Fanuel Lampiao

The in vitro effects of melatonin on human sperm function and its scavenging activities on NO and ROS

Various systems of antioxidants exist endogenously in the body to help protect it against free radical damage by scavenging excessive ROS and RNS. Melatonin, a hormone secreted by the pineal gland, and responsible for controlling the circadian rhythm, is one such endogenous antioxidant. Melatonin has been reported to be present in human seminal fluid, but its antioxidant activities in semen are rather contradictory. This study aimed at establishing the effects of melatonin treatment on human spermatozoa. Spermatozoa were incubated with 2 mm melatonin (120 min, 37 °C, 5% CO2) after which motility parameters were measured by computer aided motility analysis, while cell viability (PI), intracellular NO (DAF‐2/DA) and ROS (DCFH‐DA) were assessed using flow cytometry. In vitro melatonin treated samples (n = 12) showed a significantly higher percentage of motile, progressive motile and rapid cells, while simultaneously reducing the number of nonviable spermatozoa when compared with the control. Endogenous NO was significantly decreased, but no effect was observed on ROS levels. From these results, it can be concluded that melatonin was able to directly or indirectly scavenge NO, as indicated by the reduction in 4,5‐diaminofluorescein‐2/diacetate fluorescence. Future studies will indicate whether melatonin treatment during sperm preparation techniques could protect spermatozoa from excessive NO production.

Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H2O2 with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H2O2 concentrations (0, 2.5, 7.5 and 15 μm). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H2O2 did affect the sperm parameters. Exogenous H2O2 was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.

Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production

AIM To investigate the in vitro effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. METHODS Washed human spermatozoa from normozoospermic donors were treated with insulin (10 microIU) and leptin (10 nmol). Insulin and leptin effects were blocked by inhibition of their intracellular effector, phosphotidylinositol 3-kinase (PI3K), by wortmannin (10 micromol) 30 min prior to insulin and leptin being given. Computer-assisted semen analysis was used to assess motility after 1, 2 and 3 h of incubation. Viability was assessed by fluorescence-activated cell sorting using propidium iodide as a fluorescent probe. Acrosome-reacted cells were observed under a fluorescent microscope using fluorescein-isothiocyanate-Pisum sativum agglutinin as a probe. NO was measured after treating the sperm with 4,5-diaminofluorescein-2/diacetate (DAF-2/DA) and analyzed by fluorescence-activated cell sorting. RESULTS Insulin and leptin significantly increased total motility, progressive motility and acrosome reaction, as well as NO production. CONCLUSION This study showed the in vitro beneficial effects of insulin and leptin on human sperm function. These hormones could play a role in enhancing the fertilization capacity of human spermatozoa.

TNF-α and IL-6 affect human sperm function by elevating nitric oxide production

Many studies have reported the effects of cytokines on human sperm function, even though their role and the mechanisms involved remain unclear. The effects of increasing concentrations of the cytokines TNF-α and IL-6 on human sperm motility and viability were assessed, and the possible mechanisms involved were investigated. TNF-α and IL-6 significantly reduced progressive motility at higher concentrations in a dose- and time-dependent manner. No differences were observed in cell viability. Both cytokines increased nitric oxide production in a dose-dependent manner. TNF-α and IL-6 did not statistically differ in their detrimental effects on human spermatozoa. These results indicate that TNF-α and IL-6 have an effect on sperm function. This effect is possibly mediated via an increase in nitric oxide production.

The in vitro effects of Mondia whitei on human sperm motility parameters

Mondia whitei has been used since ancient times as an aphrodisiac even though little scientific evidence exists about its efficacy. An aqueous extract of Mondia whitei was administered to human spermatozoa in vitro and motility parameters assessed. Mondia whitei significantly enhanced total motility as well as progressive motility in a time‐dependent manner. These data may open the way for the use of Mondia whitei especially in men affected with asthenozoospermia. Copyright

Effects of tumour necrosis factor alpha and interleukin‐6 on progesterone and calcium ionophore‐induced acrosome reaction

For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-alpha and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-alpha and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR (p < 0.05) in a dose-dependent manner. TNF-alpha showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-alpha and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively.

Effects of Sperm Processing Techniques Involving Centrifugation on Nitric Oxide, Reactive Oxygen Species Generation and Sperm Function~!2009-11-11~!2010-01-07~!2010-03-30~!

This study was aimed at investigating the effects of sperm centrifugation on nitric oxide (NO) and reactive oxygen species (ROS) generation as well as sperm motility and viability. Human spermatozoa were centrifuged for 10 and 30 minutes (400 x g) in the presence or absence of the NOS inhibitor, N G -nitro-L-arginine methyl ester (L-NAME); ROS scavenger, N-(2-mercaptopropionyl)Glycine (MPG) or the combination of L-NAME + MPG. Total sperm motility was significantly decreased with 30 minutes of centrifugation whereas progressive motility and cell viability were significantly decreased with 10 and 30 minutes of centrifugation. These effects were reversed with the administration of MPG or L- NAME + MPG. Ten minutes centrifugation significantly elevated ROS and NO production (P < 0.05). Thirty minutes centrifugation elevated ROS generation (P < 0.01) whereas NO was attenuated. This study has demonstrated that 10 and 30 minutes of sperm centrifugation were detrimental to both sperm motility and viability, but generally 30 minutes centrifugation was more detrimental to sperm than 10 minutes. It has also demonstrated that 10 minutes centrifugation led to both NO and ROS elevation whereas 30 minutes centrifugation led to ROS elevation and NO attenuation. We therefore recommend that sperm separation techniques should avoid using centrifugation or prolonged centrifugation in assisted reproductive technologies.

Effects of Nitric Oxide Exposure on Human Sperm Function and Apoptosis Markers

Nitric oxide (NO) is a signaling molecule produced by intracellular nitric oxide synthase (NOS) enzymes. Studies have shown that this free radical affect sperm capacitation, a maturation step preceding acrosome reaction. This study was aimed at investigating the effects of exogenously administered NO through its donor, sodium nitroprusside (SNP) has on human sperm motility, viability and apoptosis markers. Increased concentrations of SNP (10, 30, 50, 100 � M) were administered to human spermatozoa in the presence or absence of NO synthase inhibitor, N-nitro-L- arginine methyl ester. Spermatozoa motility and viability were assessed at 60 and 90 minutes of incubation. The caspase activity was assessed after 90 minutes of incubation. SNP significantly decreased spermatozoa motility and viability in a dose and time dependent manner (p < 0.05). The caspase activity was significantly increased with increasing concentration of SNP (p < 0.05). This study therefore conclude that high concentrations of NO result in the decrease of sperm function and increase of germ cell apoptosis rate that may contribute to male infertility.

Sperm assessment: Traditional Approaches and Their Indicative Value

The traditional semen analysis is the established cornerstone of assessing male fertility, and the diagnostic management depends on a sequential, multi-step approach. Recognized reference values for normality are essential due to the relationship between sperm quality and fertility. The information provided by a semen analysis is the least invasive and most cost-effective assessment of a male’s fertility status. Despite the introduction of alternative techniques such as computer-assisted sperm analysis and the advancement of assisted conception, the prediction of fertilization in vitro is still crucial.