10 SPERM BINDING TO PORCINE UTERINE EPITHELIAL CELLS MIGHT BE LECTIN MEDIATED

Abstract

Evidence suggests that porcine spermatozoa bind to the endometrium lining after entering the uterus. In previous studies, it was verified that this binding is specific to porcine uterine epithelial cells (UEC). Here, we present trials aimed at identifying the engaged binding mechanism. Several interactions of spermatozoa with the female reproductive tract are known to be lectin mediated. The respective sugar ligands are species specific for the oviductal as well as zona pellucida surface membrane. Therefore, we hypothesised that sperm-endometrium interactions are mediated by lectins, too. To block possibly existing sugar moieties on the sperm surface, we pre-incubated spermatozoa with lectins selected for their strong binding to viable spermatozoa [wheat germ agglutinin WGA, succinylated WGA (sWGA), concanavalin A (ConA)] and subsequently undertook co-incubation studies using an established cell culture model from primary porcine uterine epithelial cells. All trials were carried out in modified Dulbecco’s Modified Eagle’s Medium containing 20% serum (D20) because the UEC monolayer proved to react sensitive to PBS as well as to semen extender. Pre-trials confirmed that in D20 sperm vitality remained unaffected and lectin binding efficiency did not differ compared with PBS. The sperm-rich fraction of the ejaculate from 3 German Landrace boars was collected in D20 and washed by centrifugation. The pellet was resuspended in D20 and the concentration adjusted to 100 × 10⁶ spermatozoa mL^–1. Then, the samples were incubated with 1 of the 3 lectins (10 µg mL^–1) for 15 min at 37°C. As a control, 1 aliquot of the sperm suspension was incubated without lectins. After another washing step, the pellet was resuspended in D20. For co-incubation with UEC, 500 µL of lectin-incubated sperm was released onto a UEC monolayer and the binding activity observed under a phase contrast microscope. The binding density was quantified by area under view and compared to results from the control incubation with untreated sperm. Images (2 repeats/boar and lectin) were divided into fields of 61.6 µm² and the fields with and without sperm were counted. For statistical analysis, a Tukey Test was carried out. Sperm treated with WGA and sWGA, both binding to glucosamine molecules, showed significantly reduced binding activity to the UEC (P < 0.05), whereas ConA-treated and untreated sperm bound in the dense pattern as shown in previous trials. The results indicate that the sperm-UEC binding mechanism is mediated by glucosamine moieties on the sperm surface, which interact with matching lectins placed on the UEC membrane surface. Investigations for further confirmation of the suggested binding mechanism between porcine sperm and the uterine epithelial layer are ongoing.

Supported by IMV Technologies, L’Aigle, France.