Characterization of a Fucose-Binding Protein from Bull Sperm and Seminal Plasma That May Be Responsible for Formation of the Oviductal Sperm Reservoir1

Abstract

Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.

Regulation of sperm storage and movement in the mammalian oviduct

“…Affinity purification using Lewis-a as the trap identified PDC109 (also known as BSPA1/A2) as a protein responsible for binding bull sperm to oviductal epithelium (Gwathmey et al, 2003;Ignotz et al, 2001). PDC109 is a small (approximately 16 kDa) acidic, heparin-binding protein, consisting primarily of two fibronectin type II domains.…”

“…Capacitated bull sperm show reduced binding to oviductal epithelium as well as to the carbohydrate ligand involved in sperm binding (Ignotz et al, 2001;Revah et al, 2000). This can be accounted for by a shedding of the adsorbed seminal plasma protein PDC109 from the sperm head during capacitation, because binding can be restored in capacitated sperm by adding back purified PDC109 to sperm (Gwathmey et al, 2003).…”

Glycobiology of fertilization in the pig

“…Current understanding (Revah et al, 2000;Ignotz et al, 2001;Ekhlasi-Hundrieser et al, 2005) For on blot deglycosylation (Zhou et al, 2004), aliquots of the OF and OAMF fractions were blotted onto PVDF membranes. Alternatively, protein bands were excised and prepared for in-gel digestion (Küster et al, 1997).…”

A Role for Acp29AB, a Predicted Seminal Fluid Lectin, in Female Sperm Storage inDrosophila melanogaster

“…Carbohydrates mediate the adherence of sperm to the epithelium, with different sugars playing important roles in different species (e.g., Demott et al 1995;Lefebvre et al 1995Lefebvre et al , 1997Ekhlasi-Hundrieser et al 2005). In cows, for example, biochemical studies suggest that fucose residues on oviductal annexins act as receptors for sperm, with several sperm-bound seminal proteins recognizing the fucose moiety (Ignotz et al 2001(Ignotz et al , 2007Gwathmey et al 2006).…”