Analyses of spermatozoa surface proteins using different separation techniques
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.