Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopyranoside (anti-CESP) and aga inst proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 11 1 kDa with different regionalization throughout the epididymis. The stronge st epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same r egions after testosterone supplementation, indicating that they were secret ed by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were hig hly enriched in the Triton X-114 detergent phase and could be extracted fro m the cauda epididymal fluid by a chloroform-methanol mixture. These protei ns were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the prote in in the cauda epididymal sperm extract and immunolocalized it on the sper m flagellum membrane and at the luminal border of all cells in the cauda ep ididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic proper ties could be directly integrated in a specific domain of the sperm plasma membrane.