264 PORCINE SPERM-HEAD RECEPTOR INTERACTION WITH PROTEINS PERIPHERALLY BOUND TO THE OVIDUCTAL LUMEN

Abstract

Here we show that cell–cell interaction between boar spermatozoa and the oviductal lumen surface are mediated by specific receptor–ligand binding. We have previously demonstrated increased sperm viability following incubation of boar spermatozoa with apical plasma membrane (APM) proteins from sow oviductal epithelial cells (Fazeli A et al. 2003 Reproduction 123, 509–517). Fresh intact oviducts were internally flushed with PBS and filled with Sulfo-NHS-LC-biotin (Pierce Biotechnology, Inc., Rockford, IL, USA) in PBS (pH 8.0). Each end was clamped and incubated for 30 min at RT. Unbound biotin was quenched with 50 mM ammonium chloride for 10 min, and biotinylated soluble APM (sAPM-B) preparations were prepared. Percoll-washed boar spermatozoa (25 × 10⁶ mL⁻¹) were incubated with sAPM-B (150 μg mL⁻¹) for 40 min at 39°C in 5% CO₂, and unbound sAPM-B was removed. The sperm pellet was resuspended in 0.5% SDS and incubated for 90 min at RT. Solubilized proteins were isolated by centrifugation at 14,000g for 5 min. The proteins were separated by SDS-PAGE alongside non-biotinylated APM and untreated “sperm-only” samples. Biotinylation was detected by NeutrAvidin/HRP. In addition, sAPM-B treated spermatozoa were smeared onto slides for the detection of biotinyl groups by anti-biotin/Alexa Fluor (Molecular Probes, Leiden, NL). NeutrAvidin/HRP Pierce detected a biotinylated sAPM band migrating to approximately 100 kDa in the sperm/sAPM-B sample. This band was not present in the “sperm-only” sample. Detection of in situ-labelled sAPM-B on spermatozoa showed that fluorescence was specific to the sperm head. We conclude that an oviductal protein of approximately 100 kDa is a potential viability-enhancing ligand for a sperm receptor that is mainly located over the acrosomal region.

This study was funded by the BBSRC (UK).