169 MATURE OOCYTES TRIGGER THE RELEASE OF BOVINE SPERM FROM AN IMMOBILIZED OVIDUCT GLYCAN

Abstract

After mating, sperm move from the site of semen deposition towards the oviduct, where sperm are stored before fertilization. Much of the literature indicates that oviduct glycans bind sperm to retain them in the oviduct reservoir. We have discovered that a specific oviduct glycan on the epithelium (sulfated Lewis A trisaccharide; suLeA) binds bovine sperm and maintains sperm viability. But the way in which sperm are released from oviduct epithelial glycans to fertilize oocytes is enigmatic. In this study, we tested the hypothesis that oocytes signal the release of sperm from an oviduct glycan (suLeA) on a bead and that the released sperm could fertilize the oocytes. Sepharose beads with avidin on their surface were used to immobilize biotinylated suLeA. After coupling, the beads were washed. Frozen bovine semen was thawed, and sperm were washed and allowed to bind to suLeA-coated beads for 1 h in capacitating medium at 39°C. Mature bovine oocytes were then prewashed with the same medium (without heparin) and added to droplets with sperm bound to beads and incubated for 18 h at 5% CO₂ at 38.5°C. Bound sperm (mean = 4 per bead) were enumerated before and after oocyte addition. A control using dead (fixed) oocytes was also used. Three independent replicates were tested using 125 oocytes for each treatment. Data were analysed by ANOVA. The addition of live oocytes released 68.1% of the sperm bound to beads, whereas only 18.8% of sperm were released by addition of dead oocytes (control; P < 0.05). If heparin for sperm capacitation (3.125 g mL⁻¹ final concentration) was added concurrently with the addition of live oocytes to sperm bound to beads, the majority of bound sperm were released (87.7%), whereas dead oocytes (control) released fewer sperm (67.3% sperm, P < 0.05). Thus, both heparin and oocytes released sperm from beads independently but, together, they released the greatest number of sperm. Altogether, our results indicate that, in addition to heparin, mature oocytes induce sperm release from immobilized oviduct glycans for fertilization. This is the first demonstration that oocytes can signal sperm release from oviduct glycans in any mammal.