Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa.

Mitochondrial membrane potential (ΔΨm) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC-1 has been widely used to assess sperm ΔΨm; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with ΔΨm. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm ΔΨm. The results showed that TMRM accurately detects sperm populations displaying either high or low ΔΨm. Moreover, TMRM was able to measure sperm ΔΨm under the experimental conditions in which JC-1 had previously presented difficulties. Differences in ΔΨm according to sperm and semen quality were properly detected and a positive correlation between ΔΨm and conventional semen parameters was observed. Finally, a positive correlation was found between the ΔΨm measurement by TMRM and by the widely used JC-1. In conclusion, TMRM is a simple, time-effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in ΔΨm with efficiency comparable to JC-1 without its limitations.

Nitrosative stress by peroxynitrite impairs ATP production in human spermatozoa.

The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS.

Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

STUDY HYPOTHESIS: Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? STUDY FINDING: Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. WHAT IS KNOWN ALREADY: MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study in male infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: The study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT (F.T.). The authors declare no conflict of interest.

Effect of mitochondrial calcium uniporter blocking on human spermatozoa.

Calcium (Ca(2+) ) regulates a number of essential processes in spermatozoa. Ca(2+) is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 µm Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer-aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence-based method, mitochondrial membrane potential (ΔΨm) using JC-1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.

Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.

Distinct isolates of uropathogenic Escherichia coli differentially affect human sperm parameters in vitro.

Sperm motility and vitality are decreased in male genital tract infection. Uropathogenic Escherichia coli (UPEC) are frequently associated with sperm parameter loss, but there are no reports to date regarding the effects of different E. coli isolates on human spermatozoa. The aim of this work was to compare the effect in vitro of different E. coli isolates on human sperm parameters. Normal spermatozoa were incubated with E. coli isolated from nine men with urinary tract infection. After 1 h of incubation, sperm motility, vitality and mitochondrial membrane potential (ΔΨm) were measured. The E. coli isolates were serotyped with specific antisera. Sperm motility was decreased with five of nine E. coli isolates. Two UPEC were typed as O6 strains, and they did not decrease sperm motility in the same experimental conditions as the other five isolates, despite the described high pathogenicity of the O6 strain in urogenital infections. Neither UPEC analysed affected vitality or ΔΨm. UPEC isolates were shown to be heterogeneous in their effects, suggesting the need to characterise the pattern defining the pathogenicity of E. coli on human spermatozoa.

Sperm membrane functionality in the dog assessed by flow cytometry.

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.

Effects of seminal fluid fractions on plasma and acrosome membrane integrity and mitochondrial membrane potential determined by flow cytometry in chilled canine spermatozoa.

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third[prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential(Δψm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm-rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5C for 72 h in egg yolk-TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT-SF) or third (EYT-PF) ejaculated fractions. After cold storage,groups EYT-SF and EYT-PF showed significantly higher percentages of sperm cells with an intact acrosome[68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1%respectively]. Only in EYT-SF was PS translocation significantly reduced compared to EYT-PF and EYT [3.9 ± 0.4%,10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT-SF and EYT-PF compared to EYT[36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in Δψm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.

[Intraprostatic BCG vaccine in patients with prostatic cancer. Expression of interferon gamma and interleukin 4 (a preliminary study)]. / Vacuna BCG intraprostática en pacientes con cáncer de próstata. Expresión de gamma interferón e interleukina 4. (Aportación preliminar).

UNLABELLED: The BCG treatment in the superficial bladder cancer is utilized since 1976. There are some animal experiences with BCG intraprostatic in dogs with prostatic cancer. GOAL: determine if insitu BCG vaccine increase the prostatic immunity in patients with prostatic cancer. MATERIAL AND METHOD: Three patients with D stage prostatic cancer where injected with 0.3-0.5 mg of BCG vaccine in the prostate. An histological picture where made in the some place before and after the injection. A RT-PCR study was made for gamma Interferon and Interleukin-4. RESULTS: In two patients we observed gamma interferon expression (67%) respect the control, this produce an increase of the cytotoxic T lymphocytes, natural killer cells and macrophagos, that have antitumoral action. One patient increased the interleukin-4 that means production of T helper lymphocytes and B lymphocytes. There wasn't complications. CONCLUSIONS: The BCG vaccine injected in prostatic tissue of patients with prostatic cancer, produces an increase of cytotoxic T lymphocytes and natural killer cells, with antitumoral action.

Vacuna bcg intraprostática en pacientes con cáncer de próstata. Expresión de gamma interferón e interleukina 4. (Aportación preliminar) / Intraprostatic BCG vaccine in patients with prostatic cancer, expression of Interferon gamma and Interleukin 4

El tratamiento BCG en el cáncer superficial de vejiga es utilizado desde 1976. Hay algunas experiencias en animales referente a producción de necrosis del tumor, inyectando BCG intraprostático en perros con cáncer de próstata. Objetivo: Determinar si la vacuna BCG in situ, produce aumento de inmunidad en la próstata de pacientes portadores de adenocarcinoma prostático. MATERIAL Y MÉTODO : A tres pacientes portadores de cáncer prostático etapa D2, se les realizó biopsia prostática del lóbulo derecho para obtener tejido de control, luego se inoculó por vía transrectal vacuna BCG en dosis de 0,3 a 0,5 mg en el mismo lóbulo prostático y posteriormente a los 15 días se extrajo nueva biopsia del lóbulo inyectado. Se realizó estudio RT-PCR para expresión de gamma interferón e interleukina-4 en las muestras pre y post BCG de cada paciente en estudio. RESULTADOS: Se demostró expresión de gamma interferón por el RNA mensajero en dos pacientes (67 per cent), con respecto al control pre-BCG, lo cual significa un aumento en la activación de linfocitos T citotóxicos, de las células natural killer y macrófagos, que tienen acción antitumoral. En un paciente se demostró expresión de interleukina-4 (33 per cent) en relación al control, lo cual significa producción de linfocitos T helper y linfocitos B con producción de anticuerpos inespecíficos. No hubo complicaciones. CONCLUSIONES: La vacuna BCG inyectada al tejido prostático de pacientes con cáncer prostático, produce estimulación inmunológica, especialmente de linfocitos T citotóxicos activados y células natural killer, los cuales tienen acción antitumoral, y de moléculas del complejo principal de histocompatibilidad tipo II, que permite que lo anterior se lleve a cabo. Mayores estudios se requieren para determinar si con esta respuesta se logrará en el futuro, eliminar o frenar, un cáncer prostático inicial, en reemplazo de la cirugía radical (AU)