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 378C 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 (DCm) by JC-1 staining. Viability and DCm 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, peroxynitriteinduced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.

This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.

Mitochondrial membrane potential (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.

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.

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