206 ALPHA6, BETA1, AND BETA3 INTEGRINS EXPRESSED BY SPERM MAY BE INVOLVED IN CATTLE FERTILIZATION

Abstract

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 the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of integrin subunits on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to integrin subunits. Frozen–thawed sperm, donated by ABS Pecplan, were centrifuged at 700g for 10 min and washed once in warm PBS (Nutricell®, Campinas, São Paulo, Brazil). 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 × 106 spermatozoa, were incubated overnight at 4°C with a monoclonal anti-integrin α6 immunoglobulin G (IgG), monoclonal anti-integrin β1 IgG, and monoclonal anti-integrin β3 IgG (Chemicon®). The next day, 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 a slide, air-dried, mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen), and covered with a coverslip for analysis. Frozen–thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with a monoclonal anti-integrin α6 IgG (2), FM with monoclonal anti-integrin β1 IgG (3), and FM with monoclonal anti-integrin β3 (4). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 10 × 104 washed pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Immunoreactive α6, β1, and β3 were 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. Addition of anti-integrin α6, anti-integrin β1, and anti-integrin β3 decreased fertilization (P < 0.05) compared with the control: (1) 91.2 ± 2.0%; (2) 17.4 ± 2.0%; (3) 14.3 ± 2.0%; (4) 24.3 ± 2.0%. These findings show that α6, β1, and β3 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization. This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.