Magister Scientiae (Medical Bioscience) - MSc(MBS) / Passage of spermatozoa through the female reproductive tract is essential for the regulation of fertilization, ensuring that healthy sperm reach the oocyte. Previous studies were devoted to morphological selection of sperm cells by the cervical mucus. However, research prove that the loss of integrity of the sperm plasma membrane is associated with infertile men, irrespective of their normal semen parameters. This indicates that the sperm plasma membrane plays an important role in fertilization. Further studies indicated that sperm surface proteins assist penetration through the female reproductive tract and would therefore provide useful insight in understanding other factors associated with male infertility. The aim of this project was to determine if there are any differences between sperm surface proteins of fertile donor samples in relation to infertile patient samples using different separation techniques and different detergents. Three different sperm separation techniques were employed, including wash, swim-up (SU) and Percoll density gradient centrifugation (DGC).Parallel to this, the deoxy-ribose nucleic acid (DNA) fragmentation of these cells were analysed for comparison of the extent of DNA damage induced due to different separation techniques used. This provided evidence that the best separation technique is the DGC as it minimises the amount of DNA fragmentation caused. Four different detergents were used in the process of extracting the membrane proteins from spermatozoa, namely sodium dodecyl sulphate (SDS), saponin,cetyl-trimethyl-ammonium bromide (CTAB), and TWEEN-20. The membrane proteins were then separated on a12% SDS poly-acrylamide gel electrophoresis (PAGE), and analysed by Coomassie blue and silver staining techniques as well as densitometry. Due to the different chemical nature of the detergents that extracted different surface proteins, CTAB (cationic) and SDS (anionic) extracted the most because of its strong solubilising abilities as non-ionic detergents. Common proteins that were extracted in donor samples included; 115, 92.5, 89, 61, 55.5, 51.5, 47, 44.5, 43, 38.5, 34 and 28 kDa proteins. In patients, commonly occurring proteins included; 92.5, 74.5, 70, 60.5, 51.5, 50, 44.5, 43, 36, 29.5, and 25.5 kDa proteins. Marked differences were found between membrane proteins extracted from donor samples in comparison to patient samples. Identification of these proteins was done using the SwissProt database and a literature search. Mostly non-genomic progesterone receptors were identified; others included oestrogen receptor, a phosphotyrosyl protein, P34H, equatorial segment protein, mannose lectin receptor, human guanylylcyclase receptor, epididymal protease inhibitor receptor, PH30 and estradiol binding protein. The function of the membrane surface proteins identified in this study plays a vital role in fertilization. A few of these functions include sperm attachment and binding to the oocyte as well as penetration thereof. Others play a role in signalling events such as capacitation, hyperactivation and acrosome reaction. The absence of these proteins in patient sperm possibly accounts for the functional inability to successfully achieve fertilization suggesting that this provides molecular insight to reasons for infertility amongst men. In addition to this, proteins presented by patient samples that were absent in healthy donors may too account for their infertility status. Estradiol binding protein and PH30 are two proteins presented only in patient samples. Their function plays a role in the inhibition of the acrosome reaction and sperm-egg fusion, respectively. In conclusion, these differences in protein expression between fertile donors and patients may form the molecular basis of infertility amongst men and indicates possibilities for novel proteonomic approaches to improve andrological diagnosis in future.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm. For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa. The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068). The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation. In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting. / AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak. Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon. Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068). Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering . Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.