PARENT SESSION
Uterus/Oviduct

IDENTIFICATION OF A FUCOSE-BINDING PROTEIN FROM BULL SPERM AND SEMINAL PLASMA THAT PARTICIPATES IN FORMING THE OVIDUCTAL SPERM RESERVOIR.

Ignotz, George¹, Lo, Margaret¹, Perez, Carissa¹, Gwathmey, TanYa¹, Suarez, Susan¹, ¹

ABSTRACT- Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle, the reservoir is formed when sperm bind in a calcium-dependent fashion to fucosylated moieties on the surface of oviductal epithelium which resemble the Le^a trisaccharide (-L-Fuc[1,4]--D-Gal[1,3]-D-GlcNAc). 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-buffered saline. Extracts were subjected to affinity chromatography using immobilized Le^a. Two-dimensional PAGE of affinity chromatography eluates revealed a protein of approximately 16.5 kDa and a pI of 5.8. An 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 recovered by affinity chromatography. Epifluorescence microscopy revealed that live, uncapacitated sperm labeled over the acrosome with a fucose-BSA-FITC conjugate, while capacitated sperm did not. Treatment of capacitated sperm with the Le^a-binding protein restored labeling. When oviductal explants were pretreated with the purified Le^a-binding protein, sperm binding to epithelial cells was significantly reduced. Partial amino acid sequence derived from tryptic fragments of the Le^a-binding protein identified it as PDC-109 (BSP A-1/A-2), a protein abundant in seminal plasma and produced by seminal vesicle epithelial cells. PDC-109 is known to adsorb to sperm membrane choline phospholipids and has been proposed to play a role in regulating capacitation. PDC-109 was purified from seminal plasma by ion exchange and heparin affinity chromatography. Ligand blots of PDC-109 also demonstrated its binding affinity for Le^a which was blocked by fucose or EGTA. PDC-109 occurs in seminal plasma as glycosylated (A1) and non-glycosylated (A2) forms in roughly equal amounts. Although purified PDC-109 was shown to contain carbohydrate, none could be demonstrated on the Le^a-binding protein extracted from sperm. These results suggest that, in cattle, the non-glycosylated form of PDC-109 mediates the formation of the oviductal sperm reservoir. (Supported by USDA grant no. 97-35203-4734).

KEY WORDS: bos taurus, sperm-reservoir, oviduct, fucose