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Prenatal development of the epididymis was studied in bovine fetuses ranging from 10 to 90cm crown-rump length (CRL) (75-285 pcd). The studies aimed to apply both glycohistochemistry and immunohistochemistry for the detection of the differentiation of the developing prenatal epididymis. Both conventional histological and histochemical techniques were applied on paraffin sections of the epididymis from different fetal stages. Establishment of the urogenital junction between the extra-testicular rete testis and the mesonephric duct, via the growing efferent ductules (ductuli efferentes) was first evident in fetuses with 10cm CRL. At the fetal age of 110 pcd (24cm CRL), the mesonephric duct began to lengthen and coil forming three distinct regions (caput, corpus and cauda). In addition to the macroscopical modifications in the extra-testicular excurrent duct system, histological differentiation involved both the tubular epithelial and the peritubular mesenchymal cells. The epithelium lining the efferent ductules was differentiated into ciliated and non-ciliated columnar cells. The simple epithelium of the epididymal duct increased in height and developed stereocilia on the apical surface. Additionally, some basal cells first appeared at 185 pcd (56cm CRL), within the epithelium lining the cauda only. Lectin histochemistry (WGA, PNA, GSA-I) showed early immunostaining in epithelium of the efferent ductules and in peritubular mesenchymal structures. Immunoreactivity for different proteins (S-100, fibroblast growth factor-1 and factor-2, angiotensin converting enzyme, laminin, alpha-smooth muscle actin) was evident, both in the epithelial and in the peritubular mesenchymal cells as early as at 75 pcd. On the basis of our histochemical observations, we conclude that both glycohistochemistry and immunohistochemistry are useful tools to demonstrate that the differentiation in the peritubular structures and efferent ductular epithelium begins earlier than other components., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The emerging insights into glycan functionality direct increasing attention to monitor core modifications of N-glycans and branch-end structures. To address this issue in histochemistry, a panel of lectins with respective specificities was devised. The selection of probes with overlapping specificities facilitated to relate staining profiles to likely target structures. The experiments on fixed sections of adult murine testis and epididymis were carried out at non-saturating lectin concentrations to visualize high-affinity sites with optimal signal-to-background ratio. They revealed selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis. Leydig cells, for instance, were reactive with the Sambucus nigra agglutinin and human siglec-2 (CD22), two lectins also separating principal from basal and apical cells in the caput segments I-III of the epididymis. Apical cells were reactive with the Maackia amurensis agglutinin-I, and basal cells with the erythroagglutinin of Phaseolus vulgaris. The reported differences support the concept of lectin staining as cell marker. They thus intimate to study glycogene (genes for glycosyltransferases and lectins) expression and cellular reactivity with tissue lectins. These investigations will be instrumental to assign a role as biochemical signals to the detected staining properties., (© 2010 Blackwell Verlag GmbH.)

A histochemical study using fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in the efferent ductules and the three segments of the ductus epididymis (initial, middle and terminal segment) of dogs was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E and PHA L). The lectin-binding pattern in the canine epididymis presents similarities and differences to those observed in other mammalian species. The ductuli efferentes distinctly stained with most of the lectins used, whereas in the ductus epididymis a segment specific staining pattern was observed. Whereas principal cells of the ductus epididymis stained clearly with several FITC-labelled lectins (WGA, UEA and PHA-L), basal cells showed only a significant binding of Con A.

Originally defined as a lymphokine inhibiting the random migration of macrophages, the macrophage migration inhibitory factor (MIF) is an important mediator of the host response to infection. Beyond its function as a classical cytokine, MIF is currently portrayed as a multifunctional protein with growth-regulating properties present in organ systems beyond immune cells. In previous studies, we detected substantial amounts of MIF in the rat epididymis and epididymal spermatozoa, where it appears to play a role during post-testicular sperm maturation and the acquisition of fertilization ability. To explore its presence in other species not yet examined in this respect, we extended the range of studies to the bull. Using a polyclonal antibody raised against MIF purified from bovine eye lenses, we detected MIF in the epithelium of the adult bovine epididymis with the basal cells representing a prominently stained cell type. A distinct accumulation of MIF at the apical cell pole of the epithelial cells and in membranous vesicles localized in the lumen of the epididymal duct was obvious. In the fetal bovine epididymis, we also detected MIF in the epithelium, whereas MIF accumulation was evident at the apical cell surface and in apical protrusions. By immunoelectron microscopy of the adult bovine epididymis, we localized MIF in apical protrusions of the epithelial cells and in luminal membrane-bound vesicles that were found in close proximity to sperm cells. Although the precise origin of the MIF-containing vesicles remains to be delineated, our morphological observations support the hypothesis that they become detached from the apical surface of the epididymal epithelial cells. Additionally, an association of MIF with the outer dense fibers of luminal spermatozoa was demonstrated. Data obtained in this study suggest MIF release by an apocrine secretion mode in the bovine epididymis. Furthermore, MIF localized in the basal cells of the epithelium and in the connective tissue could be responsible for regulating the migration of macrophages in order to avoid contact of immune cells with spermatozoa that carry a wide range of potent antigens., (Copyright (c) 2006 S. Karger AG, Basel.)

Spermadhesins are proteins with various functions in sperm capacitation and zona pellucida binding. In this study the cellular localization of the spermadhesin AWN-1 has been examined in the equine male genital tract. Results obtained by immunohistochemical methods reveal that in the horse AWN-1 is synthesized in spermatogonia, in the rete testis, the ductus epididymidis and the seminal vesicles. These findings indicate that the cellular origin of spermadhesins is species-specific.

The present lectin histochemical comparison of cauda epididymal and ampullary bovine sperm was conducted to investigate whether ampullary secretions are involved in altering the plasma-membrane glycoconjugates of sperm. A marked redistribution of glycoconjugates between sperm from these two regions was indeed revealed on the basis of changes in binding patterns of the following fluoroscein-isothiocyanate (FITC)-labelled lectins: wheat-germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia I agglutinin (GS I) and Bauhinea purpurea agglutinin (BPA). This was evidenced in the first three cases by a relative reversal of staining intensity between the acrosomal and post-acrosomal regions, and by a pronounced increase in the staining of the midpiece. Changes in the distribution of BPA binding sites were limited to the latter phenomenon. The results are compared with previous findings, discussed in the context of the hypermotility characteristic of ampullary sperm and related to previously reported differences in the lectin-binding patterns of the luminal and glandular epithelia.

cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue-specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HE1-homologue was up-regulated markedly only at puberty, the HE4-homologue was already present at moderate levels prepubertally. The distribution of the HE1-homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3-4-week-old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4-homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.

Mammalian spermatozoa that leave the testis are neither motile nor fertile. Maturation of spermatozoa occurs during epididymal transport. Among the changes during epididymal passage alterations of the surface properties of spermatozoa appear especially interesting. In this report we describe ultrastructural localization of Con A and WGA-binding sites on bovine spermatozoa from caput and cauda epididymides using a indirect lectin-horse-radish-peroxidase gold technique. With Con A the plasma-membrane covering the sperm head was heavily labelled with gold granules in caput as well as in cauda epididymal spermatozoa. A different distribution was observed for WGA-binding sites. The acrosomal region of caput and cauda spermatozoa was heavily labelled with gold, both in caput and cauda spermatozoa. The postacrosomal region was only sparsely marked in caput sperm whereas in sperm cells originating from the cauda a calix like membrane area displayed intense labelling. Some differences in the number of binding sites were also seen on sperm tails: those of caput spermatozoa show generally more Con A binding sites that those from cauda epididymal spermatozoa. No changes in the number of WGA-binding sites on sperm tails was observed during epididymal passage. The technical aspects of the indirect lectin-horse-radish-peroxidase gold technique are briefly discussed.

The histochemical localization of some oxidoreductases was investigated in the epididymides of adult tomcats. Succinate dehydrogenase, lactate dehydrogenase, beta-hydroxybutyrate-dehydrogenase revealed their highest activity in corpus and cauda epididymidis whereas glucose-6-phosphate-dehydrogenase was strongest in the caput. The activity of the diaphorases and of cytochrome oxidase in the epididymal epithelium increased from caput towards the cauda epididymidis. The reaction for isocitrate dehydrogenase was distinct throughout the whole length of the ductus epididymidis. Our findings were compared with the biochemical results of other authors and the functions of the oxidoreductases in the epididymal epithelium were briefly discussed.

Modifications in bull sperm plasmamembrane during epididymal passage were investigated by the use of four different lectins: Concanavalin A (Con A); Ricinus communis I (RCA1); Wheat germ agglutinin (WGA); Ulex europaeus agglutinin I (UEA1). During sperm passage from caput to cauda epididymidis agglutination by RCA1 and WGA distinctly increased. Similar but somewhat less pronounced difference in the agglutinability was found for Con A. No agglutination was observed with UEA1. Ultrastructural examination of Con A binding sites on sperm plasma membrane with a Con A-horseradish peroxidase-gold technique (Con A-HRP-G) revealed a significant increase in the number of gold granules on the sperm tails during the epididymal passage of spermatozoa. No change in WGA-binding sites was observed between caput and cauda spermatozoa using a WGA-peroxidase method.

The histochemical localization of 6 lysosomal enzymes was studied in the epididymis of adult tomcats. A weak to distinct reaction for acid phosphatase, leucyl-amino-peptidase and non--specific esterase could be observed in the epithelium of the ductus epididymidis in all three segments. Among the glycosidases, N-acetyl-beta-glucosaminidase displayed the strongest activity. alpha-man and alpha-fuc could not be demonstrated. For N-Acetyl-beta-glucosaminidase, beta-Galactosidase, acid phosphatase and non-specific esterase, an increase of enzyme activity from the initial segment towards the terminal segment was seen. Intracellularly, the maximum of enzyme activity of those four enzymes was supranuclear. This histochemically enstablished pattern of enzyme activity in the epididymis of the tomcat was compared with those of other mammals. The possible functions of enzymes in the epididymis was briefly discussed.

Localization and activity of five hydrolases (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, nonspecific esterase and leucylamino-peptidase) were evaluated histochemically in the epididymides of mature dogs. In the ductuli efferentes, cilia and apical parts of the epithelial cells displayed high activity of alkaline phosphatase and adenosine triphosphatase. Strong activity of acid phosphatase, nonspecific esterase and leucylamino-peptidase was present in the basal and supranuclear zones of the epithelium of the ductuli efferentes. Stereocilia of all three segments of the ductus epididymidis showed a high activity of alkaline phosphatase. Positive adenosine triphosphatase reaction was confined to the stereocilia of the initial segment. A complex pattern of acid phosphatase activity was observed in the middle segment. The subdivision of the middle segment in four subsegments was therefore suggested. In the epithelium of the initial segment only a few nonspecific esterase-positive cells were seen. The infranuclear and basal areas of the epithelium in the middle segment and the supranuclear zone of the terminal segment displayed distinct nonspecific esterase activity. The possible contribution of the hydrolases to the function of the epididymis is discussed.

The histochemical localization of five glycosidases was studied in the epididymis of mature dogs. beta-Galactosidase showed a distinct to strong reaction in the epithelium of the ductuli efferentes and throughout the whole length of the ductus epididymidis. beta-N-Acetylglucosaminidase reactivity was weak in the initial segment, but increased significantly in the middle and terminal segment. The maximum beta-glucuronidase activity was found in the ductuli efferentes and in the initial segment. The alpha-mannosidase reaction was weak in all segments except the middle segment where a distinct activity was seen. With the method employed, no alpha-fucosidase activity could be detected. The physiological role of the glycosidases in the epididymis is discussed briefly.

The ultrastructure of the epididymal duct and ductuli efferentes in the dog has been studied by electron microscopy. The epididymidis can be separated into the classical divisions of caput, corpus and cauda epididymidis on the basis of general morphology and ultrastructure. The ductuli efferentes have a low epithelium with pronounced cilia at the apices of cells and appear to provide primarily a transport role for spermatozoa. In the epididymis proper the caput region is characterized by an extremely large Golgi apparatus with large numbers of lysosomes and nuclear inclusions. Secretory activity appears to be most common in the corpus region. Absorption and secretion are most active in the first two segments while in the cauda epididymidis the long-term storage of spermatozoa in the lumen is associated with many dense crystalline bodies formed in the epithelial cells within the Golgi apparatus and possibly deriving from absorbed macromolecular material from the lumen. The theory of whole sperm cell resorption by the epididymal duct is not supported by this study.