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-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest 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 regions after testosterone supplementation, indicating that they were secreted 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 highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins 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 protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.
Gatti
J L
JL
URA 1291 INRA-CNRS, Institut National de la Recherche Agronomique, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Monnaie, France. gatti@tours.inra.fr
Druart
X
X
Syntin
P
P
Gúerin
Y
Y
Dacheux
J L
JL
Dacheux
F
F
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D007150:Immunohistochemistry; D008297:Male; D017830:Octoxynol; D010446:Peptide Fragments / Q000032:analysis; D011092:Polyethylene Glycols; D012756:Sheep; D012995:Solubility; D013079:Sperm Maturation / Q000187:drug effects; D013094:Spermatozoa / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D006023:Glycoproteins; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D013739:Testosterone; D017830:Octoxynol; C010615:Nonidet P-40