Glycohistochemical, Immunohistochemical and Ultrastructural Studies of the Bovine Epididymis

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.