302 EFFECT OF MONOSACCHARIDES ON SPERM ABILITY TO BIND TO OVIDUCTAL EPITHELIAL CELLS

Abstract

The attachment of sperm to the oviductal epithelium is one of the major factors responsible for successful in vivo fertilization. Carbohydrates are reported to be involved in this attachment. In the present study, sperm were separated into 2 fractions based on their ability to bind to the oviductal epithelial cells (OECs; sperm that do not bind to OECs, UB-sperm; sperm that bind to OECs, B-sperm). Subsequently, the fertilization competence of these sperm fractions was examined. Furthermore, the effect of various monosaccharides on the ability of sperm to bind to OECs was investigated. Oviducts of cows whose ipsilateral ovaries demonstrated new clot formation (ovulation fossa) were collected from a slaughterhouse, and those that contained ovulated oocytes were further selected for experimentation. OECs were collected from these oviducts. OECs and frozen-thawed semen were each suspended in a medium (synthetic oviduct fluid, SOF, supplemented with 5 mg mL^-1 of BSA and heparin), at a final concentration of 1 × 10⁶/mL. These suspensions were mixed and centrifuged to separate the 2 abovementioned fractions. Immediately after the suspensions of sperm and OECs were mixed, d-mannose, d-fucose, or n-acetyl-d-glucosamine (GlcNAc) was added (final concentration of each monosaccharide, 5 mM). Next, the number of live sperm in each sperm population was estimated at 0 and 3 h. In addition, the oocytes collected from the ovaries were fertilized with the UB-sperm or non-separated (control) sperm, and the percentage of fertilization was examined. The percentage of fertilization and the number of sperm binding to the zona pellucida (ZP) were lower (P < 0.05) for the UB-sperm than for the control sperm. When sperm (10⁶) were separated in the presence of GlcNAc, the number of motile sperm attached to OECs was significantly higher than that among sperm that were separated in the absence of monosaccharides (0.49 × 10⁶ cells vs. 0.16 × 10⁶ cells). The motility of B-sperm that were separated in the absence of monosaccharides was maintained for a longer time than that of the control sperm (percentage of motile sperm: 74.6% vs. 38.4%, P < 0.05). The B-sperm separated in the presence of GlcNAc or d-fucose lost their motility to a greater extent than the B-sperm separated in the absence of monosaccharaides (27.3% vs. 49.9%, P < 0.05). Further, when GlcNAc (5 mM) was added to the suspension for sperm cryopreservation, the number of sperm that bound to the OECs also increased. In conclusion, it was shown that sperm with low ability to bind to OECs have low fertilization competence, and GlcNAc increased the number of sperm binding to the OECs.