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Alkafafy, Mohamed (2005): Glycohistochemical, Immunohistochemical and Ultrastructural Studies of the Bovine Epididymis. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

In the present work, efferent ductules and epididymal duct from male foetuses as well as from sexually mature bulls were investigated using conventional light and electron microscopical techniques as well as glycohistochemical and immunohistochemical staining techniques. The prenatal development of the bovine epididymis was studied in foetuses ranging from 10 cm CRL (75 pcd) to 90 cm CRL (285 pcd). In foetuses with 10 cm CRL (75 pcd) the main event was the establishment of the urogenital junction between the extratesticular rete testis and mesonephric duct via the growing efferent ductules. At the foetal age of 110 pcd (24 cm CRL), efferent ductules underwent a strong coiling. At the same time the mesonephric duct began to lengthen and coil, forming three distinct regions, namely caput, corpus and cauda epididymidis. The coiling was much more distinct in caput and cauda than in corpus epididymidis. At 130 pcd (30 cm CRL) and upwards efferent ductules were organized in lobules which are then arranged in groups separated from each other by connective tissue septa. A similar organization involved the highly convoluted epididymal duct, particularly in the head and tail regions. In addition to the macroscopical modifications in the morphology of extratesticular excurrent duct system, histological differentiation involved both the tubular epithelium and the peritubular mesenchymal cells. The epithelium of efferent ductules was differentiated into ciliated and nonciliated columnar epithelium. The simple epithelium of the epididymal duct increased in height and developed stereocilia on its apical surface. Distribution of WGA-, PNA- and GSA-I-binding sites on luminal surface of the epithelium of efferent ductules, but not of epididymal duct may indicate earlier differentiation of the former. WGA-binding to the peritubular and interstitial mesenchymal cells, but not to the epididymal epithelium indicated that the mesenchymal structures differentiate before epithelial ones. S-100, FGF-1, FGF-2, ACE, laminin and GT were immunolocalized in the epithelium both of efferent ductules and epididymal duct as early as at 75 pcd (10 cm CRL). Also ?-SMA was immunolocalized in the peritubular mesenchymal cells at 75 pcd (efferent ductules) and at 95 pcd (epididymal duct, CRL 18 cm). The epithelium of the adult bovine efferent ductules is simple columnar including ciliated and nonciliated cells as well as some scattered intraepithelial leucocytes. On the basis of their cytological characteristics, nonciliated cells could be categorized into three sub-types. The epididymal duct of the adult bull is lined with pseudostratified columnar epithelium. It consists mainly of tall, slender, stereocilia-bearing columnar cells and small basal cells. On the basis of several morphometric parameters like epithelial height, luminal diameter and width of peritubular muscle coat the epididymal duct could be subdivided into six segments. Ultrastructural studies revealed a well developed Golgi apparatus, numerous profiles of sparsely granulated endoplasmic reticulum and mitochondria as well as rER in the cytoplasm of principal cells particularly in those of the first three segments. Apical surfaces of principal cells particularly those of the proximal segments were equipped with long stereocilia and their apical cell membrane and cytoplasm displayed a well developed endocytotic apparatus. The narrow basal extensions of principal cells were crowded with numerous pleomorphic mitochondria, lysosomes, heteromorphic electron dense granules and residual bodies. Basal cells were insinuated between the narrow basal extensions of principal cells and the basal lamina. They possessed kidney-shaped, mostly deeply-invaginated nuclei and were characterized by a paucity of organelles. Apical mitochondria-rich cells were frequently found in segments II and III and rarely in segments IV and V. Their hyaloplasm was lighter than that of the neighbouring principal cells and their apical surfaces were provided with short microvilli. Apart from a reasonable number of mitochondria, small Golgi apparatus and sporadic strands of rER, they displayed a paucity of organelles. Intraepithelial macrophages were occasionally encountered in the basal third of the epithelium. They possessed many mitochondria, well developed Golgi apparatus and rER as well as small heterochromatic nuclei. Various profiles of lysosomes and dark residual bodies were found in their cytoplasm. Intraepithelial lymphocytes were characterized by their heterochromatic, round and mostly indented nuclei and narrow peripheral cytoplasmic rim. They were often encountered in immediate proximity to subepithelial capillaries. Fluoresceinisothiocyanate (FITC)-labelled lectins (GSA-I, PNA, ECA, WGA, Con A, LCA, PSA, DBA, HPA, SBA, VVA, LTA and UEA-I) were also used for the study of the regional distribution of saccharide groups in adult bovine epididymal tissues. WGA, Con A, LCA, PSA, DBA and HPA bound distinctly to stereocilia of principal cells in the different segments. However, DBA- and HPA-binding sites were confined to stereocilia in caput region. WGA, LCA, PSA, DBA and HPA possessed distinct binding sites in Golgi zone of principal cells, mostly of the caput epididymidis. Basal cells reacted distinctly with WGA, Con A, LCA, PSA and HPA. Intraepithelial leucocytes displayed moderate binding sites for PNA, WGA, LCA and PSA. The basal membrane reacted moderately only with WGA. Epididymal connective tissue showed weak to moderate binding only with ECA and WGA. GSA-I bound distinctly to vascular endothelium and could be applied as a good marker for bovine endothelium. Sperm cell mass bound WGA and PNA distinctly. No binding sites could be found for VVA, LTA or UEA-I. Immunohistochemical studies used the Avidin-Biotin-peroxidase Complex (ABC) method for localization of S-100, FGF-1, FGF-2, ACE, GT, VEGF, ?-SMA, laminin, connexin 43, CD4, CD8 and CD68 in the epididymis. The epithelium of the efferent ductules showed intense immunoreaction for S-100, FGF-1 and FGF-2 and a moderate immunostaining for ACE and GT. Principal cells of the first three epididymal segments exhibited a distinct immunostaining for S-100. They also showed a distinct immunoreactivity for FGF-1 throughout the different segments. Principal cells in the first, second and sixth segment displayed intense immunostaining for ACE. Immunostaining for GT in Golgi zone of the principal cells was intense (segments II and III), distinct (segments IV and V) and moderate (segments I and VI). Basal cells showed moderate (FGF-1) or intense (FGF-2) immunostaining in different epididymal segments. Intense immunostaining for ACE, laminin and ?-SMA was found respectively in the endothelium, endothelial basal lamina and smooth muscle cells of blood vessels. The basal lamina of the epithelium and the peritubular smooth muscle cells displayed a moderate immunoreactivity for laminin. The peritubular smooth muscle cells manifested an intense immunostaining for ?-SMA. CD4+ T cells and CD68+ macrophages were found within the epithelium and in the interstitium. Mast cells were conventionally stained with Alcian blue and Toluidin blue. They also displayed a distinct immunostaining for VEGF and FGF-2. In conclusion, my study supports the previously proposed 6-segment scheme of bovine epididymis. Moreover, lectin histochemistry and immunohistochemistry were not only helpful tools in emphasising this scheme but also in correlating specific functional activities to certain regions. Lectins- and GT-binding sites as well as ultrastructural characteristics point to high synthetic and secretory activities of principal cells in the first three segments, as indicated by the well developed Golgi apparatus. Ultrastructurally, principal cells of the proximal three epididymal segments displayed a well developed endocytotic apparatus. This was reinforced by intense immunostaining for ACE in this region, which reflects extensive absorptive activities in this region. Existence of mast cells in the epididymal interstitium and T-lympho-cytes and macrophages in the interstitium and within the epithelium may reflect their harmonized co-operation in the induction of immune tolerance in the bovine epididymis.