Intraluminal androgen binding protein alters 3H-androgen uptake by rat epididymal tubules in vitro

Previous experiments have shown that androgen binding protein (ABP) and androgens exist in high concentrations in the tissue and the lumen of the rat caput epididymis. The present experiments were performed to determine whether or not intraluminal APB affects tubule net uptake of androgens. Caput epididymal tubules were dissected into 2-cm segments, subjected to microperfusion into the tubule lumen, and incubated for 2.5 h in 35 degrees C minimum essential medium (MEM) containing 2.0 ng tritiated testosterone (3H-T) per ml. 14C-polytheylene glycol [PEG] was included as a contamination marker. In the first series of experiments, caput tubules were perfused with a control, artificial perfusion (MKB) containing no ABP or fresh rat rete testis fluid (RTF), which is known to contain ABP. Tubules incubated while containing RTF took up 138% of the tritiated androgens taken up by control tubules. In the second series of experiments, tubules were perfused with fresh caput epididymal lumen content, MKB alone, MKB containing either 5.0 ng purified rat ABP/microliters or 50 ng ABP/microliters. Tubules incubated while containing perfused MKB took up only 47% of the tritiated androgens taken up by tubules containing perfused native lumen content. Increasing intraluminal ABP concentrations in the MKB medium increased 3H-androgen uptake in a stepwise fashion. Intraluminal ABP at a concentration of 50 ng/microliters was associated with a 71% return of 3H-androgen uptake towards that amount of 3H-androgen taken up by tubules perfused with native lumen content. Intraluminal ABP enhances net androgen uptake by caput epididymal tubules from their surrounding medium in vitro.     

Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley-Liss, Inc.     

Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Evidence for the presence of oxytocin in the ovine epididymis

The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function.     

Changes in protein composition of the luminal fluids along the epididymis of the tammar, Macropus eugenii

Micropuncture samples of luminal fluid were collected from the rete testis and along the epididymis. Quantitative analyses showed that the ductuli efferentes reabsorb about half the protein leaving the testis. Considerable protein is secreted by the caput epididymidis (initial segment) and there is a net loss of protein from the corpus and cauda epididymidis. Denatured, polyacrylamide gel electrophoresis showed that there are 5 proteins in rete testis fluid which are not present in blood (Mr of 14,700, 22,800, 24,100, 43,000 and 44,800). One of these proteins (Mr 14,700) is lost from plasma in the ductuli efferentes and 2 (Mr 43,200 and 44,800) are lost in the corpus epididymidis. Twelve proteins appear in the epididymal plasma and are not present in rete testis fluid or blood: 6 appear in the caput epididymidis (Mr 30,000, 31,000, 32,300, 17,400, 18,700 and 21,400), 3 in the corpus epididymidis (Mr 12,800, 39,800 and 90,600) and 3 in the cauda epididymidis (Mr 10,900, 56,300 and 63,000). A protein with the same molecular weight as a blood protein (149,500) accumulates in the corpus and cauda epididymidis. None of the samples of luminal fluid contained particulate matter other than spermatozoa, indicating that the tammar is a useful animal for micropuncture studies.     

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin)

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.     

Postnatal development and regulation of proteins secreted in the boar epididymis

The number of proteins secreted by the boar epididymis increased progressively from 1 mo of age to the adult period. The first specific secretory activity was revealed at 2 mo in the distal caput (hexosaminidase, clusterin, and lactoferrin) and in the corpus (train O/HE1). Train A and glutathione peroxidase specific to the proximal caput, and trains E and M specific to the corpus, appeared at 4 mo. At 5 mo, secretion of procathepsin L occurred in the middle caput and that of mannosidase and E-RABP in the distal caput. Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions. The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.     

Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

The concentration of carnitine in the luminal fluid of the testis and epididymis of the rat and some other mammals.

Luminal fluid was collected by micropuncture techniques from the testis and epididymis of the rat, hamster, rabbit, boar and ram and the concentration of free L-carnitine in the fluid was estimated using enzymic methods. Carnitine was present in the testicular fluid of the rat in concentrations less than 1 mM but increased down the epididymis to reach 53 mM in luminal fluid from the cauda epididymidis, approximately 2000 times higher than in blood plasma. A high concentration was first found in the luminal fluid from the distal caput epididymidis, at about the point where the spermatozoa become motile. Carnitine was also present in the epididymal luminal fluid of the other species studied; the amounts were not as high as those in the rat but were still higher than those in blood plasma.     

Characterization of a 22 kDa protein with widespread tissue distribution but which is uniquely present in secretions of the testis and epididymis and on the surface of spermatozoa

The purification is reported of a 22 kDa protein which was first identified as one of the major components of the luminal secretion of the rat testis and epididymis. Antibodies against the 22 kDa protein cross-reacted with a protein of the same molecular weight in cytosolic extracts of other tissues from both male and female rats. However, since the protein could not be detected in blood, peritoneal fluid, saliva, milk, uterine fluid, seminal vesicle secretion, coagulating gland secretion or prostatic secretion, it would appear that the testis and epididymis may be unique in containing the protein in a soluble form within their luminal secretions. Proteins with slightly lower molecular weight were detected by the antibodies in cytosolic extracts of tissues from other animals (mice, rabbits, sheep, pigs, cattle), indicating that the protein may be conserved in a variety of species. However, in contrast to the rat, the protein was apparently not present in the testicular and epididymal secretions of these species. In addition to the occurrence of the 22 kDa protein as a soluble moiety in rat testicular and epididymal fluids, the protein was also located on sperm plasma membranes where its distribution was restricted to the surface of the flagellum. Amongst sperm surface proteins, the 22 kDa protein was the major protein containing sulphydryl groups and one of the major entities containing disulphide bonds. These properties may be of importance in the maintenance of sperm viability.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.     

Binding of epididymal proteins to rat spermatozoa in vivo.

The secretion of epididymal proteins and their binding to spermatozoa in rats were examined after retrograde perfusion of the superior and inferior epididymal arteries with [35S]methionine. PAGE revealed that the pattern of radioactive proteins in the luminal fluid was markedly different from the well-characterized pattern of secretory proteins obtained by in vitro incubation of epididymal minces with labeled methionine. Of the proteins secreted into the lumen, about 1% were associated with Percoll-purified spermatozoa. More proteins were associated with the spermatozoa in the corpus epididymidis than in the caput. Sequential extraction of spermatozoa with an isotonic buffer, a high-salt buffer, Triton X-100, and SDS revealed that almost half of the radiolabeled proteins could be extracted with the isotonic buffer. The firmly bound radioactive proteins remaining, which were extracted with Triton X-100 or SDS, consisted of one major band of 25 kDa and two minor bands of 30 kDa and 32 kDa. Analysis of the sperm-associated proteins at various times after the isotope was administered indicated that tight binding of proteins to spermatozoa occurs within 3 h after isotope injection.     

Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa

Ejaculated spermatozoa were washed and extracted with 0.6 M NaCl (2 h at 0 degree C) and the extract used to immunize rabbits. The crude antibody reacted with epididymal fluid and cytosol and with prostatic cytosol but did not recognize blood serum and testicular cytosol. After adsorption with prostatic proteins, the serum was specific for epididymis. Using immunoelectrophoresis and affinity chromatography, it was found that the antibody reacted with antigens which co-electrophoresed with androgen-dependent proteins (mobility relative to albumin, Ra) 0.3, 0.43 and 1.0, previously identified in human epididymis. Weak immunofluorescence in the epithelium of proximal caput tubules was detected on tissue sections. In contrast, distal caput and corpus tubules displayed a strong fluorescence in the cytoplasm of basal and principal cells as well as in spermatozoa present in lumen. Intense fluorescence was limited to the luminal content and the apical border and sterociliae of principal cells in caudal tubules. When applied to isolated spermatozoa, the reaction was negative for testicular sperm, while 49%, 82% and 100% of spermatozoa from caput, corpus and cauda, respectively, had a fluorescent acrosomal cap. An apparent gradient of increasing fluorescent intensities was also observed in this sequence. The reaction was strongest over the acrosomal cap, apparently absent in the postacrosomal region and weaker over the midpiece and principal piece. These results are interpreted as suggestive of the progressive coating of human spermatozoa with androgen-dependent epididymal proteins during epididymal transit.     

Sperm concentration in different segments of the goat epididymis

Spermatocrit values of fluid collected by micropuncture from the caput, corpus and cauda of goat epididymides as well as rete testis fluid and testicular fluid were determined for eight animals. The values indicated that 98.02% of fluid is absorbed in the epididymis and that the cauda epididymis contains the lowest amount of fluid, significantly different from the rete testis, corpus and caput.