275 EVIDENCE FOR α₅ AND α_V INTEGRINS ON SPERM PLASMA MEMBRANES AND THEIR ROLE IN BOVINE 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 α₅ and α_V integrins on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to these integrin subunits. To determine if integrin subunits were present on sperm, sperm plasma membrane proteins were subjected to 1-dimensional SDS-PAGE and Western blot analysis. Frozen-thawed sperm, donated by ABS Pecplan, were centrifuged at 700 × g for 10 min, washed twice with warm PBS (Nutricell^®, Campinas, Sao Paulo, Brazil), and resuspended in Jones buffer (0.4% deoxyclolic acid, 8.9 M sucrose, 0.1 M Tris, pH 8.5) for 60 min at 4°C to solubilize sperm plasma membranes. Plasma membrane proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The resulting blots were probed with α_V integrin antibody (Calbiochem^®, San Diego, CA, USA) or α₅ integrin antibody (Calbiochem) and developed using ECL. 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 anti-integrin α_V IgG (2), and FM with anti-integrin a5 IgG (3). In vitro-matured cattle oocytes were incubated (39°C, 5% CO₂ in air) with 1 × 10⁵ 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. Each experiment was repeated 4 times and data from each experiment were pooled. Approximately 80 to 90 oocytes per treatment for fertilization were evaluated in each replicate. Weighted least squares means were used to analyze fertilization data (SAS software, SAS Institute, Cary, NC, USA). The significance level for all tests was P < 0.05. Both antibodies for α₅ (35 kDa) and α_V (34 kDa) integrins showed immunoreactivity on Western blots of sperm membrane proteins. Addition of anti-integrin α_V, and anti-integrin α₅ decreased fertilization (P < 0.05) compared with the control: (1) 94.1 ± 1.0%; (2) 18.2 ± 1.0%; (3) 12.2 ± 1.0%. These findings show that α_V and α₅ 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.