Characterization and androgen-dependence of proteins associated with luminal fluid and spermatozoa in the rat epididymis

Abstract

The proteins of epididymal luminal fluid and of spermatozoa recovered from different regions of the rat epididymis were examined by polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions. Albumin (A) and four major pre-albumin bands (B-E) were observed in epididymal fluid from the cauda on non-denaturing gels. By comparing the migration of these bands with that of standard globular proteins on denaturing gels, the molecular weight of Bands B and C was estimated to be 16 000, Band D was 30 000 and Band E was 32 000. Bands D and E were apparently glycoproteins since they stained with periodic acid-Schiff's reagent and were bound by an affinity column of Concanavalin A. The pre-albumin proteins (B-E) were of epididymal origin since they (a) were not detected in blood serum, (b) were not detected in testicular extracts and (c) were still found after ligation of the efferent ducts. From the incorporation of radioactive methionine, Bands B and C were shown to be synthesized in the initial segment and caput. The regional distribution of luminal proteins indicated that protein D was added in the caput and cauda and protein E in the cauda. This regional origin of luminal proteins was confirmed by the altered protein profiles consequent upon the reduced fluid flow through the epididymis brought about by ligation of the efferent ducts. The androgen-dependence of epididymal protein synthesis was also investigated using radioactive methionine. Castration had little effect on total protein synthesis but resulted in the specific reduction of the synthesis of proteins B and C. Several changes were observed in the relative amounts of specific proteins extracted from spermatozoa from different regions of the epididymis and several of these proteins had molecular weights identical with those in luminal fluid. However, there was no evidence for any substantial binding to spermatozoa of the pre-albumin proteins (B-E) of luminal fluid.

Analysis of surface proteins of rat spermatozoa during epididymal transit and identification of antigens common to spermatozoa, rete testis fluid and cauda epididymal plasma

“…This is the case for the androgen-dependent epididymal-specific secretory proteins B, C, D, and E, whose addition to spermatozoa we have demonstrated previously (Brooks & Tiver, 1983). However, unlike the Mr 42 000 protein, only small amounts of these proteins become associated with the spermatozoa whilst large amounts accumulate in the epididymal luminal fluid (Brooks & Higgins, 1980; present study).…”

“…This is the case for transferrin, albumin and a pair of proteins of Mr 22 000 (pi 5-5 and 5-3). Transferrin arises in rete testis fluid as a result of synthesis and secretion by Sertoli cells (Skinner & Griswold, 1980 ;Wright, Musto, Mather & Bardin, 1981 (pi 5-5) corresponds to the protein A' which we have described previously (Brooks & Higgins, 1980 Although our present results have demonstrated major differences in the polypeptide composition of the sperm plasma membrane during the course of epididymal transit, future studies will need to be directed towards establishing where these proteins are located on the sperm surface and ultimately towards identifying specific roles for particular proteins. Our own work with affinity-purified polyclonal antibodies (Brooks & Tiver, 1983) and that of others with monoclonal antibodies (Feuchter, Vernon & Eddy, 1981;Myles, Primakoff & Bellvé, 1981;Schmell, Yuan, Gulyas & August, 1981 ;Gaunt, 1982;Schmell, Gulyas, Yuan & August, 1982;Crichton & Cohen, 1983;Gaunt, Brown & Jones, 1983;Primakoff & Myles, 1983) reveal that the sperm plasma membrane is a highly differentiated structure with particular antigens being restricted to specific domains of the sperm surface.…”

Characterization and hormonal regulation of tissue and fluid proteins in the mouse epididymis

“…Identification of androgen-responsive proteins in epididymal fluid has been performed by electrophoresis in the rat (Brooks and Higgins, 1980 ;Jones et al, 1980 ;Brooks, 1981 ;1983), the rabbit (Jones et al, 1981 ), the monkey (Arslan et al, 1986) and man (Tez6n et al, 1985), but in the mouse, only 15 to 20 cauda luminal peptides have been detected without any mention of their androgen-dependence (Flickinger et al, 1986). Although radioautographic studies have evidenced the pathway and kinetics of protein secretion in the various segments of the mouse epididymis (Fain-Maurel et al, 1981 ;Flickinger, 1981 ) (Brooks and Higgins, 1980) and the ram (Dacheux and Voglmayr, 1983) because of its expression in all the epididymal segments.…”

“…Although radioautographic studies have evidenced the pathway and kinetics of protein secretion in the various segments of the mouse epididymis (Fain-Maurel et al, 1981 ;Flickinger, 1981 ) (Brooks and Higgins, 1980) and the ram (Dacheux and Voglmayr, 1983) because of its expression in all the epididymal segments. The 30 Kd peptide most likely corresponds to the one previously designated CP 27 (27 Kd) (Flickinger etal., 1986) …”

Co‐expression and interaction of cubilin and megalin in the adult male rat reproductive system

“…Intrinsic factor is secreted by the chief cens of the gastric glands and is strictly localized within the digestive tract thus excluding it as a cubilin ligand in the epididymis (Maeda et aL, 1995). Albumin, on the other hand, is found in large quantities testicular and epididymal fluid and although its function remains unknown, it is surely a candidate ligand not only for cubilin but also for megalin, which also shares tms ligand (Fouchecourt et al, 2000;Brooks and Higgins, 1980;Cui et al, 1996). Transferrin is a particularly interesting candidate not oruy because is present in testicular and epididymal fluid but also because it has been found to be endocytosed proximally and much greater distally in the epididymis (Djakiew et aL, 1986;Veeramachaneni and Amann, 1991).…”