discriminating the degree of cellularity, both negative and positive for each indicators.. Because of the study design was found low quantification in semen parameters, motility, morphology and sperm concentration. The average expression of cells [DNA-PI(+) / dUTP-FITC(+)] (quantification of TUNEL) and [Annexin-V(+) / PI(-)] was 36.5 +/- 17.4% and 31.2 +/- 17.4%, respectively. By comparing the expression of TUNEL without the effect of M540 bodies (36.3 +/- 1.7% vs. 36 +/- 1.7%) a significant difference was not determined.. This study shows that there is a remnant of the primary processes of spermiation, which can take an important role in apoptotic and functional processes of the sperm. However, its expression does not affect measurement of biomarkers of apoptosis seminal, whose determination changed the diagnosis and functional perception of reproductive parameters in the sperm.

Topics: Annexin A5; Apoptosis; Artifacts; Awards and Prizes; Cross-Sectional Studies; DNA Fragmentation; False Positive Reactions; Fertilization; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluorometry; Gynecology; Heterochromatin; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Membrane Lipids; Mexico; Nucleosomes; Obstetrics; Phosphatidylserines; Prospective Studies; Pyrimidinones; Sperm Motility; Spermatozoa; Staining and Labeling