Labelling of membrane glycoproteins on rat spermatozoa collected from different regions of the epididymis (original) (raw)
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Identification of a major secretory glycoprotein from rat epididymis: interaction with spermatozoa
Biology of Reproduction, 1989
A polypeptide with molecular mass of 1 7 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-13-N-acetylglucosaminidase H without prior denaturation or pro tease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 X M1 and I 7,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GPI 7. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.
Identification of a Major Secretory Glycoprotein from Rat Epididymis: Interaction with Spermatozoa1
Biology of Reproduction, 1989
A polypeptide with molecular mass of 1 7 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-13-N-acetylglucosaminidase H without prior denaturation or pro tease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 X M1 and I 7,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GPI 7. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.
Biochemical Characterization of Two Ram Cauda Epididymal Maturation-Dependent Sperm Glycoproteins1
Biology of Reproduction, 2000
Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl--D-glucopyranoside (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.
Biology of Reproduction, 1987
Rat spermatozoa were recovered from the cap Ut, Corp US, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of 13-glucosidase, #{216}-galactosidase, 13-N-acetylglucosaminidase, and 13-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for j3-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.
Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies
Molecular Reproduction and Development, 1990
The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. lmmunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bonds of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.
Biology of Reproduction, 2000
Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains ϳ12 kDa carbohydrate and, following chemical deglycosylation, migrates as a ϳ52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.
Biology of Reproduction, 1990
The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of #{176}5S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and spermassociated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.
Interactions between rat epididymal epithelium and spermatozoa
The Anatomical Record, 1991
We studied in the rat epididymis the presence of membranebounded vesicles in the stereociliar areas of the epithelial cells. The intimate contact between principal cell stereocilia and luminal spermatozoa was also explored.