Mahadevan Rajasekaran

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism.

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.

Oxidative stress and interleukins in seminal plasma during leukocytospermia

OBJECTIVEnTo quantify the levels of reactive oxygen species, superoxide dismutase (SOD), and interleukins (IL) 2 and 8 in seminal plasma of infertile patients as well as to examine the possible relationship between oxidative stress and proinflammatory cytokines.nnnDESIGNnSemen collected from normal fertile donors, infertile men without symptoms of genitourinary (GU) inflammation, and infertile men with symptoms of infection-inflammation of the GU tract was evaluated for the levels of granulocyte elastase, reactive oxygen species, SOD, IL-2, and IL-8. Any correlation between the levels of reactive oxygen species and other parameters in these population was analyzed statistically.nnnRESULTSnSignificantly high levels of granulocyte elastase (18.32 +/- 1.52 U/L), reactive oxygen species (6 x 10(5) cpm), IL-8 (3.7 +/- 0.10 microgram/L), and IL-2 (18.32 +/- 1.47 ng/L) were observed in semen of infertile patients with leukocytospermia compared with the other two groups. In leukocytospermic samples, the activity of SOD was significantly lower (624.89 +/- 41.16 NU/mL) compared with nonleukocytospermic samples (787.85 +/- 24.26 NU/mL) or fertile donors (816.29 +/- 50.16 NU/mL). A significant positive correlation was observed between the levels of reactive oxygen species and IL-8.nnnCONCLUSIONSnThese findings suggest that increased oxidative stress observed during leukocytospermia may modulate the level of proinflammatory cytokines. The increased oxidative stress may be due to a defect in the reactive oxygen species scavenging system.

EX VIVO EXPRESSION OF NITRIC OXIDE SYNTHASE ISOFORMS (eNOS/ iNOS) AND CALMODULIN IN HUMAN PENILE CAVERNOSAL CELLS

PURPOSEnNitric oxide (NO) synthesized by nitric oxide synthase (NOS) is recognized as the central mediator of penile erection. This process appears to be mediated mainly by neuronal NOS (nNOS), which is localized to the nonadrenergic, noncholinergic innervation of the penis. However, the role of non-neuronal penile constituents (specifically the cavernosal smooth muscle), as well as other NOS isoforms in NO production in the human penis is not well understood. The present study evaluates the expression of non-neuronal (inducible and endothelial) isoforms of NOS in human penile cavernosal smooth muscle cells in culture.nnnMATERIALS AND METHODSnPrimary culture was initiated with explants of human corpora cavernosa. For gene expression studies, total RNA was extracted from cavernosal cells and subjected to reverse transcriptase polymerase chain reaction (RT-PCR). For NADPH-diaphorase histochemistry, the cells were incubated with 1 mM beta-NADPH and 0.5 mM nitrobluetetrazolium at 37C for 3 hours. For indirect immunofluorescence and electron microscopy, cells were incubated overnight at 4C with specific primary (eNOS; calmodulin) and secondary antibodies. A conventional avidin biotin complex technique was used for electron microscopy.nnnRESULTSnThe mRNA expression studies revealed that these cells express both endothelial (eNOS) and inducible (iNOS) forms. Localization studies showed positive signals for NADPH-diaphorase, eNOS, and calmodulin. The electron microscopic evaluation confirmed the localization of eNOS to the cytoplasm and small vesicles in the cells.nnnCONCLUSIONSnThese findings support the hypothesis that human cavernosal smooth muscle cells express both endothelial and inducible forms of NOS, which may significantly contribute to NO production in the penile architecture during the erectile process.

POTENTIATION OF ERECTILE RESPONSE AND cAMP ACCUMULATION BY COMBINATION OF PROSTAGLANDIN E1 AND ROLIPRAM, A SELECTIVE INHIBITOR OF THE TYPE 4 PHOSPHODIESTERASE (PDE 4)

PURPOSEnPhosphodiesterases (PDEs) are an important component of the signal transduction pathway during the erectile response. To determine the PDE isoforms in the corpora cavernosa in the cat and to establish the functional presence of PDE 4 in human cavernosal tissue, the erectile response to intracavernosal phosphodiesterase (PDE) inhibitors alone and the combination of PDE inhibitors and prostaglandin E1 (PGE1) was evaluated in the anesthetized cat. The in vitro formation of cAMP and cGMP in human cavernosal smooth muscle cells (HCSMCs) treated with PGE1 and rolipram in primary culture was also measured.nnnMATERIALS AND METHODSnIn pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal injections of (i) the type 3 cAMP-specific, cGMP-inhibitable PDE inhibitor, milrinone, (ii) the type 4 cAMP-specific PDE inhibitor, rolipram, (iii) the type 5 cGMP-specific PDE inhibitor, zaprinast, and (iv) the combination of rolipram and PGE1. Systemic arterial pressure was concurrently assessed in these experiments. All responses to PDE inhibitors were compared with a control triple-drug combination comprised of papaverine (1.65 mg.), PGE1 (0.5 microg.), and phentolamine (25 microg.). HCSMCs were incubated with PGE1 (3 microM) and rolipram (10 microM) individually or in combination up to 2 hours at 37C. The intracellular cAMP and cGMP was extracted by cold absolute ethanol and measured (pmol./10(6) cells) by a commercially available EIA kit.nnnRESULTSnMilrinone (3 to 100 microg.), rolipram (3 to 100 microg.), and zaprinast (3 to 100 microg.) induced dose-dependent increases in intracavernosal pressure and penile length (p <0.05) when administered intracavernosally. The maximum increase in cavernosal pressure in response to zaprinast was associated with no significant change in systemic arterial pressure. When rolipram was combined with PGE1 (0.1 microg.), the increases in intracavernosal pressure and the duration of erectile response were significantly higher (p <0.05) and longer (p <0.05) than those observed when rolipram alone was injected intracavernosally. PGE1 (3 microM) and rolipram (10 microM) produced significant increases (p <0.05) in the accumulation of intracellular cAMP levels in HCSMCs in primary culture above those of the baseline values while intracellular levels of cGMP did not change.nnnCONCLUSIONSnPDE inhibitors administered intracavernosally caused dose-dependent increases in cavernosal pressure in the cat. When a specific cAMP PDE inhibitor was combined with PGE1, the erectile response was enhanced and intracellular levels of cAMP were increased in HCSMCs in primary culture. These data suggest further exploration of the combination of various PDE inhibitors and PGE1 in the pharmacologic treatment of erectile dysfunction and provide functional evidence for the presence of PDE 4 isoenzyme in human penile cavernosal cells.

Spermicidal activity of an antifungal saponin obtained from the tropical herb Mollugo pentaphylla

The ethyl acetate fraction of Mollugo pentaphylla, a tropical herb, contains an antifungal saponin (mollugogenol-A). We report here the spermicidal effects of this saponin. Washed sperm (> 100 x 10(6) with > 50% motility) from normal volunteers were incubated with varying concentrations (0-300 micrograms/ml) of mollugogenol-A at 30 degrees C. Sperm motility, velocity and viability were assessed at 0, 30, 60 minutes both manually and by using computer assisted semen analysis (CASA). Samples collected at 0 and 60 minutes were evaluated for membrane lipid peroxidation, superoxide dismutase (SOD) activity and transmission electron microscopy. A dose- and time-dependent effect of this saponin on sperm motion and viability was observed. The maximal spermicidal effect (4-5 fold decrease in motility and viability) was observed with 300 micrograms/ml dose of saponin. A three-fold increase in sperm membrane lipid peroxidation with corresponding inhibition of SOD activity were observed after 60 minutes incubation with this spermicidal agent. Transmission electron microscopy of saponin-treated samples revealed significant damage to the sperm membrane in both head and tail regions, and the acrosomal membranes were notably swollen and disrupted. These results indicate that this natural saponin has a potential spermicidal effect besides its known antifungal activity. The likely mechanism of its action involves sperm membrane damage by increased lipid peroxidation.

Quantitative Assessment of Cytokines (GROα and IL‐10) in Human Seminal Plasma During Genitourinary Inflammation

PROBLEM: Mechanisms involved in infertility due to genitourinary (GU) inflammation are unknown. The production of pro‐inflammatory (GROα) and anti‐inflammatory (IL‐10) cytokines in seminal plasma is monitored in this study.

Sperm-damaging effects of electric current: possible role of free radicals

The generation of reactive oxygen species (ROS) and sperm damage was evaluated in (a) samples obtained during electroejaculation (EE) of men with spinal cord injury and (b) in electrolyzed HAMs F-10 medium subjected to electric current in vitro. Chemiluminescence data showed a significant increase in ROS in the ejaculates (6 x 10(7) photons/ml) collected immediately after EE and in the electrolyzed medium (3 to 7 x 10(6) photons/ml) when compared to the control (4 to 7 x 10(4) photons/ml). Incubation of normal human sperm with the electrolyzed medium resulted in a significant threefold decrease in percent motility and a twofold decrease in percent viability. Sperm subjected to direct electric stimulation in vitro exhibited a significant twofold decrease in percent motility and percent viability. Superoxide dismutase (SOD) activity decreased significantly in sperm subjected to direct electric current in comparison to the control or the sample incubated with electrolyzed medium. These studies indicate that in vitro and in vivo electrical stimulation generate reactive oxygen species and affect SOD activity, which in part are responsible for decreased sperm motion and viability.