Cumene hydroperoxide induced changes in oxidation-reduction potential in fresh and frozen seminal ejaculates.

Oxidation-reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (reactive oxygen species-ROS) and reductants (antioxidants) that reflects oxidative stress in a biological system. This study measures ORP and evaluates the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP in fresh and frozen semen using the MiOXSYS Analyzer. Semen samples from healthy donors (n = 20) were collected and evaluated for sperm parameters. All samples were then flash-frozen at -80°C. Oxidative stress was induced by CH (5 and 50 µmoles/ml). Static ORP (sORP-(mV/106 sperm/ml) and capacity ORP (cORP-µC/106 sperm/ml) were measured in all samples before and after freezing. All values are reported as mean ± SEM. Both 5 and 50 µmoles/ml of CH resulted in a significant decline in per cent motility compared to control in pre-freeze semen samples. The increase in both pre-freeze and post-thaw semen samples for sORP was higher in the controls than with 50 µmoles/ml of CH. The change from pre-freeze to post-thaw cORP was comparable. The system is a simple, sensitive and portable tool to measure the seminal ORP and its dynamic impact on sperm parameters in both fresh and frozen semen specimens.

Characterisation of pomegranate juice effects on human corpus cavernosum.

Pomegranate (POM) juice may benefit the erectile process, but the scientific evidence is lacking. This study evaluates the molecular characterisation and confirmation of POM's action on human corpus cavernosum (HCC) obtained from patients (n = 16) undergoing penile prosthesis implantation. After phenylephrine contraction, the relaxant effects of POM with various inhibitors in the presence and absence of palmitic acid (PA)-induced acute oxidative stress were investigated. Electrical field stimulation (EFS)- and acetylcholine (ACh)-induced relaxation were performed using organ bath preparation. Expression of neuronal nitric oxide synthase (nNOS), endothelial (eNOS), phosphodiesterase (PDE)-5A and cGMP levels were assessed in cells from ex vivo organ cultures of HCC, using RT-PCR, ELISA and immunohistochemistry techniques. POM induced marked relaxation of HCC (maximum response: 97.0 ± 3.1%) and reversed the PA-induced decrease of EFS (20 Hz). nNOS transcription was increased by 7-fold in POM-treated cells without influencing eNOS and PDE5A expressions. We conclude that POM induced marked relaxation of HCC via: (i) nNOS stimulation, and (ii) downstream relaxation stimulated by nNOS and cGMP and bypassing the NO and PDE5. This action provides a rationale for the therapeutic or preventative use of POM in men with erectile dysfunction who do not respond well to PDE5 inhibitors.

Effect of avanafil on rat and human corpus cavernosum.

We compared the activity of a new phosphodiesterase-5 inhibitor (PDE5i) avanafil with sildenafil and tadalafil in human and rat corpus cavernosum (CC) tissues. The effect of avanafil with several inhibitors and electrical field stimulation (EFS) was evaluated on CC after pre-contraction with phenylephrine. With the PDE5i, sildenafil and tadalafil, concentration-response curves were obtained and cyclic guanosine monophosphate (cGMP) levels were measured in tissues. Avanafil induced relaxation with maximum response of 74 ± 5% in human CC. This response was attenuated by NOS inhibitor and soluble guanylate cyclase (sGC) inhibitor. Avanafil potentiated relaxation responses to acetylcholine and EFS in human CC and enhanced SNP-induced relaxation and showed 3-fold increase in cGMP levels. When compared with sildenafil, avanafil and tadalafil were effective at lower concentrations in human CC. In addition, Sprague-Dawley rats underwent in vivo intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements. Avanafil increased ICP/MAP that was enhanced by SNP and cavernous nerve (CN) stimulation in rat CC tissues. Also avanafil showed maximum relaxation response of 83 ± 7% in rat CC with 3-fold increase in cGMP concentration. Taken together, these results of our in vivo and in vitro studies in human and rat suggest that avanafil promotes the CC relaxation and penile erection via NO-cGMP pathway.

Survey of patient and partner satisfaction following collagenase Clostridium histolyticum treatment for Peyronie's disease.

Intralesional injection of collagenase Clostridium histolyticum (CCH) is a minimally invasive, Food and Drug Administration-approved, effective treatment for Peyronie's disease (PD). To assess the satisfaction of patients and their female sexual partners (FSP) following CCH therapy for PD, we conducted a retrospective review of the records of all patients treated with CCH for PD between 04/2014 and 03/2016. Collected variables included demographics, pre- and post-treatment sexual function, penile curvature, penile vascular findings, and treatment outcomes. Patients and their FSPs were subsequently contacted by telephone and queried regarding their ability to have intercourse and their satisfaction with treatment. A total of 24 couples responded to our questionnaire and constitute the subjects of this analysis. Patient and FSP satisfaction with treatment were 67% and 71%, respectively. Significant predictors of FSP satisfaction with treatment included recall of penile trauma during prior sexual intercourse, improved ability to have sexual intercourse following treatment, and absence of post-procedural glans hypoesthesia. In conclusion, CCH imparts a significant benefit on a couple's sexual health. Partner satisfaction with treatment is correlated with improved ability to have sexual intercourse and absence of patient glans hypoesthesia.

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport.

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10-12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S-adenosyl-[methyl-3H]methionine resulted in the formation of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N]dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The Km for S-adenosylmethionine was 1.10(-4) M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4-5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na+-dependent uptake of either D-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.

The characterization, current medications, and promising therapeutics targets for premature ejaculation.

Premature ejaculation (PE) is the most prevalent male sexual dysfunction. This is associated with negative personal and interpersonal psychological outcomes. The pharmacologic treatment of PE includes the use of antidepressants, local anesthetic agents, and phosphodiesterase type 5 inhibitors. While numerous treatments can control PE, only antidepressants and topical anesthetic creams and sprays have recently been shown to be more effective. This review focuses on the physiology and pharmacology of ejaculation, the pathophysiology of PE and the most effective pharmacological treatment of PE. Pharmacotherapy of PE with off-label short-acting selective serotonin reuptake inhibitors (SSRIs) is common, effective, and safe. Dapoxetine, a SSRI with a short half-life, has been recently evaluated for the treatment of PE by several countries and results are promising. In clinical practice, follow-up side effects are an important part of the management strategy for PE. The understanding of etiology, pathophysiology, and treatment modalities of PE would be beneficial to clinician in helping patients with this disappointing sexual problem.

The direction and severity of penile curvature does not have an impact on concomitant vasculogenic erectile dysfunction in patients with Peyronie's disease.

Although the association between Peyronie's disease (PD) and erectile dysfunction (ED) is well established, limited data are available correlating penile curvature and penile hemodynamic parameters. We sought to examine this association in a cohort of PD men undergoing penile duplex Doppler ultrasound (PDDU). PD patients were retrospectively evaluated to correlate the extent and direction of penile curvature with measured vascular parameters. Demographic variables, disease characteristics and PDDU parameters were tabulated and statistically compared based on extent (≤ 45° and >45°) and direction (dorsal, ventral, lateral, ventrolateral, dorsolateral) of curvature. A total of 220 PD patients (mean age of 55.0 ± 9.2 years) underwent PDDU at one institution from January 2008 to December 2010. Overall, 69.5% of patients were found to have vasculogenic ED (arterial insufficiency (AI): 10%; veno-occlusive dysfunction (VOD): 43.2%; AI + VOD: 16.4%). Mean curvature was similar among all PDDU groups (AI: 41.7 ± 5.2°; VOD: 41.3 ± 2.5°; AI+VOD: 37 ± 4.1°; no-ED: 37.3 ± 3°; P > 0.85). No significant differences were noted in the presence or type of ED among various directions of curvature (P = 0.34) or when curvatures were stratified by ≤ 45° and >45°. The direction and extent of penile curvature are not associated with altered rates of vasculogenic ED on PDDU in PD patients.

Failure to attain stretched penile length after intracavernosal injection of a vasodilator agent is predictive of veno-occlusive dysfunction on penile duplex Doppler ultrasonography.

Penile duplex Doppler ultrasound (PDDU) assesses the etiology of erectile dysfunction. Peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI) are common PDDU parameters. We assessed whether stretched penile length (SPL) in the flaccid state and measured penile length at peak erection after intracavernosal injection (ICI) of a vasodilator during PDDU correlated with the etiology of erectile dysfunction. We performed a retrospective review of 93 patients who underwent PDDU for erectile dysfunction. Normal and stretched penile length were measured, both at a flaccid state prior to ICI and at peak erection during PDDU. Collected data included patient demographics, vascular, and anatomic parameters. The mean age was 52 years. SPL was equivalent to peak penile length after ICI in 60 patients (65%, group 1) and did not match in 33 (35%, group 2). There were no significant differences between the two groups in terms of flaccid, stretched, and post-ICI erect penile lengths, IIEF score, PSV, percent rigidity or tumescence, and vasodilator dose used. Patients in group 2 had less of a change in penile length from flaccid to erect state (36% vs. 44%, p = 0.02), higher EDV (12.0 vs. 8.5, p = 0.041), lower RI (0.6 vs. 1.0, p = 0.046), and more veno-occlusive dysfunction (82% vs. 53%, p = 0.001). On multivariate analysis, failure to reach maximum SPL at peak ICI erection (OR 2.255, CI 1.191-4.271, p = 0.0126), EDV (OR 1.281, CI 1.115-1.471, p < 0.001) and RI (OR 0.694, CI 0.573-0.723, p = 0.009) predicted veno-occlusive dysfunction. Failure to reach maximal SPL during PDDU using ICI with a vasodilator agent predicted veno-occlusive dysfunction, which is independent of both penile rigidity and tumescence. This measurement could serve as another diagnostic tool for predicting veno-occlusive dysfunction when PDDU is not readily available. Limitations include the subjective nature of penile measurements and different PGE1 doses used.

Differential expression of novel biomarkers (TLR-2, TLR-4, COX-2, and Nrf-2) of inflammation and oxidative stress in semen of leukocytospermia patients.

Chronic genitourinary inflammation results in Leukocytospermia (LCS), an elevated number of white blood cells (WBCs) in semen, which, in association with oxidative stress, may suppress sperm function, and manifest as male factor infertility. The current clinical diagnosis of LCS employs manual enumeration of WBCs and requires complex staining and laboratory skills or measurement of inflammatory cytokines and chemokines levels. Many patients with idiopathic infertility are asymptomatic. In search of better inflammatory markers for LCS, we evaluated expression of toll-like receptors 2 and 4 (TLR-2/4), cyclooxygenase-2 (COX-2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) in semen samples of age-matched infertile patients with and without LCS. We employed the usage of specific Western blot evaluation, cytokine array; immunofluorescence microscopy (IFM) followed by computer-based analysis, and other molecular approaches. As compared with non-LCS patients (n = 38), semen samples from LCS patients (n = 47) displayed significantly lower total sperm count (p < 0.01), motility (p < 0.0001), normal head count (p < 0.0001), and a significantly higher white blood cell count (p < 0.0001). Differential cytokine profiling of seminal plasma by antibody array revealed up-regulation of several pro-inflammatory chemokines in LCS samples. Western blot analysis of LCS seminal plasma (n = 15) also showed a significant increase in expression of TLR-2 (p < 0.001) and 4 (p < 0.01), COX-2 (p < 0.001), and Nrf-2 (p < 0.001) as compared with semen samples from non-LCS patients (n = 15). Computer-based objective IFM analysis of spermatozoa from LCS patients showed increased expression of TLR-4 (p < 0.001), Cox-2 (p < 0.01), and (Nrf-2) (p < 0.01). Significant differences in the subcellular localization of these proteins were evident in the sperm head and tail segments of LCS samples. Altogether, these observations suggest that TLR-2/4, COX-2, and Nrf-2 can serve as novel biomarkers of inflammation and oxidative stress. Therefore, developing a rapid assay for these biomarkers may facilitate early diagnosis and management of LCS especially in idiopathic and asymptomatic male infertility patients.

In vitro inhibition of testosterone biosynthesis by ketoconazole.

Oral ketoconazole has been demonstrated to lower plasma testosterone in man. Measurement of blood precursors of testosterone suggest that ketoconazole may have its effect inhibiting the 17,20-desmolase enzyme within the testis. To substantiate this, a series of in vitro experiments was conducted using the rat testis to determine where in the testosterone biosynthetic pathway ketoconazole has its effect. To accomplish this, an assay system to measure 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase activities involved in the delta 4-testosterone biosynthetic pathway was developed. It was demonstrated from dose-response and time-course experiments that a dose of approximately 10 micrograms/ml ketoconazole was sufficient to inhibit in vitro testicular steroidogenesis. Using dosages between 10 and 300 micrograms/ml ketoconazole, a marked inhibition of both the 17 alpha-hydroxylase and the 17,20-desmolase activities occurred. Ketoconazole under these conditions had no effect on 17 beta-hydroxysteroid dehydrogenase activity. Ketoconazole also inhibited the increased activity of these enzymes induced by hCG (1 IU). These data confirm the observation that in vitro ketoconazole has a direct inhibitory effect on 17,20-desmolase activity. These results further suggest that ketoconazole has more than one site of action in inhibiting testosterone biosynthesis in the testis and may indeed be a suitable agent for the treatment of patients with disseminated prostate cancer.

Cyclosporine inhibits testosterone biosynthesis in the rat testis.

To determine whether the immunosuppressive agent cyclosporine (CsA) has an effect on testicular androgen function in the male rat, four groups of adult animals were treated daily for 28 days with either orange juice or three doses of CsA (7.5, 15, or 30 mg/kg X day). Twenty-four hours after the last dose of CsA, the animals were killed and the following parameters were measured: testis, seminal vesicle, and ventral prostate weights; serum levels of CsA, creatinine, testosterone (T), and LH; and intratesticular levels of pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and T. There were no differences between the control (orange juice) and the three CsA-treated groups with respect to serum creatinines and testis, seminal vesicle, and ventral prostate weights. Serum T decreased significantly in the 15 and 30 mg/kg CsA-treated groups. The intratesticular T level decreased significantly only in the 15 and 30 mg/kg CsA-treated groups. In the 15 and 30 mg/kg CsA-treated groups, the intratesticular pregnenolone, progesterone, and 17 alpha-hydroxyprogesterone levels all showed a significant decline compared to controls. There was no significant change in the androstenedione levels in any of the CsA-treated groups. To determine whether the decrease in T production by the testis exposed to CsA is via inhibition of the hypothalamic-pituitary axis, serum LH was measured in all four groups. Serum LH decreased significantly only in the 15 and 30 mg/kg CsA-treated groups. These data suggest that oral CsA administration in doses greater than 15 mg/kg X day results in diminished intratesticular T production that appears to be mediated via inhibition of pituitary LH function.

Effects of cyclosporine on the hypothalamic-pituitary-gonadal axis in the male rat: mechanism of action.

In the intact adult male rat, cyclosporine (CsA) induces a significant decrease in serum testosterone (T), serum LH, and intratesticular T. To elucidate the mechanism of action of this CsA-induced hypogonadotropic hypogonadism, castrated male rats were treated with oral CsA (30 mg/kg.day) or vehicle alone, and serum LH was measured after 1, 2, and 4 weeks of treatment. Surprisingly, serum LH was higher in these CsA-treated castrated rats at 1, 2, and 4 weeks than in castrated controls. To provide insight into the cause of this increase in serum LH, a T implant (8 mm) was inserted (at week 5 of treatment) in both CsA-treated and control castrated animals, and serum LH was measured 1 week after insertion of the T implant. Serum LH levels decreased to control values after insertion of the T implant. Subsequently, two other groups of rats received 8-mm T implant at the time of castration and were then treated up to 4 weeks with either CsA or vehicle alone. Serum LH showed a significant decrease in these CsA- plus T-treated animals compared to the vehicle- plus T-treated animals. A GnRH stimulation test performed after 2 weeks of CsA/vehicle treatment showed a significant increase in serum LH in all the rats 30 min after GnRH administration (1, 10, 30, and 100 ng/100 g BW, ip), indicating a normal pituitary response. The increase in LH after exogenous GnRH treatment was more significant in CsA-treated intact rats than in the controls. There was no difference in creatinine clearance between intact and castrate T-treated rats regardless of whether they received CsA. These studies indicate that the hypogonadotropic hypogonadism induced by CsA in intact male rats is mediated through the hypothalamic-pituitary axis, primarily at its hypothalamus end, does not seem to be due to the nephrotoxicity of CsA, and is modulated by T and/or its metabolites.

Lack of a direct effect of gonadotropin hormone-releasing hormone agonist on human testicular steroidogenesis.

In an attempt to determine whether the chronic administration of GnRH agonist (GnRH-A) has a direct inhibitory effect on testicular steroidogenesis in the human, the testes of four men with disseminated prostatic cancer who were treated with GnRH-A daily for at least 1 yr were assayed for intratesticular pregnenolone (5-pregnen-3 beta-ol-20-one), progesterone, dehydroepiandrosterone, 17 alpha-hydroxypregnenolone (5-pregnen-3 beta 17 alpha-diol-20-one), 17 alpha-hydroxyprogesterone, androstenedione, and testosterone (T). In addition, testicular 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzyme activities of the delta 4 pathway were measured. These intratesticular steroids and enzyme activities from four GnRH-A-treated patients were compared to those in five men (controls) who were orchiectomized as the primary treatment for their disseminated prostatic cancer and in three other men who were treated for 3-12 months with GnRH-A daily but received, in addition to the daily GnRH-A, 1000 IUhCG, im, every other day for 3 days immediately before their salvage orchiectomy, which was performed when their disease progressed. In the control group, the delta 5-steroids, particularly dehydroepiandrosterone and pregnenolone, represented the majority of the intratesticular steroids. Compared to control values, all intratesticular steroids except delta 4-P (for which there was no difference) were significantly lowered by treatment with GnRH-A. Intratesticular T was reduced by 98% from 328 +/- 139 (+/- SEM) ng/g testis in the control group to 8 +/- 3 in the GnRH-A-treated group (P less than 0.01). The additional treatment with hCG for 3 days in the GnRH-A-treated group reversed the inhibition of all steroids to either control or above control levels, with intratesticular T rising to 1144 +/- 273 ng/g testis. A similar trend was found for all three enzymatic activities, i.e., GnRH-A alone inhibited each of the enzymatic activities, whereas the addition of hCG reversed this inhibition by GnRH-A. These data indicate that the chronic administration of GnRH-A to elderly men results in inhibition in both the delta 4 and delta 5 pathways, with a subsequent decrease in the intratesticular T concentration. The ability of exogenous hCG to reverse both the reduction in delta 4 and delta 5 intratesticular steroid content and the intratesticular enzyme activities induced by GnRH-A treatment supports the concept that GnRH-A does not have a direct inhibitory effect on testicular T biosynthesis.

Mechanism of inhibition of human testicular steroidogenesis by oral ketoconazole.

To determine the antisteroidogenic effect of ketoconazole (KTZ) in the human testis, we measured the plasma delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, dehydroepiandrosterone (DHEA), progesterone, 17 alpha-hydroxyprogesterone, androstenedione (A), and testosterone (T) concentrations in three men with previously untreated metastatic prostate cancer at various time intervals for 24 h before and 48 h after the administration of 200 mg oral KTZ every 8 h. The adrenal glands of these three patients were suppressed (as measured by the plasma cortisol levels) by the administration of 1.0 mg dexamethasone daily for 7 days before and during the study. After six doses of KTZ, bilateral orchiectomy was performed, and the intratesticular concentration of the aforementioned seven steroids and the intratesticular activities of the 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzymes in the delta 4-steroidogenic pathway were determined. These seven intratesticular steroids and three intratesticular enzyme activities were compared to those in five men with previously untreated prostate cancer who underwent orchiectomy as primary treatment for their disease. Plasma A, DHEA, and T all significantly decreased during KTZ therapy. There was no significant change in the other four steroids in the plasma. In the testis, delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, and delta 4-17 alpha-hydroxyprogesterone were all significantly elevated, whereas intratesticular DHEA, A, and T were significantly decreased in the three KTZ-treated patients compared to levels in the five non-KTZ-treated patients. Measurement of the enzyme activities demonstrated a significant reduction in both 17 alpha-hydroxylase and 17,20-desmolase, but no change in 17 beta-hydroxysteroid dehydrogenase, in the KTZ-treated patients compared to the levels in the non-KTZ-treated patients. We conclude that oral KTZ decreases testicular T production by inhibiting the 17,20-desmolase and also the 17 alpha-hydroxylase steps in both the delta 4- and delta 5-T biosynthetic pathways.

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.

Relative impact of oxidative stress on male reproductive function.

Impairment of normal spermatogenesis and sperm function are the most common causes of male factor infertility. Abnormal sperm function is difficult to evaluate and treat. There is a lack of understanding of the factors contributing to normal and abnormal sperm function leading to infertility. Many recent studies indicate that oxygen-derived free radicals induce damage to spermatozoa. The excessive generation of these reactive oxygen species (superoxide, hydroxyl, nitric oxide, peroxide, peroxynitrile) by immature and abnormal spermatozoa and by contaminating leukocytes associated with genitourinary tract inflammation have been identified with idiopathic male infertility. Mammalian spermatozoa membranes are rich in polyunsaturated fatty acids. This makes them very susceptible to oxygen-induced damage, which is mediated by lipid peroxidation. In a normal situation, the antioxidant mechanisms present in the reproductive tissues and their secretions are likely to quench these reactive oxygen species (ROS) and protect against oxidative damage to gonadal cells and mature spermatozoa. During chronic disease states, aging, toxin exposure, or genitourinary infection/inflammation, these cellular antioxidant mechanisms downplay and create a situation called oxidative stress. Thus, a balance between ROS generation and antioxidant capacity plays a critical role in the pathophysiology of disease state. Recent efforts towards the development of new reliable assays to evaluate this oxidative stress status have resulted in the establishment of ROS-TAC score. Such assessment of oxidative stress status (OSS) may help in designing newer modes of male factor infertility treatment by suitable antioxidants.

Oxidative stress and role of antioxidants in normal and abnormal sperm function.

Defective sperm function is the most common cause of infertility, and until recently, it was difficult to evaluate and treat. Part of this difficulty was due to our incomplete understanding of the factors contributing to normal and abnormal sperm function leading to male infertility. Mammalian spermatozoa membranes are rich in high unsaturated fatty acids and are sensitive to oxygen induced damage mediated by lipid peroxidation. Limited endogenous mechanisms exist to reverse these damages. The excessive generation of reactive oxygen species (ROS) by abnormal spermatozoa and by contaminating leukocytes (leukocytospermia) has been identified as one of the few defined etiologies for male infertility. In a normal situation, the seminal plasma contains antioxidant mechanisms which are likely to quench these ROS and protect against any likely damage to spermatozoa. However, during genitourinary infection/inflammation these antioxidant mechanisms may downplay and create a situation called oxidative stress. In addition, aging and environmental toxicants are also likely to further induce this oxidative stress. Assessment of such oxidative stress status (OSS) may help in the medical treatment of this male factor infertility by suitable antioxidants.

Effect of cyclosporine on steroidogenesis in rat Leydig cells.

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.

Reversibility of cyclosporine-induced hypoandrogenism in rats.

In an attempt to determine whether the inhibition of the hypothalamic-pituitary-testicular axis induced by oral cyclosporine (CsA) is reversible, intact adult male rats were treated with 30 mg/kg oral CsA daily for 4 weeks, and then vehicle (orange juice) for the next 4 weeks. A second group of animals (control) was fed orange juice throughout the entire 8 weeks of the experiment. Serum testosterone (T) was decreased significantly (P less than 0.01) after 4 weeks of CsA treatment when compared with controls. After cessation of oral CsA for the next 4 weeks, there was no difference in serum T between the control and CsA-treated groups. Serum LH, intratesticular T, ventral prostate (VP) and seminal vesicle (SV) weights paralleled the serum T levels at 4 and 8 weeks--i.e., all values were decreased in the 4-week CsA-treated group when compared with controls, and these returned to normal at 8 weeks. Intratesticular 17 alpha-hydroxylase and 17,20-desmolase activities were significantly lower after 4 weeks of CsA treatment; following cessation of the CsA, these enzymatic values returned to normal within 4 weeks. These data demonstrate that at the duration of treatment and the dose studied, the CsA-induced inhibition of the hypothalamic-pituitary-testicular axis of the intact adult rat is completely reversible.

In vivo gene expression profile analysis of metallothionein in renal cell carcinoma.

The antiapoptotic and mitogenic responses of metallothionein (MT) have been well documented in vitro. While MT protein overexpression, frequently encountered in a number of human primary tumors, has been shown to be correlated with disease progression, little information is available on the in vivo isoform expression of MT. In this study we have demonstrated the occurrence of MT proteins and further defined their differential expression profile in human primary renal cell carcinoma (RCC). Pooled normal human kidney RNA and paired biopsy specimens (tumor and control) obtained from 11 patients diagnosed with RCC with tumor grade ranging from 1-3 and a pathological staging of T2-T3 (N0M0) were used for the study. Samples were analyzed for the presence of MT protein using immunohistochemical (IHC) analysis and for MT isoform-specific mRNA expression by reverse transcriptase polymerase chain reaction. Metallothionein protein assumed both cytoplasmic and nuclear staining in cancer cells and was detected in eight of 11 samples (72%) with polyclonal antibodies. The immunoreactivity of MT protein, but not its cellular localization, in RCC specimens suggests a relationship between and advanced disease. While alterations in the basal level of expression of MT-1E, MT-1F and MT-1X genes remained unchanged, significant up-regulation of MT-2A and down-regulation of MT-1A and MT-1G transcripts was observed in RCC tissue specimens when compared with controls. Intriguingly, the paired RCC biopsy specimens had lower MT-1H transcripts than pooled normal human controls. We here provide the first report of the differential expression of MT isoforms in human RCC and that this data further support the role of MT-2A in tumorigenesis.