Characterization of galactose-binding proteins in equine testis and spermatozoa

Abstract

Carbohydrate-binding proteins are thought to be involved in a myriad of sperm functions including sperm–oviductal and sperm–zona interactions. Recent studies in our laboratory have characterized galactose-binding proteins on equine spermatozoa as possible candidate molecules for sperm adhesion to oviduct epithelial cells. In the current study, equine sperm membrane proteins were subjected to galactose-affinity chromatography, and bound proteins were eluted with excess galactose in a calcium-free buffer. The eluted fraction recovered after galactose-affinity chromatography was used for generation of a polyclonal antibody which was immobilized on an affinity column to recover a purified protein from equine sperm extracts. Several protein bands of approximately 70, 25, and 20–18 kDa were detected with a major band at 25 kDa on immunoblots which was subjected to N-terminal amino acid sequencing. These galactose binding proteins (GBP) were specific to sperm and testis and were absent in all the somatic tissues tested. Based upon immunocytochemistry, GBP were localized over the sperm head. In noncapacitated sperm, fluorescent labeling was observed over the rostral sperm head as well as the postacrosomal area; whereas in capacitated sperm, the labeling was localized primarily in the equatorial segment. Immunohistochemistry of equine testis demonstrated abundant staining in the adluminal region of the seminiferous tubules corresponding to round spermatids. In summary, this study demonstrates the presence of testis- and sperm-specific galactose binding proteins in the horse. The function of these proteins remains to be determined.