Mechanism of Sephadex Trapping of Capacitated Stallion Spermatozoa1

Abstract

Results of assay of frozen stallion semen with niters containing Sephadex and glass wool are well-related to fertility. Capacitated spermatozoa do not pass Sephadex niters. The objective of this investigation was to identify seminal protein(s) attaching to Sephadex to possibly identify the trapping mechanism. Testicular tissue, cauda epididymal and ejaculated sperm, rete testis fluid, cauda epididymal fluid, and seminal plasma from 6 stallions were used. Proteins were extracted from sperm with octyl-p-glucopyranoside (OBG). Analysis was done with SDS-PAGE and Western blots stained for ram anti-clusterin and rat anti-sulfated glycoprotein-2 (SGP-2) activity. Protein analysis was repeated on material that had passed through a Sephadex column and was subsequently flushed with urea. In addition, Bouin-nxed testicular sections were stained with anti-clusterin antibodies. As expected, SDS-PAGE revealed major differences between rete testis fluid, cauda epididymal, and seminal plasma protein composition. OBG extracts of washed ejaculated sperm contained several seminal plasma proteins not detected in cauda sperm. Immunostaining with ram anti-clusterin, but not anti-rat,SGP-2, revealed a band at-42 kDa in all samples except seminal plasma, where the band appeared at ~66 kDa. Major proteins migrating at-16.5, 19, and 23 kDa, and minor bands at ~20 and 42 kDa were decreased or missing in sodium citrate eluates of OBG extracts of sperm from the Sephadex columns. The major low molecular bands migrating at ~16.5 and 19 kDa were probably of seminal plasma origin. The retained bands were found in urea extracts from the Sephadex column but the ~42-kDa band not consistently. Immunoreactivity to the ram anti-clusterin antibody was found in the lumen of the seminiferous tubule and in the epithelium, particularly in the Sertoli cell cytoplasm. We conclude that binding of sperm membrane proteins migrating at ~20, 23, and 42 kDa on reducing gels, the larger one of which may be clusterin or a clusterin-like protein, may constitute one mechanism by which capacitated sperm bind to Sephadex. Seminal plasma protein fractions are likely to cover this molecule in uncapacitated sperm.