Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model.
The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx).
Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins.
Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation.
The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle.
In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/196485 |
| Date | January 2013 |
| Creators | Huang, Wenxin, 黃聞馨 |
| Contributors | Chiu, CN, Ho, PC, Pang, RTK |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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