Magister Scientiae (Medical Bioscience) - MSc(MBS) / The incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).

Several studies have highlighted the possibility that radio-frequency electromagnetic fields (RF-EMF) used in mobile phone technology could influence DNA integrity of male germ cells as well as sperm motility. Current knowledge concerning the influence of RF-EMF on male germ cells is extremely limited. In the present study the hypothesis that 900 MHz GSM radiation could induce the activation of stress response in human spermatozoa was investigated. Ejaculated, density purified, human spermatozoa from donors were exposed to 900 MHz GSM mobile phone radiation at specific absorption rate (SAR) levels of 2.0 and 5.7 W/kg and examined at various time points post exposure. Sperm motility and morphology were evaluated by computer-aided sperm analysis (CASA). The ability of RF-EMF exposed sperm to undergo the acrosome reaction was evaluated by flow cytometry. Sperm binding to the zona pellucida of human oocytes was determined by the hemi-zona (HZA) assay. Apoptotic markers, phosphatidylserine (PS) externalization, change in mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, caspase activation and DNA fragmentation were analysed using flow cytometry. Heat shock protein (Hsp) 27 and 70 expression and activity were analyzed using specific antibodies with flow cytometry and Western blot methods. Stress fibre stabilization (F-actin polymerization) was visualized using fluorescent dye labelled phalloidin. No effect was seen on kinematic parameters assessed at SAR 2.0 W/kg, however straight line velocity (VSL) and beat cross frequency (BCF) were significantly altered after exposure at SAR 5.7 W/kg. Sperm shrinkage (decrease in surface area) was observed at both exposure levels. RF-EMF did not influence exposed spermatozoa’s ability to undergo the acrosome reaction. A significant decrease in sperm-zona binding was observed at both exposure levels. RF radiation did not have an effect on any apoptotic markers. ROS generation increased significantly with an increase in SAR (5.7 W/kg). RF-EMF did not induce a stress response in exposed sperm (no activation of Hsp70 and 27 activity). These results cannot be ascribed to heating, as the temperature did not increase by more than 0.2 - 0.3ºC during exposure. The decrease in sperm-zona binding is the result of an alternative non-stress inducible pathway. This study should be replicated at lower SAR levels that would simulate the radiation absorption from carrying the cell phone in a pocket close to the testes. / Thesis (PhD (Reproductive Biology))--University of Pretoria, 2008. / Obstetrics and Gynaecology / unrestricted