| Abstrakt: |
Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about integrins' roles in sperm-oocyte fusion remain unsolved. To investigate the potential role of integrins in fertilization, we determined the presence of integrin subunits on catlle (Bos indicus and Bos taurus) sperm by confocal microscopy. Frozen thawed sperm, donated by ABS Pecplan, were centrifuged at 700 x g for 10 min and washed once in warm PBS. Sperm were resuspended in PBS and treated with an equal volume of cold 4% paraformaldehyde (prepared fresh from formaldehyde) for 15 min on ice. All subsequent steps were conducted at RT. Fixed sperm were washed twice with PBS (10 000 rpm, 5 min) in an IEC Micromax (Needham, MA), and the pellet was resuspended in PBS containing 5% BSA. Samples of 1 ml, each containing 5 x 106spermatozoa, were incubated overnight at 4°C with a monoclonal anti-integrin α6IgG, monoclonal anti-integrin β1IgG, and monoclonal anti-integrin β3 IgG (Chemicon;1:500 ). The next day, after being washed 3 times in PBS-BSA, sperm were resuspended in Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). After being washed 3 times in PBS-BSA as before, a drop of sperm suspension was smeared on slide, air-dried, mounted with antifade reagent with DAPI (Invitrogen) and covered with a coverslip for analysis. Sperm were viewed by a confocal microscope. Immunoreactive α6, β1, and β3were concentrated at the apical segment of the acrosome in all bulls tested. There was no detectable fluorescence in any of the controls, and not all the sperm in an ejaculate were immunoreactive. The presence of α6, β1, and β3integrins on bovine sperm indicate a possible role in sperm-oocyte adhesion and/or fusion. |
