Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Immunolocalization of PTHrP in the European bison and pine vole testis and epididymis

The present study deals with immunohistochemical localization of PTHrP in European bison and pine vole testis and epididymis. PTHrP immunoreactivity was observed in spermatogenic cells of seminiferous tubules in European bison and pine vole testis, with the strongerst reaction occurring in spermatozoa of pine vole testis and epididymal duct. We also observed PTHrP expression in vascular smooth muscle of epididymis and testis in both animal species, as well as slightly weaker reaction in endothelial cells of European bison epididymis. PTHrP was also expressed in the smooth muscle of the epididymal duct in European bison and pine vole. In conclusion, PTHrP is a multifunctional peptide showing both paracrine and autocrine action. Its presence in vascular endothelium and smooth muscle of testis and epididymis is connected with the regulation of vascular muscle tone, thus affecting blood flow in the vessels. PTHrP expression depends on a number of local factors. Moreover, we suppose that PTHrP also contributes to the proliferation and differentiation of spermatogenic cells.     

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.     

Anatomic distribution of lectin-binding sites in mouse testis and epididymis

Abstract. Testis and epididymis of sexually mature mice were studied histochemically using 25 fluorescein-isothiocyanate-labeled lectins. Several lectin-specific binding patterns were recognized. Thus, HAA, HPA, GSA-I, and UEA-I1 reacted only with spermatozoa. PNA, GSA-11, SBA, VVA, BPA, RCA-I, and RCA-I1 reacted with spermatozoa and spermatocytes. WGA, PEA, LCA, and MPA reacted with spermatogonia, spermatocytes, and spematozoa in increasing order of intensity. ConA, SUC. ConA, LAA, STA, LTA, LPA, PHA-E, PHA-L, IJEA-I, and LBA reacted with all spermatogenic cells with equal intensity. In the epididymis, 12 lectins reacted uniformly with the epithelial cells lining all segments of this organ. One lectin (VVA) did not react with epididymal lining cells. The remaining 12 lectins reacted in a specific manner with portions of the head, body, or tail, thus selectively outlining different portions of the epididymis. RCA-I and RCA-I1 selectively accentuated the so-called halo cells of the epididymis. These findings provide a detailed map of lectin-binding sites in the mouse testis and epididymis and show that certain lectins can be used as specific markers for spermatogenic cells and segments of the epididymis.     

Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain

The aims of this study were to determine the cellular and subcellular localization of aquaporin-9 (AQP9) in different rat organs by immunoblotting, immunohistochemistry and immunoelectron microscopy. To analyze this, we used rabbit antibodies to rat AQP9 raised against three different AQP9 peptides (amino acids 267-287, 274-295, and 278-295). In Cos7 cells transfected with rat AQP9, the affinity-purified antibodies exhibited marked labeling, whereas nontransfected cells and cells transfected with aquaporin-8 (AQP8) exhibited no labeling, indicating the specificity of the AQP9 antibodies. Immunoblotting revealed a predominant band of 28 kDa in membranes of total rat liver, epididymis, testes, spleen, and brain. Preabsorption with the immunizing peptides eliminated the labeling. Immunohistochemistry showed strong anti-AQP9 labeling in liver hepatocytes. The labeling was strongest at the sinusoidal surface, and there was little intracellular labeling. Immunoelectron microscopy revealed that the labeling was associated with the plasma membrane of the hepatocytes. In testes Leydig cells exhibited anti-AQP9 labeling, and in epididymis, the stereocilia of the ciliated cells (principal cells) exhibited significant labeling, whereas there was no labeling of the nonciliated cells (basal cells). This was confirmed by immunoelectron microscopy. In spleen strong labeling of cells was observed of leukocytes in the red pulp, whereas there was no labeling of cells in the white pulp. In rat brain, AQP9 immunolabeling was confined to ependymal cells lining the ventricles and to the tanycytes of the mediobasal hypothalamus. Antibody preabsorbed with the immunizing peptide revealed no labeling. In conclusion, AQP9 proteins is strongly expressed in rat liver, testes, epididymis, spleen, and brain.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

Summary The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Development of mouse testis and epididymis following intrauterine exposure to a static magnetic field

In order to test if the in utero exposure to static magnetic fields affects testis and epididymis development in mice, females were exposed to 0.5-0.7 T, generated by a permanent magnet, from day 7 of gestation to the day of birth. No significant differences were found between exposed and sham-exposed animals with respect to body weight gain of dam during the gestational period, litter size, body weight of male pups at the day of birth, and body or testis-epididymis weight gain of pups from birth to day 35. Histopathologic evaluation of testis and epididymis of pups of 1, 5, 15, and 35 days of age showed no detectable alterations due to in utero exposure to static magnetic fields.     

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.     

Functional relationships of the mammalian testis and epididymis

The development of knowledge in four areas of research in male reproductive physiology of particular interest is reviewed. The concept of the blood-testis barrier (BTB), which arose following dye exclusion from the seminiferous tubules, has now been established as the differential transfer from interstitial fluid to tubular and rete testis fluids of molecules of physiological importance. The composition of fluid collected mostly from the rete testis of several species, not only reflects the nature of the barrier, but also the secretory capacity of the Sertoli cell. The functional significance of the transfer of molecules into testicular fluid and the composition of the fluid flowing into the epididymis are discussed. Sertoli cells establish the structural basis of the BTB during puberty and divide the seminiferous epitheliuym into basal and adluminal compartments. The Sertoli cell is the prime target for follicle stimulating hormone (FSH). The responses evoked by FSH are discussed, including special mention of androgen binding protein (ABP) and the protein hormone, 'inhibin', with FSH-suppressing properties. The control of FSH in the lamb is mentioned including new evidence to support a tubular source of a feedback agent with significance during the impuberal stage. Finally, some of the biochemical properties of the epididymis and its fluid contents are reviewed and the epididymal sperm are identified as the site of the antifertility action of the 6-chloro-6-deoxy sugars.     

S-100 protein immunoreactivity in mammalian testis and epididymis

The present study deals with immunohistochemical localization of S-100 protein in mouse, bank vole and pine vole testis and epididymis. S-100 protein immunoreactivity was observed in the endothelia of capillaries and lymphatic sinusoids of pine vole testis. A reaction to S-100 protein of the same intensity as that noted in the endothelia of testicular capillaries was found in myoid cells of pine vole and bank vole seminiferous tubules. Moreover, a positive reaction to S-100 protein was observed in bank vole and mouse Leydig cells. In the epididymis, a weaker reaction to S-100 occurred in smooth muscles of pine vole and mouse epididymal duct. Despite difficult interpretation of physiological role of S-100 protein we suggest that it may be a part of the blood-testis barrier. It may also participate in the processes of transcytosis and contractility; its cellular expression is regulated by local factors. However, location of S-100 is not specific to the representatives of the same order.     

Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine-conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin-stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis-derived material were lectin-positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea-pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin-stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.     

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.     

Localization of the antigotensin-converting enzyme activity in testis and epididymis

Angiotensin-converting enzyme (ACE) has been studied in different reproductive organs of the male rat, in somatic cell lines clonally derived from both rat and mouse testes, and in isolated spermatogenic cells of the mouse. Among the various reproductive organs only testis and epididymis show high levels of enzyme activity. The testicular activity is found mainly in the isolated germinal cells and residual bodies, whereas somatic cell lines contain negligible levels of activity even after addition of hormones and growth factors. Testicular homogenates, spermatogenic cells, epididymal spermatozoa, and spermatozoan cytoplasmic droplets, when fractionated by anion exchange chromatography, contain one major and one minor activity peak, whereas epididymal homogenates and epididymal secretions reveal an additional major activity peak together with the minor peak. All forms of ACE have a similar response to different modifiers, and are equally sensitive to the specific inhibitor N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline (Enalapril). The testicular enzyme could provide a useful marker for spermatogenic maturation and/or cytoplasmic processes both in testis and epididymis. The separate epididymal peak is a secretory enzyme that may be responsible for the processing of spermatozoan plasma membrane constituents during epididymal transit, or may have a role in attacking some biologically active compounds.