The ductuli efferentes are a series of tubules that conduct
sperm from the rete testis to the epididymis. These small ducts
are unique because they are the only region of the male reproductive
tract that is lined by a ciliated epithelium. The presence of
cilia seemed to have provided the impetus for their study starting
with Becker in 1856 and 1857 who first described the cylindrical
ciliated cell in the epithelial lining. Beginning with these studies,
a panoply of functions ranging from absorption and transport to
endocrine and metabolism, has been attributed to these small ductules.
In spite of the wealth of material available on the structural
organization of the ductuli efferentes, many textbooks on reproduction
still refer to the efferent ductules as mere "conduits"
to move sperm from the testis to the epididymis (Dellmann and
Wrobel, 1981). The functional considerations of these ductules
have been largely ignored until recently. Also, there has been
no universal agreement on the terminology of these ductules. They
have been called variously as ductuli efferentes, ductuli efferentes
testis, ductuli efferenti, vasa efferentia, vasa efferentes, and
tubuli efferentes. The tubules have been considered as parts either
of the testis or the epididymis, as well as a separate organ.
The Nomina Histologica (1980) however considers the ductuli
to be a part of the caput epididymidis in deference to their common
embryologic origin from the mesonephric structures. In this review,
we will use the terms ductuli efferentes, and its Anglicized
form efferent ductules or ducts which are consistent with
the Nomina Anatomica Veterinaria (1983).
Top
The first description of the efferent ductules is attributed to
De Graaf (1668) who described these small tubes in his Tractatus
De Virorum Organis Generationi Inservientibus as the exit
of the seminiferous tubules from the testicles. As translated
by Jocelyn and Setchell (1972) De Graaf depicted them as "emerging
from the testicle not in one thick duct but in six or seven very
thin ones which coiled individually from side to side and form
the globe of the epididymis". He further stated that "these
very thin ducts, where they break out of the testicles through
the tunica albuginea, because of their thinness, are scarcely
more visible than undistended lymphatic vessels, unless they swell
with semen".
In describing the epididymis, De Graaf states "the duct of
the epididymis becomes thicker the further it proceeds from its
origin as six or seven branches at the top of the testicles. Where
the branches run together into one duct, it can be compared with
a rather thin thread which gradually enlarges until it attains
the thickness of a piece of string". Thus, by careful dissection
and unravelling of the testis and epididymis, De Graaf was able
to show that the epididymis was a single duct connected to the
seminiferous tubules by the small tubules which were named ductuli
efferentes in his illustrations. It was not until a century later
however, that Von Haller (1765) by using mercury injections confirmed
the existence of the ductuli efferentes as the connecting segment
between the testis and the epididymis.
By the 1800's, the topography of the ductuli efferentes had already
been very well described in the literature (Lauth, 1830). One
example is the following account from Gray's Anatomy, Descriptive
and Surgical (1859): "At the upper end of the
mediastinum, the vessels of the rete testis terminate in from
twelve to fifteen or twenty ducts, the vasa efferentia: they perforate
the tunica albuginea, and carry the seminal fluid from the testis
to the epididymis. Their course is at first straight; they then
become enlarged, and exceedingly convoluted, and form a series
of conical masses, the coni vasculosi, which, together, constitute
the globus major of the epididymis. Each cone consists of a single
convoluted duct from six to eight inches in length, the diameter
of which gradually decreases from the testis to the epididymis.
Opposite the bases of the cones, the efferent vessels open at
narrow intervals into a single duct, which constitute, by its
complex convolutions, the body and globus minor of the epididymis."
During the later part of the last century and the early part of
the present one, the histology of the ductuli efferentes also
has been a popular subject for many authors. This has been brought
about by Becker's discovery (1856; 1857) that the efferent ductal
epithelium is ciliated. He had shown that the efferent ducts were
histologically distinct from the epididymis because of their smaller
tubular diameter and of the height and character of their lining
epithelium. He stated that even though cilia were also present
in the epididymis (an incorrect assumption), they were not motile
unlike those found in the efferent ductules. From these observations,
he subsequently suggested that cilia in the ductuli propelled
sperm toward the ductus epididymidis.
Following Becker's discovery, the histology of the efferent ductules
along with the epididymis, particularly the epithelial lining
has been discussed and reviewed by many authors including Strickler
(1872), Schaffer (1892), Aigner (1900), Spangaro (1902), Stohr
(1903), Ikeda (1906), Benoit (1926), Cunningham (1928), Maximow
and Bloom (1930), Stieve (1930), Lucas (1932), Cowdry (1938),
Mason and Shaver (1952), Harrison (1953), MacMillan (1953), and
Reid and Cleland (1957). More recently, the ultrastructure of
the efferent ductal epithelium has been reviewed by Hamilton (1975)
and Robaire and Hermo (1988).
Early descriptions of the histology of the efferent ductules in
textbooks and manuals (Strickler, 1872; Schaffer, 1892; Stohr,
1903) had a similar theme: the epithelium consists of simple ciliated
cylindrical elements alternating with clusters of cubical cells
partly without cilia. It was believed that the epithelium is glandular
or secretory; the nonciliated cells were classified as a simple
alveolar type of gland and long branched ducts were erroneously
thought to be present extending from the lumen to the surrounding
connective tissue.
The nonciliated cells were also described to contain a varying
quantity of pigment granules as well as a variety of secretory
granules. Vesicular processes resembling drops of secretions were
also often described as found on the free surface of cells indicative
of secretory activity. A striated tunica propria and a
"membrane" of smooth muscle fibers, consisting of several
layers of circularly arranged elements interlaced with elastic
fibers, were described to complete the wall of the ductuli efferentes.
In a careful study of the morphological differences between the
ductuli efferentes and epididymis, Aigner (1900) confirmed Becker's
original finding that the cilia in the epididymis were non-motile
and only the efferent ductules possess true cilia. [It is interesting
to note however, that as late as 1989, some authors still make
no distinction between the cilia of ductuli and the stereocilia
of the epididymis (Bornman et al., 1989)]. Aigner (1900) found
the ciliary activity in the ductuli so vigorous that it moved
sperm over each other as it propelled them toward the epididymis.
Lucas (1932) later suggested that the movement of sperm by the
cilia may be augmented by the contraction of the thin layer of
smooth muscles which had been described by previous authors (Strickler,
1872; Spangaro, 1902; Stohr, 1903; Benoit, 1926 and Maximow and
Bloom, 1930). In 1926, Benoit published a paper dealing with the
histology of the epididymis in different mammalian species. This
landmark paper in male reproductive morphology was the first to
describe the considerable histological complexity of the epididymal
duct and gave rise to the concept that the initial part of the
epididymis is a separate region. As corroborated by Cunningham
(1928), Benoit depicted the ductuli efferentes epithelium to be
columnar and to have a characteristic appearance due to the presence
of two layers of nuclei, a basal layer of numerous crowded nuclei
and a distal layer of fewer nuclei located near the lumen but
at a greater distance from each other. These nuclei belong to
two kinds of cells described in detail as secretory and ciliated
respectively.
The secretory cells were characterized as more numerous and terminating
distally in a bordure en brosse or striated border, composed
of short filaments (Cunningham, 1928). Benoit did not regard these
filaments as cilia and made no mention of movement in them. The
distal nuclei belong to non-secretory cells each of which terminates
in a tuft of long cilia. In contrast to the secretory cells which
are somewhat broader towards the base than distally, the ciliated
cells have a narrow base (Cunningham, 1928).
In summary of the findings of these early comparative works on
the efferent ductules, Lucas (1932) has described the epithelium
as generally thrown into folds forming a groove that extends lengthwise
through the tube. These folds are irregular, their number is not
constant, and the depression may vary from a simple alveolus to
an open crescent (Fig.
9). Such folds are lacking in the dog and in the mouse.
In man, these folds do not appear until the onset of maturity
and persist during the period of senility. All the cells extend
to a circular membrana propria and thus the folds are produced
by cells of different heights. Ciliated cells usually are limited
to the crests of these folds but they may also be scattered throughout
the depressions.
Many of these earlier investigations state that the various cellular
structures and cytoplasmic inclusions found in both the ciliated
and secretory cells are related to cyclic transformations of ciliated
into secretory cells and vice versa. Others regard the two cell
types as distinct from each other, having in common only the same
embryonic origin (Spangaro, 1902; Ikeda, 1906). Castration and
hibernation were found to cause marked changes in the secretory
cells (i.e., loss of brush border, decrease in size of the nucleus)
but did not affect the ciliated cells (Benoit, 1926). Also in
some animals, variations in the numerical relationship between
the two cells were found to be contingent upon seasonal variations
in their reproductive cycles (Lucas, 1932).
In 1920, Mollendorff attributed another function to the epithelium
of the efferent ductule which sparked further interest to their
study. He found that after subcutaneous injection of Trypan blue,
the dye was sequestered within the epithelium of efferent duct.
It appeared that the dye was transferred from the blood stream
across the wall of the seminiferous tubules and then passed along
with the excretory products of the testis and taken up later by
the efferent ductule. Because of this reaction to Trypan blue,
Mollendorff postulated that the efferent ductal epithelium had
the capacity for reabsorption just like the epithelium of the
proximal convoluted tubules of the kidney.
Since then, numerous indirect observations of reabsorption at
the level of the efferent ducts has appeared in the literature.
Most of these studies involved ligation of the excurrent ducts
at different regions: efferent ductules, epididymis and ductus
deferens (Van Wagenen, 1924; 1925; Oslund, 1924a,b; 1926; Toothill
and Young, 1931; Young, 1933). It was found that ligation of the
efferent ductules caused a striking increase in testicular fluid
within 12 to 24 hours, followed promptly by pressure atrophy of
the germinal epithelium (Van Wagenen, 1924; Oslund, 1926; Young,
1933). In contrast, degeneration and atrophy of the testis was
found to be much slower in appearance and less severe when the
ligature was placed in the more distal parts of the excurrent
ducts (Oslund, 1924a,b; Young, 1933). These findings provided
evidence that much of the fluid produced in the testis is reabsorbed
by the ductuli efferentes and possibly by the proximal portions
of the epididymis. It was not until many years later though that
the absorptive function of the epithelium was confirmed by a direct
observation on the absorption of a tracer, Fe-59 by the nonciliated
cells (Burgos et al., 1959).
With the advent of the electron microscope in the 1950's, many
ultrastructural studies on the epithelium of the ductuli efferentes
appeared in the literature. The first electron microscopic examinations
of the ductuli efferentes appear to be those of Burgos (1957)
in the hamster and Young and Ladman (1957) in the guinea pig.
Like Mollendorff before them, these authors also have likened
the cells in the ductules, particularly the nonciliated cells,
to the cells in the kidney because of the similarity of their
ultrastructural features (e.g. microvilli, apical canaliculi,
vesicular structures, etc). Since then, the ultrastructure of
the epithelium in a wide variety of mammalian species (reviewed
by Hamilton, 1975; Jones, 1977; Robaire and Hermo, 1988) and birds
(see Table 2) has been described.
Most of these ultrastructural studies focus on the structural
correlation of absorptive function in efferent ductal epithelium.
The ductuli efferentes are the series of small tubules that
provide a vital link between the testis and the epididymis (Fig.1). These ductules
arise separately from the rete testis near the tunica albuginea.
The rete testis in the boar, bull, goat and dog are formed as
a central zone within the testis (Orsi et al., 1983; Hees et al.,
1987), but in man, rat, mouse, hamster and bird the rete testis
is found at the testis margin and usually forms an extra-testicular
portion that connects to the ductuli efferentes (Reid and Cleland,
1957; Cooper and Jackson, 1972; Budras and Sauer, 1975a; Amann
et al., 1977).
After piercing the tunica albuginea, at their initial or proximal
ends (the testicular segment), the ductuli are slightly convoluted,
embedded in the superior epididymal ligament (Shafik, 1987) and
fat, and may number between 2-33 depending on the species (Table 1). Toward the epididymis in the
rat, in the region called coni vasculosi, the ductuli become highly
tortuous, increasing the surface area facing the lumen (Hamilton,
1975; Jones, 1977). Within the coni, the ductules anastomose with
one another (Fig.1-2)
and become invested with a connective tissue capsule. As the ductules
approach the head of the epididymis, they become moderately coiled
and thinner.
|
Fig. 1. Gross anatomical arrangement of the ductuli efferentes in the rat. The ductules are encased in connective tissue and fat and begin at the rete testis that is encapsulated in the tunica albuginea of the testis. The proximal zone (P) is smaller in diameter than the conus region (C). The terminus (T) is a narrow zone that enters the connective tissue capsule covering the caput epididymidis where the single ductule changes into the initial segment (IS) of the epididymis. x 4.2. Fig. 2. Micro-dissected ductuli efferentes from the rat showing 7 proximal ends that merge in the conus region and finally form a single ductule or terminus. |
DEVELOPMENT
It was generally accepted that the efferent ductules, epididymis,
vas deferens, and seminal vesicles are mesonephric (Wolffian)
duct in origin (Setchell, 1978), with the former two regions arising
from the upper segment, the vas deferens from the middle segment,
and the seminal vesicles from the lower segment (Howards, 1983).
The development of the Wolffian duct occurs during fetal life
under the influence of testosterone and not dihydrotestosterone
(Wilson et al., 1981; Byskov and Hoyer, 1988). This influence
appears to be local because if the animal is unilaterally castrated
before sexual differentiation has occurred, the Wolffian duct
develops only on the un-operated side (Setchell, 1978).
However, as early as 1926, Wilson believed that the efferent ductules
originated from the mesoneph rictubules or glomeruli (Wilson,
1926; Stampfli, 1950; Budras and Meier, 1981; George and Wilson,
1988). This hypothesis was confirmed by immuno-histochemistry
using antibodies directed against the kidney which was shown to
cross-react only with the ductuli efferentes suggesting that the
ductuli efferentes are derived from the proximal mesonephric tubules
(Linder, 1971; Croisille et al., 1977, 1978; Croisille, 1981).
Furthermore, Marshall et al (1979) have found in rats exhibiting
congenital renal agenesis and mesonephric anomaly (ACI rats) that
a portion of the caput epididymis and the ductuli efferentes were
present while all other Wolffian duct structures were missing.
This also confirmed Wilson's original finding that these two regions
of the excurrent duct system originate from the mesonephric tubules
rather than the mesonephric duct. In fact, it has been shown in
the sheep that the efferent ducts originate from the so-called
giant nephron of the mesonephros (Zamboni and Upadhyay, 1982).
More recently, however, Takeuchi (1992) has shown using gamma
glutamyl transpeptidase as a marker that the ductuli efferentes
originate from the mesonephric duct. In the bird, the proximal
efferent ductules arise from the glomerular capsule of the mesonephros
and the distal efferent ductules develop from the tubular component
of the mesonephros (Budras and Meier, 1981).
Ciliated cells appear in the ductuli efferentes by day 5 in
the mouse and by day 28 in the rat (Jordan and Helvestine, 1923;
Reid, 1959). Differentiation of the epithelium and the formation
of the conus region begins around day 32 in the rat. In the human,
the anlage of the ductuli efferentes has been found to first appear
in embryos 13-17 mm long (6th week of development) as accumulations
of epithelial cells and primary germ cells between reducing glomeruli
and mesonephric tubules (Krutsiak and Kumka, 1988). Canalization
of this cellular accumulation takes place during the 8th week
of development. At 20-28 weeks, the mesonephric tubules enlarge
to form the ductuli efferentes and head of epididymis (de Kretser
et al., 1982). The origin of the ductuli from embryonic elements
may explain why the epithelia of both the adult kidney (metanephros)
and efferent ductules have many structural and physiological similarities
(Hinton and Turner, 1988).
DEVELOPMENTAL ANOMALIES
Spermiostasis is a common abnormality appearing in ruminants
which is usually manifested by a firm enlargement of the epididymal
head (Gustafsson and Galloway, 1988; Wu, 1981). McEntee (1976,
1990) reported that a majority of the sperm granulomas in bulls
used for artificial insemination were located in the ductuli efferentes.
In many animals, sperm stasis was present, but granulomas had
not formed. This anomaly is said to be a disturbance of efferent
tubule development from the mesonephric structures, which appears
to induce the formation of tubules connected at only one end and
thus having a blind end (Fig.
5). During puberty in some species, the blind-ended tubules
fill with spermatozoa, the wall breaks down and spermatozoa gain
access to the interstitial tissue causing a granuloma.
Blind ending tubules that originate from rudimentary mesonephric
tubules are called "ductuli aberrantes" (Blom and Christensen,
1960). Two kinds of aberrant ductuli were distinguished by Hemeida
et al. 1976: (a) blind-ending tubules that originate from the
rete testis and the testicular segment of the efferent ductules
and (b) blind aberrant ductules which arise from the epididymal
segment. Both kinds of abnormalities have been reported in the
boar, goat, ram, bull, and stallion, which, especially in the
last two animals, are present in sufficient numbers to be considered
as a factor in spermiostasis (Blom and Christensen, 1960; Hemeida
et al., 1978). In the bull, because of their location, these aberrant
ductules could be confused with the normal ductuli, but can be
distinguished histologically by their smaller diameter and lack
of dense granules and vacuoles in the epithelial cells (Goyal,
1983). It is also thought that aberrant tubules may cause spermiostasis
in the ram (Ashdown and Ford, 1967). Blind-ending tubules have
also been reported in the dog (Hess and Bassily, 1988). In this
animal, sperm granulomas were associated with the blind ending
tubules (Fig. 4)
that were swollen and contained stagnant sperm and inflammatory
cells.
| Fig. 3. Drawings that represent the species
variations in morphological connections between efferent ductules
(ED), the rete testis (RT) and the epididymis (E). Even within
a species, there may be considerable variation in the number
of efferent ductules and the number of junctions prior to entering
the epididymis (Guttroff et al., 1992). Based upon the work of
Benoit (1926); Hemeida et al. (1978); Jonté and Holstein
(1987); Saitoh et al. (1990); Yeung et al. (1991); Guttroff et
al. (1992). Fig. 4. Rete testis/ductuli efferentes region in the dog taken from serial sections of Bouin's fixed tissue. A blind-ending efferent ductule containing a forming sperm granuloma (G) is derived a short distance from the rete testis (R). Normal ductuli efferentes (DE) are observed outside the tunica albuginea capsule (TA) that surrounds the testis (T). x 50. |
|
Figs. 5-7. Blind-ending ductuli efferentes in the rat. Fig. 5. An efferent ductule that terminates as a blunt end (arrow) in a highly coiled section that was micro dissected from the connective tissue. x 55. Fig. 6. Low magnification light microscopic view illustrating how the blind-ending tubules are segregated within a connective tissue zone (noted by the dots and arrows). These smaller tubules have a collapsed lumen and are smaller in diameter than the normal ductuli efferentes (DE). x 65. Fig. 7. Histological sections of the blind-ending tubules showing a reduced luminal area that is devoid of sperm and an epithelium consisting of ciliated (C) and nonciliated (N) cells that have reduced numbers of granules and vacuoles. x 480. |
There are relatively few differences in the general light microscopic
appearance of the ductuli efferentes among the animals studied;
some cells have more vacuoles, others have more dense granules.
In addition to the principal nonciliated cell, all efferent ductal
epithelia contain a population of ciliated cells. Detailed descriptions
of the histology of the efferent ductules for a number of species,
including diverse species such as rodents, dogs, the buffalo,
elephants and man, are available (see gross anatomy section for
the animals examined, also reviews by Hamilton, 1975; Jones, 1977;
and Robaire and Hermo, 1988).
The lining epithelium of the ductuli efferentes, depending on
the species and the author's viewpoint, is classified either as
pseudostratified columnar or columnar, composed of ciliated cells,
nonciliated cells, and a few basal cells and intraepithelial lymphocytes
and macrophages (Dym and Romrell, 1975; Hamilton, 1975; Ramos
and Dym, 1977; Hoffer and Greenberg, 1978; Byers et al., 1985;
Jones and Jurd, 1987; Hess and Bassily, 1988). As an exception
to this generally accepted classification however, Goyal and Dhingra
(1975) have reported the epithelium of the water buffalo (Bubalus
bubalis) to be devoid of cilia although their pictures showed
structures which appeared to be ciliary.] The nonciliated cells
also are frequently referred to as principal cells as in the epididymis
(Burkett et al., 1987a,b; Robaire and Hermo, 1988).
The wall of the ductules is formed by a single layer of columnar
epithelium supported by a thin layer of smooth muscle and connective
tissue. In histological cross-sections, the lumen of the ductuli
are typically empty or contain few spermatozoa (Figs.
8-10) except in the terminal segment where spermatozoa
appear to become more concentrated (Talo, 1981). The delineation
between rete testis and the efferent duct is abrupt, evidenced
by the epithelium changing from low cuboidal to columnar in a
sharp transition and a marked increase in peritubular capillaries
(Amann et al., 1977; Robaire and Hermo, 1988). Nevertheless, in
some species such as the boar, there is no clear demarcation of
the beginning. Wystub et al. (1989) described a flowing transition
at different levels, such that both kinds of epithelia are recognized
in the same area of the proximal efferent ductules.
|
Figs. 8-10. Normal histology of the ductuli efferentes in the rat. The tissue was fixed by vascular perfusion (Hess and Moore, 1993) and then embedded in glycol methacrylate for sectioning. Fig. 8. Proximal zone illustrating a wide lumen, numerous nonciliated cells (N) with apical dense granules and ciliated cells (C) with proluminal nuclei. The tubule is surrounded by a thin layer of smooth muscle cells (Sm). x 525. Fig. 9. Efferent ductule from the highly coiled conus region. Note the reduced lumen diameter, the epithelial groove (arrow), and the reduction in number of dense-granules. x 525. Fig. 10. Terminus zone illustrating the smaller size tubule and reduced height of epithelium. Dense granules are rare in the epithelial cells. x 525. |
| Figs. 11-12. Ductuli efferentes in the
dog, fixed by vascular perfusion and embedded in glycol methacrylate.
A zone of epithelium containing few granules and few apical vacuoles.
Fig. 11. In light microscopy the cell nuclei are seen at multiple levels within the epithelium. x 525. Fig. 12. In electron microscopy the nonciliated cells of this zone contain an extensive tubular element (T) component of the endocytotic apparatus. The apical border is lined by short microvilli (M) and the apical cytoplasm contains a few light vesicles (V), light-staining lysosomal granules (L), and mitochondria (Mi). A junctional complex (J) separates the non-ciliated cell from the ciliated (C). Microfilaments, (F); Nucleus, (N). x 10,550. |
| Figs. 13-14. Ductuli efferentes in the
dog, fixed by vascular perfusion and embedded in glycol methacrylate.
A zone of epithelium containing cells with basal and/or apical
dense granules and few apical vacuoles. Fig. 13. In light microscopy the vacuoles appear as large clear structures and the granules are densely stained. x 525. Fig. 14. In electron microscopy the noncilated cell cytoplasm contains an extensive endocytotic tubular apparatus (E), apical vacuoles (V) and lysosomal granules (G). Mitochondria are rather long in both the nonciliated and ciliated cells (C). Nc, nucleus of the ciliated cell. Nn, nucleus of the non-ciliated cell. x 4,200. Figs. 15-16.Ductuli efferentes in the dog, fixed by vascular perfusion and embedded in glycol methacrylate. A zone of epithelium containing cells with basal dense granules and numerous apical vacuoles. Fig. 15. In light microscopy the vacuoles (V) appear as large clear structures throughout the cytoplasm. Most nuclei are located in the basal region. C, ciliated cell. x 525. Fig. 16. In electron microscopy the noncilated cell cytoplasm contains numerous large vacuoles (V) and scattered lysosomal granules. The nuclei (N) in this region contain deep invaginations and are often compressed by the large vacuoles. x 8,000. |
| Figs. 17-18. Ductuli efferentes in the
dog, fixed by vascular perfusion and embedded in glycol methacrylate.
A zone of epithelium in which the epithelial cells exhibit "apocrine"
secretion. Fig. 17. In light microscopy the epithelium is characterized by nuclei that are located more apically and the basal cytoplasm contains light staining granules. The apical secretory blebs are seen protruding from the nonciliated cell surface into the lumen (arrows). The blebs start as small protrusions, then form balloon-like bulges, which pinch off at narrow bases that form below the rounded protuberances. x 525. Fig. 18. In electron microscopy the apocrine protrusion (A) consists of a membrane-bound balloon filled with a homogeneous proteinaceous material. The protrusion is formed between microvilli (M) and apical to the endocytotic tubular apparatus (E). C, cilia. x 8,000. |
The point of transition from the ductuli efferentes to the initial segment of the epididymis also appears to be abrupt as reported in the guinea pig (Hoffer and Greenberg, 1978) and the goat (Goyal and Williams, 1988). In these two animals, the epithelium suddenly changes from ciliated columnar cells to pseudostratified columnar cells with stereocilia. This appears not to be the case in humans where the transition has been found to be gradual - islands of epididymal cells are first seen interspersed with ductuli efferentes epithelium until pure epididymal epithelium is present (Jonté and Holstein, 1987).
Little variation in the structure of ciliated cells is reported.
Histologically, the ciliated cells are recognized by their deeper
staining appearance, the apical position of their nuclei, and
the tufts of long cilia protruding into the lumen (Robaire and
Hermo, 1988). Basal bodies are also easily recognized at the apical
cell surface (Hoffer and Greenberg, 1978).
In general, there is a greater proportion of nonciliated cells
in the proximal regions of the ductuli efferentes, and an increase
in the proportion of ciliated cells in the region nearest the
epididymis. The early study of Benoit (1926) found that the ratio
of ciliated and nonciliated cells in the epithelium in different
animals generally ranged from 1:3 to 1:8. More recent data suggest
similar ratios in the rat, the ratio being 1:5 in the initial
zone and coni vasculosus and 1:2 in the common ductulus efferens
(Jones and Jurd, 1987). A different situation occurs in the bull
where ciliated cells were found to comprise about 80% of the lining
epithelium (Hemeida et al., 1978).
Top
Histochemical, cytochemical and immunocyto-chemical studies on
the epithelium of the ductuli efferentes have shown that the epithelium
is rich in acid phosphatase, thiamine pyrophosphatase, alkaline
phosphatase and other non-specific esterases, carbonic anhydrase,
glycogen, PAS positive materials, ATPases, lipids and various
forms of glycoconjugates (Alsum and Hunter, 1978; Burkett et al.,
1987a,b; Byers and Graham, 1990; Cavazos and Feagans, 1960; Tingari
and Lake, 1972; Cohen et al., 1976; Ekstedt et al, 1991, Ekstedt
and Ridderstråle, 1992; Goyal et al., 1980; Ilio and Hess,
1992; Maneely, 1955, 1958; Martan et al., 1967; Montagna, 1952;
Singh et al., 1991a,b; Sinowatz et al., 1979; Yokoyama and Chang,
1971). Novel proteins like immobilin (Hermo et al., 1992a), glutathione
S-transferase (Veri et al., 1993) and sulfated glucoprotein-1
(SGP-1) (Hermo et al., 1992b; Igdoura et al., 1993) have also
been localized in the ductuli efferentes. Certain intermediate
filaments (low molecular weight) are found in the ciliated cells
of the efferent ductules, but both ciliated and nonciliated cells
are vimentin negative in the dog (Wakui et al., 1994). In contrast,
both vimentin and low molecular weight cytokeratins are expressed
in the human ductuli efferentes (Achtstatter et al., 1985; Dinges
et al., 1991; Kasper and Stosiek, 1989).
Acid phosphatase and thiamine pyrophosphate activities are localized
in the Golgi apparatus, the former also consistently being localized
in lysosomal bodies in the supranuclear region of both ciliated
and nonciliated cells (Alsum and Hunter, 1978; Yokoyama and Chang,
1971). The localization of these enzymes in the Golgi and lysosomes
is consistent with the endocytotic activity of cells in which
material taken up by the endocytotic apparatus, such as testicular
proteins, are degraded in lysosomal bodies (Hermo and Morales,
1984). Lysosomes and acid phosphatase are associated with lipid
droplets in the nonciliated cells and the disappearance of both
of these structures is seen following ductectomy (Niemi and Kormano,
1965) or ligation (Hermo and Morales, 1984; Robaire and Hermo,
1988). The significance of lipids in the ductuli efferentes is
not understood, but several hypotheses have been promoted, including
breakdown products of residual cytoplasm of spermatids that is
released from the seminiferous epithelium (Niemi and Kormano,
1965) and steroid synthesis (Budras and Sauer, 1975b; Tingari,
1973).
The localization of alkaline phosphatase activity in the epithelium
is somewhat inconsistent. Montagna (1952) reported a weak to moderate
alkaline phosphatase activity in the epithelium. Martan et al.
(1967) however showed its activity to be strongly positive in
the apical region of the epithelial cells while virtually absent
in the basement membrane. Alsum and Hunter (1978) on the other
hand demonstrated alkaline phosphatase activity to be negative
in the epithelium but strong in the basal region, particularly
in the smooth muscle cells surrounding the tubules. No particular
function has been attributed to this enzyme except the general
function of absorption.
Studies of carbonic anhydrase activity in the excurrent ductal
epithelium of the rat (Cohen et al., 1976) and the bull (Goyal
et al., 1980) have shown that the highest activity of the enzyme
occurs in the ductuli efferentes. Cohen et al. (1976) found intense
reaction in the entire epithelium of the ductuli while Goyal et
al. (1980) found the activity confined only to the nonciliated
cells. Additionally, in the bull, the luminal border of cells
was positive only in ductules lined by type III cells. Carbonic
anhydrase is thought to play a role in the transfer or accumulation
of H+ or HCO3- ions in organs of secretion. The final cellular
product may be acidic (as in the stomach or kidney) or alkaline
(as in the pancreas) (Cohen et al., 1976). The enzyme may be responsible
for the change in pH (from 7.2 to 6.4) in the luminal contents
between the testis and caput epididymis (Levine and Marsh, 1971).
Adenosine triphosphatase (ATPase) activity has been reported to
be weak in the ductuli efferentes epithelium of the opossum (Martan
et al., 1967). However the methodology used in this earlier study
was a variant of the original Wachstein-Meisel (1957) method,
which has now been discounted because it utilizes a concentration
of lead which inhibits the enzyme (Ernst and Hootman, 1981). A
more recent and highly accurate immunological localization of
sodium and potassium activated ATPase has detected the presence
of the enzyme in the testis, ductuli efferentes and epididymis
(Byers and Graham, 1990). The staining was especially heavy in
the basolateral region of the ductuli efferentes compared to the
epididymis. In the epididymis, Na+,K+-ATPase activity has been
found to be stimulated by the mycotoxin, T-2 (Singh et al., 1985).
The strong presence of this transport enzyme in the epithelium
of the ductuli efferentes and the epididymis provides evidence
that a sodium coupled fluid transport occurs in these parts of
the excurrent duct of the testis.
The Na+,K+-ATPase enzyme was also localized ultracytochemically
and biochemically using its p nitrophenyl phosphatase activity
in ductuli efferentes of the rat (Ilio and Hess, 1992). Enzymatic
activity was demonstrated along the cytoplasmic side of the plasmalemma
of the ductal epithelial. The most intense deposition of reaction
products was found on the plasmalemma delimiting the lower lateral
and basal regions of the cells. The plasma membranes forming the
microvilli, apical junctional complexes were devoid of reaction
product while the mid-lateral membranes showed a weak reaction.
The enzyme reaction was potassium-dependent and was abolished
by addition of 10 mM ouabain to the incubation media. Enzyme activity
decreased significantly from proximal to distal regions of the
ductules. A unified model for water absorption was proposed for
efferent ductules based upon this data (Ilio and Hess, 1992) and
that of others. This model incorporates both trans-cellular and
para-cellular movements of water, where some intracellular water
is used in the reaction of carbonic anhydrase, which would result
in the production of H+ for secretion into the lumen.
The distribution of 1-glutamyl transpeptidase in the mouse epididymis
was examined recently (Agrawal et al., 1989). It was found that
the activity of this enzyme was exhibited strongly in the epithelium
of the ductuli efferentes, and was localized both in supranuclear
and basal locations. The enzyme was responsible for the removal
of L-glutamic acid from the amino terminal of peptides and proteins
or for the transfer of the amino acid to other amino acids by
its enzymatic activity. It was postulated that this enzyme may
be involved in the coating of spermatozoa with negatively charged
moieties during their transit in the excurrent duct.
PAS+ substances are frequently encountered in the apical region
of the epithelium particularly in the multiple oval granules of
different sizes (Martan et al., 1967; Alsum and Hunter, 1978).
Further studies of these substances in the mouse by lectin-horseradish
peroxidase reaction (Burkett et al., 1987a,b) have identified
several glycoconjugates with sugar moieties which appeared to
have been synthesized in and secreted by the Golgi apparatus in
both ciliated and the nonciliated cells. In the nonciliated cells,
these glycoconjugates were sialic acid, some form of galactosamines,
L-fucose, and sugars with N-glycosidic side chains. Sialic acid
and galactosamine were predominant in the ciliated cells. From
these results, the authors concluded that synthesis and secretion
of glycoconjugates that bind to spermatozoa appear to involve
more regions of the reproductive tract than was previously thought.
In particular, the finding that -D-GalNAc was stained in previously
unreactive testicular spermatozoa during their transit in the
ductuli and also in the Golgi zone and apical surface of the nonciliated
cells gives evidence that the ductuli do secrete a glycoconjugate
which coats spermatozoa as they pass from the testis to the epididymis.
Angiotensin converting enzyme (ACE) has also been localized both
by biochemical and autoradiographic methods in the testis and
ductuli efferentes (Hohlbrugger et al., 1982; Strittmatter and
Snyder, 1984; Strittmatter et al., 1985). It was postulated that
the testis and the ductuli efferentes are probable sites of synthesis
of the enzyme in the reproductive tract (Hohlbrugger et al., 1982).
The enzyme converts angiotensin I to the angiotensin II involved
in blood pressure regulation. Functional Angiotensin II receptors
also have been demonstrated in the epididymis (Grove and Speth,
1989). No clear function has been assigned to ACE and angiotensin
II in the epididymis, but they are thought to be important factors
in sperm motility and spermiogenesis as well as muscular contraction.
ULTRASTRUCTURE
The electron microscopic appearance of the epithelium of the ductuli
efferentes has been examined in a wide range of species (Table 2).
NONCILIATED CELL
Top
The fine structural features of the nonciliated cells appear to
indicate a common function in the many species examined. In general,
these cells have distinct features which suggest that they are
specialized for the uptake of particulate material and fluid from
the lumen. The apical cytoplasm is characterized by the presence
of microvilli in the apical cytoplasm and a profusion of apical
canaliculi, vesicles, and a variety of large vacuoles and membrane-bound
bodies of different shapes, sizes and staining intensities. Over
the years, with more detailed ultrastructural and kinetic studies
appearing in the literature, the nomenclature of these structures
has changed. The following description of the ultrastructure of
the epithelium, unless otherwise indicated, is primarily based
on the account of Hermo et al. (1988) and Robaire and Hermo (1988)
in the rat. The current nomenclature for the vesicular structures
in the epithelium is used.
The apical cytoplasm of the nonciliated cell typically possesses
a brush border with well-developed but clumped microvilli (Hamilton
et al., 1977). Between the bases of microvilli are large dilated
tubular elements with occasional bulbous extremities called tubular
coated pits which may extend for some distance into the apical
cytoplasm. In early studies, these pits were termed 'canaliculi'
(Burgos, 1960; Ladman, 1967; Ramos and Dym, 1977; Wrobel, 1972;
Flickinger et al., 1978; Jones et al., 1979; Aire, 1980) and were
found to be continuous with coated vesicles and large vacuoles
(Wrobel, 1972). The coated pits are lined by fuzzy material on
their luminal aspect and with a clathrin-like lattice coat on
their cytoplasmic aspect (Montorzi and Burgos, 1967; Robaire and
Hermo, 1988).
The membrane-bound apical tubules are found immediately beneath
the cell surface. These tubules vary in length but appear circular
in cross section, with a constant diameter. They are fairly straight,
and have a uniform, moderately dense-staining content that fills
their lumen. In confirmation of the earlier studies, images of
transition between the tubular coated pits and apical tubules
were found, suggesting that these tubular pits give rise to apical
tubules (Hermo et al., 1988).
Endosomes which are larger dilated membranous vacuoles (described
in early studies as coated vesicles) also are found in the apical
region. These have pale-stained lumina, sometimes containing a
fine flocculent material. Apical tubules are occasionally seen
connected to endosomes (Wrobel, 1972; Robaire and Hermo, 1988).
Deeper in the apical region are multivesicular bodies showing
either a pale moderately dense or dense staining matrix. Many
small vesicular profiles are often seen around these bodies. In
addition, small coated and uncoated vesicles of similar sizes
also are found in this region.
In the supranuclear region are numerous spherical membrane-bound
bodies that stain with different intensities. In the initial zone
of the ductules, these membrane-bound bodies are pale and contain
a fine flocculent material while in the terminal zone, such bodies
have a homogenous dark-staining pattern. These membrane-bound
bodies stain positive with acid phosphatase and thus are considered
to be lysosomes.
Appropriate markers for electron microscopy have demonstrated
that the coated pits, apical tubules, endosomes, multivesicular
bodies and lysosomes are components of an elaborate endocytotic
apparatus which is capable of fluid-phase and adsorptive (Morales
and Hermo, 1983; Hermo and Morales, 1984; Hermo et al., 1985)
and possibly receptor-mediated endocytosis (Byers et al., 1985;
Veeramachaneni and Amann, 1991; Veeramachaneni et al., 1990).
Testicular fluid is taken up sequentially by the endocytotic apparatus
from coated pits to multivesicular bodies then to lysosomes and
broken down by means of hydrolytic enzymes (Yokoyama and Chang,
1971; Wrobel, 1972; Hermo and Morales, 1984).
The following route of endocytosis has been proposed by Hermo
et al. (1988). Tubular coated pits invaginate from the apical
plasma membrane, pinch off, and undergo constriction accompanied
by the gradual loss of their bristle cytoplasmic coat to form
apical tubules. The average time required for this process is
5 minutes. Apical tubules fuse to form endosomes; 30% of the apical
tubules recycle back to the apical plasma membrane; the rest partake
in the transformation of endosomes to multivesicular bodies to
secondary lysosomes. The average time required for an apical tubule
to fuse with an endosome is 2 min. Recycling of apical tubules
back to the apical plasma membrane requires an average turnover
time of 39 minutes. Functionally, the endocytotic activity of
the epithelium has been implicated in the regulation of the composition
and quantity of the intraluminal fluid (Morales and Hermo, 1983).
The differential staining characteristic between lysosomes of
the initial zone, where these granules are pale-staining, and
the terminal zone where they are deeply osmiophilic, is suggestive
of regional differences in the endocytotic activity along the
duct. Thus, it is believed that nonciliated cells in the initial
zone take up more fluid while these cells in the terminal zone
take up more particulate matter (Robaire and Hermo, 1988). In
contrast, an opposite situation occurs in the bull and goat in
which more fluid is taken up in the terminal zone where type III
cells with light staining vacuoles abound (Goyal and Williams,
1988; Goyal and Hrudka, 1980,1981).
Aside from lysosomes, other organelles such as mitochondria, rough
endoplasmic reticulum and Golgi apparatus are also found in the
supranuclear region. The Golgi appears to show no evidence of
secretory granule formation (Robaire and Hermo, 1988). Thus in
the rat, the nonciliated cells are not thought to be secretory
(Hoffer, 1972; Robaire and Hermo, 1988) in contrast to the situation
reported for the bull and the goat in which the granules and vacuoles,
presumably of Golgi origin, are thought to be secretory (Goyal
and Hrudka, 1980; 1981; Gray et al., 1983). Instead, from an electron
microscope stereoscopic study, the Golgi has been determined to
actively produce lysosomal enzymes destined for the numerous secondary
lysosomes in the supranuclear cytoplasm of the cell (Rambourg
et al., 1987). Peroxisomes of unknown function also are found
in the supranuclear area of the cell.
| Figs. 19-21. Lipid droplets in the epithelium
of rat proximal ductuli efferentes. Fig. 19. Cryostat sections stained with Oil Red O show a heavy localization of lipids in the basal cytoplasm. x 650. Fig. 20. Hematoxylin and eosin stained section of ductuli efferentes embedded in glycol methacrylate. One side of the tubule contains nonciliated cells with numerous lipid droplets in the basal cytoplasm (arrows), while the opposite side contains relatively few lipid droplets. x 330. Fig. 21. A cryostat section stained with Oil Red O showing a region that is devoid of lipid, with only a few droplets along one side (arrows). x 650. |
Top
The electron microscopic appearance of the ciliated cells is less
studied. These cells have been characterized as unremarkable,
possessing the organelles and organization typical of ciliated
cells elsewhere (Hoffer, 1972). These were also found to possess
in reduced amounts, vesicular structures that are involved in
the uptake of material from the lumen through adsorptive as well
as fluid-phase endocytosis (Hermo et al., 1985). Thus, aside from
the function of moving fluid and spermatozoa through the duct,
ciliated cells appear to be capable of modifying the composition
of the luminal fluid by the process of endocytosis. In the dog,
most ciliated cells are typical in appearance, having long cilia,
short rootlets and an apical cytoplasm filled with mitochondria,
microtubules and small vesicular elements (Fig.
22). In some regions the nucleus is displaced more apically
than in the noncilated cells (Fig. 15) and may contain lysosomal
granules and vacuoles.
| Fig. 22. Ciliated cell from the ductuli efferentes in the dog. The long motile cilia contain a distinct axonemal complex (A), well developed basal body (B) and short striated rootlets (R) that extend straight into the cytoplasm. Coming off the basal body is a foot process (Fp) to which microtubules (Mt) appear to be attached. Long narrow mitochondria (M) are typical in the apical cytoplasm. D, desmosome. x 25,600. |
Multinucleate Epithelial Cell
In an ultrastructural study of ductuli efferentes from 40 adult
men, Nistal et al. (1990) found a different type of cell in all
the subjects they studied. These cells appeared in the luminal
protrusions of epithelial folds and corresponded to either principal
or ciliated cells. The ultrastructure did not differ from that
of their respective mononucleate cells except that they contained
3-20 closely juxtaposed nuclei.
Junctional Complexes
Both ciliated and nonciliated cells are attached at their boundaries
to adjacent cells by a juxtaluminal junctional complex composed
of three distinct components: (a) the zonula occludens (tight
junction) adjacent to the luminal surface; (b) below this, the
zonula adherens (intermediate junction) and (c) macula adherens
(desmosomes) (Jones, 1977; Ramos and Dym, 1977; Yokoyama and Chang,
1971). From freeze fracture studies in the rat (Suzuki and Nagano,
1978), the tight junctions between adjacent nonciliated cells
have been found to be segmented or incomplete. Instead, belt-like
gap junctions are seen associated with or replacing the poorly
developed tight junctions. In contrast, well-developed tight junctions
are found between two adjacent ciliated cells.
The presence of junctional complexes that are of the leaky type
suggests that the permeability barrier of this epithelium is weak
and may facilitate bulk fluid movement (Pudney and Fawcett, 1984).
However, a weak barrier might not be the case at all for the lumen
to blood direction of flow since most trace studies using horseradish
peroxidase in rodents (Morales and Hermo, 1983; Hermo and Morales,
1984) have not demonstrated the transfer of tracer from lumen
to blood; instead egress of tracer has only been demonstrated
from the blood to lumen direction (Suzuki and Nagano, 1978). This
leaky type of barrier could instead suggest that the ductuli efferentes
may be the primary site of antibody invasion along the excurrent
duct which can lead to sperm agglutination and ductal occlusion
(Dym and Romrell, 1975; Tung and Alexander, 1980).
Basolateral Membranes
The lateral plasma membranes below the junctional complexes are
often straight, however, in the basal and supranuclear regions
the lateral membranes often show interdigitations with one another
(Hoffer, 1972; Ramos and Dym, 1977) sometimes occluding the dilated
intercellular spaces (Jones and Jurd, 1987). These interdigitations
can be complex, forming a well-localized "tubular network"
(Robaire and Hermo, 1988; Ilio and Hess, 1992). The intercellular
spaces have also been found to be dilated especially in the basal
regions when absorption is active (Pudney and Fawcett, 1984).
The occurrence of these membrane amplifications and putative dilated
intercellular channels strongly suggests that fluid transport
in this part of the tract may be coupled to active solute transport
(Suzuki and Nagano, 1978). However, despite the formation of tubular
networks between epithelial cells, the lateral membranes show
considerably less amplification (Ilio and Hess, 1992) than is
normally found in resorptive epithelium such as the proximal convoluted
tubules of the kidney.
Sub-Epithelial Layer
The epithelium of the ductuli efferentes rests on a well-defined
basement membrane consisting of an amorphous component of moderate
electron density and fibrils of the subadjacent connective tissue
(Montorzi and Burgos, 1967). In the monkey however, the basal
lamina is characterized as a very thick (about 2-8 µm),
homogeneous, amorphous component of moderate electron density.
Below the basal lamina is the periductal connective tissue in
which fibrocytes and abundant collagen fibers are recognized.
One or more layers of smooth muscle cells alternating with interstitial
layers of collagen and elastic fibers are also found (Lopez and
Breuker, 1986).
Blood capillaries near the epithelium have thin endothelium and
are equipped with fenestrations and vesicles in conformation with
the absorptive capacity of the ductules (Montorzi and Burgos,
1967; Suzuki, 1982). A rich sympathetic innervation comprising
of short adrenergic neurons, particularly in the smooth muscle
layer of the coni vasculosi, has been demonstrated in marsupials
(Maruch et al., 1989). The nerve supply in rats was described
as double adrenergic-cholinergic innervation forming perivascular,
subepithelial and muscle plexuses (Garnacho et al., 1989). Adrenergic
innervation however appears not to be a constant feature in higher
mammals (El-Badawi and Schenk, 1967). In the boar, Kaleczyc et
al. (1993) have shown that the ductuli efferentes are weakly innervated.
DUCTULI EFFERENTES IN CULTURE
Top
In vitro epithelial systems are intrinsically difficult
to establish so that the tissue is maintained in a "normal"
functional state. Two basic approaches have been used for the
culture of efferent ductal epithelium: a) organ cultures and b)
cultures of isolated epithelial aggregate or fragments. The isolation
of individual epithelial cells for culture has never been reported
for this organ. Rozewicka et al. (1985) demonstrated the growth
of a mixture of cells and aggregates; however, high resolution
histological and ultrastructural confirmation of cell types was
not presented.
Organ cultures have been useful in demonstrating that sperm maturation
in the caput epididymidis is androgen-dependent (Orgebin-Crist
et al., 1976; Orgebin-Crist & Ménézo, 1980;
Klinefelter & Hamilton, 1984), specifically for dihydrotestosterone
(Orgebin-Crist et al., 1976). However, the disadvantage of such
cultures, regardless of the addition of a continuous flow system,
is that the epithelium depends upon diffusion for movement of
nutrients and waste products. Another difficulty with organ culture
is the interpretation of data. It is impossible to separate responses
due to epithelial function versus those coming from the surrounding
connective tissues. Also, in organ cultures, the luminal surface
of the epithelium is not bathed with potentially important factors
that may be essential for normal activity, particularly in the
head of the epididymis where androgens bound to ABP are taken
up by endocytosis (Turner, 1988; Robaire & Hermo, 1988). Microcannulation
of the efferent ductules in culture would avoid this problem;
however, use of this method has never been reported for these
tubules, possibly because of their small diameter and the difficulty
in isolating such tortuous ductules.
The most recent methods used for the culture of epididymal tissue
have depended upon the isolation of tubular aggregates or fragments.
This method requires carefully designed, enzymatic digestions
of dissected tubules and the plating of cell aggregates on an
extracellular matrix (Byers et al., 1985; Klinefelter, 1992; Raczek
et al., 1992). Using this new procedure, efferent ductal epithelium
was grown on plastic for the first time in a manner that supported
ciliary beat for up to 7 days (Byers et al., 1985). This system,
however, did not encourage polarization of the cells; the cells
assumed a flattened, squamous appearance although they contained
a complement of organelles similar to cells in vivo. In
contrast, epithelial sheets maintained on permeable extracellular
matrix supports were polarized and exhibited a cuboidal/columnar
appearance (Byers et al., 1985). In addition, cells maintained
in this system also exhibited endocytosis of androgen binding
protein by coated pits. ABP was then sequentially seen in uncoated
endosomes, multivesicular bodies and lysosomes.
Klinefelter (1992) has taken a similar approach to tissue isolation
by combining collagenase digestion, pipetting and washing of epithelial
fragments and then plating the fragments on an inner-chamber filter
coated with an extracellular matrix made from rat tail collagen.
This novel method is now being used in our lab to culture efferent
ductal epithelium (Fig.
23) and should provide an ideal system for the study of
factors that regulate ductal function. Using as similar method
of isolation, but also including hyaluronidase in the final digestion,
human efferent ductal epithelium has also been maintained in a
similar manner for nearly one month (Raczek et al., 1992). They
found that ciliary beat could be maintained for 25 days and that
the monolayers supported typical epithelial ultrastructure.
| Fig. 23. Tissue culture of epithelium from the rat ductuli efferentes. The epithelium was isolated using the Klinefelter method (1992) by first uncoiling the tubules by microdissection and then digesting small segments of the isolated tubules.The digested sections were then pipetted repeatedly, washed, and re-digested twice. After a final pipetting, the epithelial fragments were washed and re-suspended in defined media. The cell plaques were added to the apical compartment of culture inserts coated with Type I collagen plus MatrigelTM. The cells were incubated at 34°C in atmosphere and 5% C02. These cells have grown in culture for 7 days. The cells migrate (arrows) away from the original placque (P) to form a thin monolayer and have maintained their ciliary beat for up to 15 days. x 65. |
MODULATION OF THE DUCTULI EFFERENTES
The regulation of ductuli efferentes epithelium is not well understood.
The primary focus of the few studies that have appeared in the
literature has been the effects of androgen withdrawal. In addition,
several articles have examined aging effects as well as the effects
of certain reproductive toxicants on the structure of the ductuli
efferentes.
Hormonal Regulation
Androgen control of the ductuli efferentes epithelium has been
studied following ligation of the ductuli in the goat and bull
(Goyal and Hrudka, 1980; Gray et al., 1983) and castration in
the bull (Goyal and Hrudka, 1980). In the bull, after bilateral
castration, the most obvious changes in the epithelium were the
involution of microvilli and endocytotic apparatus and the decrease
in alkaline phosphatase activity (Goyal and Hrudka, 1980). All
of these properties were restored to normal levels upon testosterone
administration. Furthermore, ligation of the ductuli did not affect
endocytosis nor alkaline phosphatase activity although the microvilli
became less abundant. It was concluded that while luminal androgens
are essential for regulation of epithelial structure, they are
not necessary for the maintenance of the reabsorptive apparatus
of the nonciliated cells.
In the goat, Gray et al. (1983) demonstrated that deprival of
androgen-rich rete testis fluid caused a marked decrease in the
number of large vacuoles in the type III cells and the appearance
of more type I cells in the epithelium. These workers concluded
that the reabsorptive apparatus of the epithelium is dependent
on the presence of luminal androgens, a conclusion that is opposite
that of Goyal and Hrudkas (1980) for the bull.
It also is interesting to note that in the initial segment of
the epididymis in the rat, while exogenous androgens fail to prevent
regression of the epithelium upon ligation, deprival of rete testis
fluid did not affect the endocytotic apparatus (Fawcett and Hoffer,
1979) and even increased its activity upon castration (Moore and
Bedford, 1979). Whether these differential responses to androgen
withdrawal are due to species differences is not known.
Studies in our laboratory suggest that the activity of Na+,K+-ATPase
in the different regions of the ductuli and epididymis is dependent
on circulating androgens, at least in the rat. Castration markedly
reduced enzyme activity in all the regions examined by about 50%
(Fig.24). Administration
of testosterone and dihydrotestosterone to castrated animals increased
enzymatic activity, but only in the caput was activity returned
to normal levels. This result is in parallel with the observed
abolition of water absorption by castration and its reversal by
androgen administration in the cauda epididymidis (Wong and Yeung,
1977b, Wong et al., 1978a,b, 1979). The effect of estrogen on
Na+,K+-ATPase activity in the ductuli efferentes was also studied.
Administration of 17ß-estradiol to intact animals significantly
lowered enzyme activity in the proximal ductuli and caput epididymis
(Fig. 25). When
castrated animals were treated with high doses of estrogen, no
significant changes were seen in the enzyme activity except in
the conus where the activity was elevated to 138% of castrate
values (Fig.24).
Estradiol when given in combination with testosterone generally
diminished the enhancing effect of testosterone but the overall
effect was still significantly higher than castrate values. These
results suggest that there may be a selective modulation of enzyme
activity by estrogen along the different regions of the excurrent
duct of the testis.
|
Fig. 24. Effects of castration and hormone treatment on Na+, K+-ATPase activity in the ductuli efferentes and epididymis of the Sprague-Dawley rat. Enzyme activity was measured as reported by Ilio and Hess (1992) for micro-dissected efferent ductules. Shams were treated surgically without castration or injections. Castration was performed by midline abdominal incision under anesthesia without disturbing |the surrounding vasculature and 5 days prior to hormone treatments. After castration the animals received daily hormone injections subcutaneously: either corn oil as vehicle (0.1 ml/kg); testosterone (T; 2 mg/kg); 5 dihydrotestosterone (DHT) (2 mg/kg); 17ß-estradiol (E2; 25 µg/kg) for a period of 5 days. Number of animals: three to five per group. Significant differences between treatment effects are indicated by different letters within a ductal region (P < 0.05). Fig. 25. Effects of estrogen treatment on Na+, K+-ATPase activity in the ductuli efferentes and epididymis of the Sprague-Dawley rat. The animals were given sham operations without castration or ductal ligations 5 days prior to treatment with 17ß-estradiol (E2) (25 µg/kg) for a period of 5 days. Enzyme activity was measured as reported by Ilio and Hess (1992) for micro-dissected efferent ductules. Number of animals: 3-5 per group. Significant differences between treatment effects are indicated by (*) within a ductal region (P < 0.05). |
| Fig. 26. Effects of efferent ductal ligation on Na+, K+-ATPase activity in the ductuli efferentes and epididymis of the Sprague-Dawley rat. The ductuli efferentes were ligated at their rete testis origins without disturbing the surrounding vasculature and 5 days prior to determining enzyme activity. Shams were treated surgically but without ligation. Enzyme activity was measured as reported by Ilio and Hess (1992) for microdissected efferent ductules. Number of animals: three to five per group. Significant differences between treatment effects are indicated by (*) within a ductal region (P < 0.05). |
Top
The postnatal maturation of efferent ductules has been studied
in the rat (Francavilla et al., 1986) and the bull (Goyal and
Hrudka, 1981). It was found in the rat (Francavilla et al., 1986)
that the endocytotic apparatus is already extant by the first
week of life. Cilia were first noticed at the third week of life
concomitant with muscle cell development. The appearance of the
vesicular structures for endocytosis, cilia and muscular elements
preceded the canalization of the seminiferous cords which materializes
at the end of the second week. This suggested to the authors that
maturation of the efferent duct is not triggered by the passage
of testicular fluid and was complete by 35 days after birth.
In the bull, the study of postnatal development focused on the
differentiation of the absorptive apparatus, the synthetic machinery
and the regional differences (Goyal and Hrudka, 1981). It was
found that the difference in tubular diameter between the proximal
and distal regions is expressed shortly after birth, that morphological
features of reabsorption were established by 25 weeks, and that
the formation of granules and vacuoles were not observed until
35 and 50 weeks of age.
Effects of Reproductive Toxicants
Administration of certain infertility agents having known degenerative
effects on the seminiferous epithelium also has been shown to
alter the organization of the efferent ductal epithelium. In a
study of ductuli efferentes topography in rats, Cooper and Jackson
(1972) showed that administration of ethylenedimethane sulfonate
(EDS) caused hyperplasia in the ductuli efferentes epithelium
as well as the formation of small intratubular sperm retention
cysts during the early stages of exposure. These small formations
later coalesced causing some cellular reaction in the stroma.
In the same study, large doses (120 mg/kg) of -chlorhydrin were
also shown to form intratubular spermatoceles in the ductuli efferentes,
with larger formations near the rete testis than in the more distal
regions. The epithelium in these spermatoceles was attenuated
and fragmented; sometimes spermatozoa were seen to escape into
the connective tissue resulting to the formation of granulomata.
Eventually, hyalinization and ductal fibrosis supervened. A relatively
high dose of -chlorhydrin induced the blockage of ductuli efferentes
in the conus region, resulting in back-pressure atrophy of the
testis (Jones, 1978).
An even higher dose of -chlorhydrin (140 mg/kg) given to rats
orally produced obstructive lesions in the initial segment of
the epididymis and stimulated the nonciliated cells of the ductuli
efferentes to form pseudopodia and to phagocytize sperm (Hoffer
and Hamilton, 1974; Hoffer et al, 1975). The epithelium of the
ductuli however was found to be otherwise normal in appearance
in contrast to earlier findings of Cooper and Jackson (1972).
Dinitrobenzene, a chemical widely used in the manufacture of dyes,
plastics, and explosives, has also been reported to cause a limited
number of obstructions in the ductuli efferentes, some eventually
proceeding to fibrotic obliterations, calcifications and granulomatous
formations (Linder et al., 1988a). However, no detailed description
of the changes in the ductuli efferentes epithelium was offered.
Cadmium also produces lesions in the ductuli efferentes and caput
epididymidis (Mason and Young, 1967). The pathological changes
following cadmium exposure included sperm stasis and impaction,
ciliary loss, epithelial hyperplasia, edema, and phagocytosis
of sperm. Smoking also has been implicated to affect epithelial
structure by causing detachment of the ciliary tufts in the efferent
ductules or the epididymis (Bornman et al., 1989). Similarly,
high dosage of fluoride also produces loss of cilia as well as
peeling off of the epithelial lining in the ductuli efferentes
and epididymis (Susheela and Kumar, 1991).
Occlusions of the ductuli efferentes are a common observation
following treatment with the fungicides Benomyl and carbendazim
in rats (Hess et al., 1991; Nakai et al., 1992). The fungicides
are important environmental chemicals for controlling mold, fungus,
and other organisms that infest laws and garden and orchard plants.
However, these chemicals are known reproductive toxicants in males.
Work from our laboratory has shown that the primary mechanism
for the induced testicular atrophy is the occlusion of the efferent
ductules of the testis (Hess et al., 1991; Nakai et al., 1992).
At moderately high dosages, sperm and sloughed germ cells from
the seminiferous epithelium became compacted, causing the ductuli
efferentes to swell, which resulted in the accumulation of fluid
within the seminiferous tubules and subsequent "back pressure"
atrophy of the testis (Carter et al., 1987). Inflammation of the
occluded ductules then induced permanent lesions, such as fibrosis
and abnormal regrowth of the epithelium (Figs.
27, 28). The observation of efferent ductal occlusions
after benzimidazole exposure is highly significant in the study
of male reproductive toxicology. If occlusions occur during the
early period of a subchronic or subacute test, then all subsequent
exposures to the toxicant will have little to do with the resulting
testicular atrophy because regression will be a direct result
of the rapidly formed occlusion. In light of this mechanism of
toxicity, studies in which testicular atrophy is found after chronic
or subchronic exposures may need to be re-examined for terminal
lesions in the efferent ductules following acute exposures.
| Fig. 27. Ductuli efferentes of adult rats
exposed to a single dose of the fungicide benomyl (400 mg/kg).
On day 2 post treatment, the testis and reproductive tract were
fixed by vascular perfusion and embedded in glycol methacrylate
resin for histological evaluation. The ductules are occluded
and swollen with compacted sperm and sloughed seminiferous epithelial
cells. Numerous leukocytes have surrounded the ductal epithelium
(arrows). x 140. Fig. 28. Ductuli efferentes (DE) 70 days post treatment with a single dose of the benomyl metabolite carbendazim (400 mg/kg). Note the abnormal growth of the ductal epithelium (arrows) at the former site of occlusion. x 120. |
FUNCTION OF THE DUCTULI EFFERENTES
Top
From the previous description of the morphology of the ductuli
efferentes, a number of functions have been alluded and ascribed
to the ductuli efferentes. The first function, that of moving
sperm and fluid from the testis to the epididymis has been a subject
of great debate since the last century. Next, the capacity to
reabsorb a large volume of fluid entering from the rete testis
appears to result in a multi-fold increase in the concentration
of spermatozoa. While this function of the epithelium is generally
accepted, there is still controversy on the mechanism of this
process. Also, through the use of particulate markers, the ductuli
efferentes have been implicated to internalize a number of molecules
found in the luminal fluid. Additionally, secretion as a function
has been suggested, although the data for this particular function
is scarce.
Other studies have focused on the ability of the general excurrent
ductal epithelium to (a) support intermediary metabolism; (b)
synthesize and metabolize steroids; and (c) synthesize and metabolize
other compounds. References are made to specific contributions
by the efferen ductal epithelium. Finally, the function of phagocytosis
of sperm has also been discussed, particularly in epithelium perturbed
with reproductive toxicants.
Conduit For Spermatozoa
The time interval which spermatozoa require to pass along the
length of the ductuli efferentes has been determined to be 45
minutes in the rat (English and Dym, 1982), but little is known
for other species. As discussed in the previous sections, it is
believed that the main forces that propel spermatozoa along the
ductuli efferentes are the motility of cilia and the contraction
of smooth muscle cells surrounding the epithelium (Lucas, 1932).
Evidence for this function is the observation that the smooth
muscle layer contracts in vivo (MacMillan and Harrison,
1955; Risley, 1958) and that tracers injected into the ductuli
lumen are transported even when the fluid flow from the testis
is prevented by a ligature (MacMillan and Harrison, 1955; MacMillan
and Aukland, 1960).
There appears, however, to be some controversy surrounding this
simplistic mechanism of sperm transport. Talo (1981) has observed
that although ciliary beat moved single spermatozoa and round
cells, the beat is not exclusively in the epididymal direction;
cilia situated on opposite sides of the lumen beat in opposite
directions. The cilia have been in fact postulated to reduce the
flow of fluids by creating a reflux (Winet, 1980). Hess (unpublished
observation using video microscopy of micro-dissected ductules)
has observed ciliary beat that appeared to serve a function of
simply stirring the fluid, possibly for homogeneous reabsorption
of fluids. Also, although contractions appeared to spread from
the terminal ductule toward the caput epididymis, moving the luminal
contents forward, sperm clumps in preterminal ductules were observed
to move back and forth but with negligible net movement (Talo,
1981).
In addition, other factors have been proposed to influence fluid
flow and sperm transport along the ductules: 1) constant secretion
of fluid by the seminiferous epithelium (Mason and Shaver, 1952);
2) contraction of the myoepithelial layer of the seminiferous
tubule and tunica albuginea of the testis (Hargrove et al., 1977);
3) vacuum created by the ejaculation of sperm from the lower tract
and by the absorption of fluid (Mason and Shaver, 1952); and 4)
increased pressure due to the pattern of branching and convergence
of ductuli (MacMillan, 1953; Talo, 1981).
In the first two instances, during the contraction of the myoepithelial
layer and tunica albuginea in the testis, a pressure gradient
was created in the excurrent duct, the upstream pressure being
greater than the downstream pressure (Hargrove et al., 1977).
Measurement of pressure events along the duct however has shown
the opposite to be true (Johnson and Howards, 1975; 1976); that
in fact, there is a positive pressure in the distal portions of
the duct. Winet (1980) has considered that although pressure can
indeed be generated by a testis pump, this pre-rete upstream pump
cannot maintain the rate of ductal flow against such a positive
pressure. He also stated that a pump created by absorption along
the ductuli and the initial segment of the epididymis will also
generate a negative pressure downstream, which is again the reverse
of the one measured.
Talo (1981), in his examination of the electrical activity in
the ductuli efferentes, found five to eight ductuli converging
into one common duct in the terminal zone. With this arrangement,
he stated that the cross sectional area of the lumen was decreased,
thus effectively increasing the speed of flow in the terminal
zone, at least in theory. Around the point of convergence of the
ductules, however, clumps of spermatozoa were seen with minimal
net movement; thus, Talo theorized that the balance of flow here
may have been compensated for by the reabsorption of fluid. Reid
and Cleland (1957), who also observed sperm clumps, believed that
the accumulation of sperm at this junction is purely mechanical,
"similar to the accumulation of driftwood in the narrows
of a flowing stream."
Using complex mathematical and physical formulae, a cilio-peristaltic
model for the mechanism of flow in the ductuli efferentes was
proposed by Winet (1980). This model was shown to be consistent
with the positive pressure gradient toward the epididymis. The
model predicted that the major contributor to fluid flow was smooth
muscle contraction. The cilia, in contrast, may reduce flow by
creating reflux. This effect may be reduced considerably with
an increase in fluid viscosity, which is in turn a function of
sperm concentration.
Absorption
Many physiological and micropuncture studies on the proximal
segments of the excurrent ducts in different species have confirmed
the original findings of Crabo (1965) that more than 90 % of the
fluids secreted by the seminiferous epithelium is reabsorbed in
the ductuli efferentes. The reported values nonetheless vary from
50 96% (Djakiew and Jones, 1983; Howards et al., 1975a,b; Jones,
1981; 1987; Jones and Jurd, 1987; Levine and Marsh, 1971; Turner,
1984).
Although the efferent ductules are now recognized as the major
site for rete testis fluid absorption, the underlying mechanisms
for absorption remains unsettled. Hoffer et al. (1973) and Flickinger
et al. (1978) and later supported by Goyal and co-workers (1980;
1981; 1988), proposed that endocytosis is the main mechanism by
which fluid is moved across the epithelium. Hamilton (1975), Hohlbrugger
(1980) and Jones and Jurd (1987) on the other hand have stated
that fluid transport in this region may be coupled to the active
transport of electrolytes.
According to Hoffer et al. (1973) and Flickinger et al. (1978),
there are no morphological manifestations such as prominent basal
folds and dilated intercellular spaces to indicate that fluid
reabsorption is accomplished by pumping solutes to create a standing-gradient
in the intercellular clefts deep to the occluding junctions as
dictated in the standing-gradient osmotic flow model of Diamond
and Tormey (1966). To Hoffer and her co workers, the vesicular
mechanism which is present in both the ductuli efferentes and
epididymis is quantitatively sufficient to account for the fluid
absorbed considering their length and the daily volume of fluid
transported.
Goyal and Hrudka (1980; 1981) and Goyal and Williams (1988) have
further stated that because of the preferential capacity of type
III nonciliated cells to take up the horseradish peroxidase, these
cells in the bull and the goat, can be considered as specialized
cells for absorption. These authors also have suggested that transepithelial
movement of material could occur since they found horseradish
peroxidase in basolateral spaces and in macrophages in the adluminal
compartments.
Robaire and Hermo (1988) however, have expressed doubt whether
a functionally important amount of water is taken up in the efferent
ductules (and epididymis) by fluid-phase endocytosis. Experiments
that would distinguish between the uptake of water by fluid phase
endocytosis or by any other means are still lacking.
Hamilton (1975) has proposed a two step model for the absorption
of fluids in the ductuli efferentes. Endocytosis, according to
this model, may play a role in the initial stage of water movement,
not only by the uptake of macromolecules, but also of water and
ions as well. The macromolecules would then be sequestered in
lysosomes and digested while the ions and water moved out of the
cell by some unknown pumping mechanism. Moreover, Hamilton stated
that endocytosis could not explain fluid transport which directs
water movement from the luminal and epithelial compartments into
the interstitial vessels. He further adds that if the standing-gradient
theory of Diamond and Tormey (1966) is correct, a solute pump
not seen morphologically, exists along the basolateral plasmalemma
of the epithelial cells. This pump would then produce the requisite
osmotic gradient that forces water out of the cell. He also states
that this theory could very well be operative in the ductuli efferentes
and the epididymis as he found dilated intercellular spaces in
the epithelium which have also been confirmed by Pudney and Fawcett
(1984) and Jones and Jurd (1987).
Calculations of the volumes of the vesicles and vacuoles by Jones
and Jurd (1987), based on electron microscopic stereological examination
of the endocytotic apparatus, have shown that endocytosis could
not account for the rate of fluid absorption in the ductuli efferentes.
They also have calculated that the rate of absorption in this
part of the excurrent duct is greater than in the proximal convoluted
tubules of the kidney. In addition, dilated intercellular spaces
also were detected; however, the spaces were sometimes occluded
by interdigitations. Because of these findings, these authors
suggested that the main mechanism of fluid transport in the ductuli
efferentes involves the coupling of water and active salt transport.
Data from physiological and micropuncture studies on the ductuli
efferentes and the epididymis highly indicate that fluid reabsorption
in these ducts is coupled to the active transport of sodium similar
to that which occurs in other absorptive epithelia.
Crabo et al., (1964), Crabo (1965), Montorzi and Labiano (1970),
Levine and Marsh (1971), Jenkins et al., (1980), Turner (1979;
1984) and Hinton and Turner (1988) among others, have shown that
sodium concentration from the rete to the caput epididymidis declines
because of its reabsorption in the ductuli efferentes. This decline
in the concentration of sodium was found to be concomitant with
increasing fluid absorption as evidenced by increasing sperm concentrations.
Levine and Marsh (1971) and Jesse and Howards (1976) have further
demonstrated that sodium reabsorption occurs against its electrochemical
gradient, thus necessitating an energy-dependent pump. These authors
inferred that the resorption of water was secondary to the active
transepithelial transport of sodium. Hohlbrugger (1980) has in
fact demonstrated in the rat that water reabsorption is a consequence
of an active transepithelial electrolyte transport process. However,
it was not clear from his study to which electrolyte water transport
is coupled, although sodium was implicated since it was shown
that water absorption was inhibited by intraluminal application
of amiloride. Amiloride is known to block sodium channels in membranes
facing the lumen (Schneyer, 1970).
Hinton and Turner (1988) have also raised the possibility that
fluid transport in the efferent ductule might be secondary to
chloride transport as has been shown by Wong et al. (1978) in
the rat caput epididymidis. Chloride also has been shown to decrease
in concentration along the ductal length (Crabo, 1965; Levine
and Marsh, 1971), and like sodium, it was transported against
an electrochemical gradient. Its reabsorption is also found to
be concomitant with absorption of water (Hohlbrugger, 1980). Studies
of kidney tubules however have shown that active chloride absorption
is also the result of a sodium coupled transport process (Frizzel
et al., 1979).
Perfusion experiments in the epididymis performed by Wong and
co-workers (Au et al., 1978; Wong, 1990; Wong and Yeung, 1977a,b,c,d;
Wong et al., 1978a,b, 1979) concluded that water absorption in
the epididymis is due in part to passive diffusion secondary to
electrolyte transport. It was found in these experiments that
the driving ion in the cauda is sodium, while in the caput, chloride
was the responsible ion. Wong (1990) further discussed the relationship
between abnormal reabsorption of fluid in the epididymis and dysfunctional
chloride channels. He pointed out that in patients with Young's
syndrome and Cystic fibrosis (a disease resulting from a defective
Chloride channel) infertility and occlusions in the head of the
epididymis are common.
In the epididymis, sodium and water transport was abolished by
castration and restored by administration of testosterone, indicating
that absorption is dependent on circulating androgens (Wong et
al., 1978a). However, experiments to determine adrenal gland control
of absorption provided conflicting results. Wong and Yeung (1977b,c)
found that adrenalectomy did not affect the rate of fluid reabsorption
but Au et al. (1978) reported reduced absorption when the adrenals
were removed. Turner and Cesarini (1983) later showed that adrenalectomy
and inhibition of aldosterone by spironolactone decreased absorption
while hormonal replacement restored absorption to normal values.
Wong and his colleagues have also found that some reproductive
toxicants like -chlorohydrin and cytoproterone acetate reduce
sodium and water absorption (Wong and Yeung, 1977d; Wong et al.,
1978). Wong and Yeung (1977d) demonstrated a reversible inhibitory
effect of low doses of -chlorohydrin on fluid absorption in the
cauda epididymis. They indicated that impairment of fluid absorption
may not be due to structural damage to the epithelium, but to
interference with specific transport mechanisms, particularly
that of sodium. Ouabain, a specific inhibitor of the sodium pump,
was also found to have decreased absorption by more than 50 per
cent (Wong and Yeung, 1977a). This last finding, coupled with
the finding that dilated intercellular spaces were found during
maximal absorption (Wong et al., 1978b), raised the possibility
that the standing gradient model of water absorption with the
sodium pump playing a central role could operate in the epididymis
as first proposed for the gall bladder (Diamond and Tormey, 1966).
Whether these mechanisms demonstrated in the epididymis also function
in the ductuli efferentes is not known. Localization of the enzyme
manifestation of the sodium pump, Na+,K+-ATPase, in the ductuli
efferentes (Byers and Graham, 1990; Ilio and Hess, 1992) nonetheless
gives an indication that a fluid transport coupled to the sodium
pump does exist in the epithelium.
Besides sodium and chloride, other ions and small organic molecules
have been found to be absorbed in the ductuli efferentes. In general,
potassium concentration is increased along the ductuli efferentes
(Levine and Marsh, 1971; Turner, 1984). However, it has been demonstrated
that an actual net resorption of potassium, not secretion, actually
occurs between the rete testis and the caput epididymidis (Hinton
and Turner, 1988; Turner, 1984). Hinton and Turner (1988) also
have discussed the possibility of bicarbonate ions being absorbed
in the epithelium in connection with the regulation of acid-base
balance in the lumen.
The absorption of protein in the ductuli efferentes has been demonstrated
in many micropuncture and gel electrophoretic studies. Evidence
for this is the disappearance of certain bands of proteins from
the rete testis fluid between the ductuli efferentes and the initial
segment of the epididymis owing to their absorption in the ductuli
and/or the initial segment (Jones, 1987; Koskimies and Kormano,
1975; Olson and Hinton, 1985). In the tammar, a species of marsupial,
it was calculated that about half of the total protein leaving
the testis was absorbed in the ductuli efferentes (Jones, 1987).
Structurally, the capacity of the efferent ductal epithelium to
take up large molecules has been confirmed by many studies, starting
with Sedar's (1966) demonstration of the uptake of colloidal particles
by the nonciliated cells. Recently, the process of endocytosis
through fluid-phase, adsorptive and perhaps even through receptor-mediated
means has been shown to be the main mechanism for the uptake of
large particles from the testicular fluid (Morales and Hermo,
1983; Hermo and Morales, 1984; Hermo et al., 1985). More specifically,
androgen-binding protein (ABP) has been localized by immunocytochemistry
in the endocytotic apparatus of cells in the terminal segment
of the ductuli efferentes and initial segment of the epididymis
(Pelliniemi et al., 1981a; Byers et al., 1985). It was postulated
that ABP is selectively taken up by endocytosis in these regions
most probably through a receptor-mediated mechanism. It was also
suggested that the maintenance of the distal region of the ductuli
and the initial segment of the epididymis requires intraluminal
ABP for androgen trans-membrane transport.
It has been explicitly demonstrated by Veerama-chaneni and Amann
(1991) that the principal site for endocytosis of rete testis
fluid (RTF) proteins is the efferent ductules. These authors calculated
that about 80 mg of proteins enter the ductuli efferentes daily
in the ram. Using colloidal-gold conjugated to RTF proteins as
tracers, they have found that the proteins were selectively endocytosed
in the ductuli efferentes since rete testis proteins were not
detected in fluid from the proximal caput epididymidis. Veeramachaneni
et al. (1990) showed that a portion of this protein uptake appeared
to be specific, and determined that 145 times more protein was
endocytosed in the ductuli efferentes than in the caput epididymidis.
In contrast to the findings of Pellienimi et al. (1981a) and Byers
et al. (1985), however, it was reported that ABP was spared from
endocytosis along with the bulk protein and conserved for functions
in epididymal regions far distal to the site of bulk protein loss.
It would not be surprising if it is found that the efferent ductules
synthesize their own ABP, rather than failing to endocytose the
ABP from rete testis fluid.
Another type of protein that is taken up by the efferent ductules
is the sulfated glycoproteins (SGP-1 and SGP-2), major secretory
proteins of the Sertoli cell (Sylvester, 1993). These proteins
are found in rete testis fluid and are bound to the sperm. After
being released from the plasma membrane they are endocytosed by
the nonciliated cells of the ductuli efferentes (Hermo et al.,
1991,1992b; Igdoura et al., 1993). Interestingly, mRNA for SGP-1
(also known as saposin) is also present in the nonciliated cells
, and a new efferent ductal form of the protein is synthesized
and secreted in the ductules (Garrett et al., 1991; Igdoura et
al., 1993).
Secretion
Top
The secretory function of the ductuli efferentes epithelium has
already been alluded to in earlier sections. Histochemical localizations
of various glycoconjugates (Agrawal et al., 1989) and enzymes
(Burkett et al., 1987a,b; Byers and Graham, 1988; Cohen et al.,
1976; Goyal et al., 1980) have inferred secretions of glycoconjugates
and amino acids from the epithelium destined for sperm coating
and ions in connection with absorption and acid-base balance.
Also, structural studies in various animals also have shown granules
and apical blebs and protrusions in the nonciliated cells which
have the appearance of being extruded or secreted into the lumen
(Bakst, 1980; Goyal and Hrudka, 1981; Gray et al., 1983; Hess
and Bassily, 1988; Wystub et al., 1989).
It has been argued for a number of years that apocrine secretion
does not occur in the male reproductive tract, but that blebs
form in the ductal epithelium during improper fixation (Hamilton,
1975). However, there is considerable variation in efferent ductal
structure between species, and in larger mammals and birds the
appearance of apocrine secretory blebs is commonly reported (Bakst,
1980; Goyal and Hrudka, 1981; Gray et al., 1983; Hess and Bassily,
1988). Even after fixation by vascular perfusion these secretory
blebs have been observed in certain regions of the ductuli efferentes
in the goat (Gray et al., 1983) and in the bull (Goyal, 1985).
Perfusion fixation in the dog has also revealed the distinct appearance
of apocrine-like secretions in nonciliated cells of the mid-region
of the ductuli efferentes (Figs. 17, 18). This activity was noted
only in certain cell types in specific regions of the tract. The
secretory blebs were also present in the ductal lumen distal to
the site of secretion (Fig.
29). These data, along with the fact that the tissue was
fixed optimally by a rapid vascular perfusion method using a fixative
with high osmolality, strongly support the conclusion that in
some species apocrine secretions do occur in the epithelium of
the ductuli efferentes.
| Fig. 29. Ductuli efferentes of the dog showing a large number of the apocrine secretory droplets (A) in the lumen of the tubule. S, spermatozoa. x 400. |
Endocrine Activity
The possibility that the excurrent ductal epithelium participates
in the production of hormones was assessed by histochemical and
immunohistochemical techniques. Fetissof et al. (1988) used a
panel of antibodies to test for amine and polypeptide hormones
in different regions of the ductal epithelium. Only cells immunoreactive
to serotonin were detected in the ductuli efferentes, but they
were rarely observed. No other endocrine cells in other parts
of the excurrent duct were seen. More recently, protein gene product
9.5, a soluble protein localized in neurons and endocrine cells
has also been found in the cytoplasm of some columnar cells of
the ductuli efferentes (Santamaria et al., 1993).
Pudney and Fawcett (1977) demonstrated by electron microscopy
putative endocrine cells, albeit in the subepithelial layer, of
the ductuli efferentes in squirrels. These cells had similar ultrastructural
features to Leydig cells and were assumed to have steroidogenic
function. Steroidogenic enzymes have also been detected by histochemistry
in the efferent ductules of the bird (Tingari, 1973; Budras and
Sauer, 1975). In fact, activities for 3ß- and 17ß-hydroxysteroid
dehydrogenases were highest in the ductuli efferentes compared
to all other regions of the male reproductive tract. In contrast
to the initial segment epididymidis where there is significant
metabolism of testosterone by 5-reductase (Klinefelter &Amann,
1980a; Robaire &Hermo, 1988), there is little or no 5-reductase
activity in the efferent ductules (Hess and Ganjam, unpublished
data; Roselli et al., 1991).
Although the efferent ductules do not appear to be an active endocrine
organ in mammals, there is considerable evidence to suggest that
this region is a major target for steroid hormones. Receptors
for several hormones have been identified in the efferent ductules
suggesting that the ductuli may be a possible site of hormone
action. Androgen receptors have been localized mainly in the principal
cells of the epithelium of the ductuli efferentes and epididymis
(Schleicher et al., 1984; Tekpetey et al., 1990; Roselli et al,
1991; Cooke et al., 1991a). However, estrogen receptors have also
been localized in the ductuli efferentes of the mouse, monkey
and human (Schleicher et al., 1984; Sapino et al., 1987; West
and Brenner, 1990; Cooke et al, 1991b; Iguchi et al., 1991; Greco
et al., 1992, 1993). Hypoplastic testes from male-to-female transsexuals
removed during sex-reversal surgery, after administration of estrogens,
were studied histologically and by immunohistochemistry to locate
estrogen receptors (ER) and related antigens (Sapino et al., 1987).
They found that the efferent ductules and rete testis proved to
be the only structures where estrogen receptors were located,
with the highest concentration in the ductuli efferentes confirming
an earlier radioisotope study by Scheicher et al. (1984). The
immunological data, in parallel with the observed biological effects
indicate that these structures are the main target of estrogen
in the human male reproductive tract.
In a similar immunocytochemical localization for estrogen receptors
(ER) in the testis and excurrent ducts in the monkey, West and
Brenner (1990) confirmed the previous findings that high concentrations
of ER are present in the ductuli efferentes. They showed that
specific staining was confined only to the nonciliated cells of
the epithelium and that concentration did not differ from that
in the oviduct. It is interesting to note that this recent immunocytochemical
localization of estrogen receptors in the efferent ductules is
in direct contrast to the earlier findings that these receptors
are mostly found in the epididymis (Mulder et al., 1974; Danzo
et al., 1977). West and Brenner (1990) had expressed the fact
that if the ductuli efferentes are not carefully dissected away
from the caput epididymidis, then biochemical assays of the homogenized
tissue would give spuriously high values for epididymal estrogen
receptors because of the attached ductuli. The functional significance
of estrogen receptors in the efferent ductal epithelium remains
to be determined. However, it was recently discovered that developing
spermatids and epididymal sperm are capable of converting androgens
to estrogens because they contain active aromatase (Nitta et al.,
1993; Janssen et al., 1994). This new data raises the possibility
that estrogen produced by sperm during transit through the efferent
ductules is targeted toward the ductal epithelium via apical transport
systems for the purpose of modifying the androgen responsiveness
of these cells proportional to the number of sperm being transported.
Recently, soltriol [1,25(OH)2-vitamin D3] binding sites were localized
in male reproductive tract. Its greatest intensity was found to
be the epithelium of the efferent ducts (Stumpf et al., 1987;
Schleicher et al., 1989). Its labeling pattern was found to overlap
with [3H]-dihydrotestosterone and [3H]-estradiol binding sites.
The presence of vitamin D receptors in the efferent ductules raises
the possibility of hormonal regulation of fluid absorption and
local calcium concentrations in the efferent ductules. In addition,
opioid receptors, which are similar to those in the pituitary
and other endocrine tissues, also have been localized in the testis,
with their highest density found in the efferent ductules (Wolfe
et al., 1989). The function of these opiate receptors in the efferent
ductules is not known but has been postulated to probably aid
in the transit of sperm. The mRNA for proenkephalin peptide is
also found in the ductuli efferentes and initial segment epididymidis
(Garrett et al., 1990; Douglass et al., 1991) and appears to be
regulated by a factor present in rete testis fluid. Oxytocin (Veeramachaneni
and Amann, 1990) and inhibin (Veeramachaneni et al., 1989) have
also been localized in the epithelium of the ductuli efferentes.
Their function in this region remains unknown.
Spermiophagy
Another function which has been attributed to the ductuli efferentes
epithelium is the function of phagocytosis of spermatozoa. Generally,
this occurs in cases of epididymal obstruction (Orgebin-Crist,
1969; Hoffer and Hamilton, 1974; Hoffer et al., 1975; Koyama,
1987) but also when abnormal spermatozoa are produced in the testis
(Crabo et al., 1971; Hess et al., 1982).
It has been found that phagocytosis of spermatozoa by the ductuli
efferentes epithelial cells in drug induced obstruction (e.g.
-chlorhydrin and carbendazim) is not necessarily the effect of
the drug itself but a consequence of obstructions which arise
from the chemical toxicity (Hoffer et al., 1975; Nakai et al.,
1992; Nakai et al., 1993).
Top
Many cases of infertility or reduced fertility in man and animals
arise from obstructive lesions in the ductuli efferentes (Wakeley,
1944-1945; Gustafsson and Galloway, 1988; Rajalakshmi et al.,
1990). Obstruction of the ductuli efferentes, unless attenuated
within a short period of time, will initiate extensive degeneration
of the seminiferous epithelium of the testis that may persist
for long periods of time and lead to irreversible infertility.
This can occur in many experimental and clinical conditions including:
1) ligation (Van Wagenen, 1925; Cunningham, 1928; Harrison,
1953; Smith, 1962; Kuwahara, 1976); 2) ablation of the epididymal
blood vessels (MacMillan, 1953); 3) congenital agenesis
(Dubin and Amelar, 1971; Makler and Hampel, 1975) and maldevelopment
(Ashdown, 1985; Shafik and El-Sibaei, 1987; Gustafsson and
Galloway, 1988); 4) congenital occlusions (Ikadai et al.,
1987); 5) varicocele in humans (Nistal et al., 1984; 1987);
6) vasectomy (Tung and Alexander, 1980); 7) vitamin
E deficiency (Mason and Shaver, 1952); 8) Young's syndrome
(Hendry et al., 1990); 9) granuloma, sperm stasis and cysts
(Blom and Christensen, 1960; Hemeida and McEntee, 1984; Hamm et
al., 1988); and 10) chemical toxicoses; ethylene dimethane
sulfonate and -chlorhydrin (Cooper and Jackson, 1972; 1973), benzimidazole
carbamate (Hess et al., 1991; Nakai et al., 1992; Nakai et al.,
1993), cadmium (Mason and Young, 1967; Nagy, 1985) and dinitrobenzene
(Linder et al., 1988a).
The structural changes in the testis which occur in many of these
conditions are remarkably similar. In the simple case of ligation
of the ductuli efferentes in rats (Smith, 1962; Baillie, 1962),
there first occurs an initial dilatation of the seminiferous tubules
associated with fluid accumulation and an increase in testicular
weight. Thereafter, tubular diameter and testicular weight decrease
progressively, the shrinkage being associated with varying degrees
of degeneration of the seminiferous epithelium. The pattern of
degeneration of germ cells largely depends on their developmental
stage: spermatocytes and spermatids show signs of more severe
damage than spermatogonia or mature spermatozoa. By 28 days after
ligation, the weight of the testis has been reduced to one half
its original weight and the seminiferous epithelium is limited
to a single row of cells. It had been found earlier that if the
obstruction to the ductuli is incomplete, normal spermatogenesis
continues in at least some parts of the testis (Harrison, 1953).
Many cases of varicocele also show seminiferous tubule alterations
consisting of anomalies in spermatid maturation and sloughing
of immature spermatozoa and spermatids (Nistal et al., 1984).
In varicoceles persisting for a long time, spermatocytes and even
spermatogonia are found to be detached from the seminiferous epithelium,
leaving the epithelium lined only by Sertoli cells (the "Sertoli-cell
only syndrome"). The seminiferous tubules are decreased in
diameter with the establishment of peritubular fibrosis. Obstruction
at the level of either the tubulus rectus or the ductuli efferentes
due to dilated veins has been postulated to be responsible for
the lesions in varicocele leading to testicular atrophy.
These patterns of lesions are repeated in cases of ablation of
the epididymal blood vessels in the rat (MacMillan, 1953) and
in vasectomized rhesus monkeys (Tung and Alexander, 1980). It
is not clear though whether the orchitis and testicular atrophy
after long term vasectomy are the results of ductuli efferentes
granuloma or are a separate immunologic sequelae to a damaged
blood testis barrier. It has been postulated though that granulomas
in the ductuli efferentes and epididymis which occur in vasectomized
monkeys represent a physical response to vasal occlusion, and
not to an immunologic process.
Sperm blockage in the ductuli efferentes resulting in testicular
swelling has been found to occur immediately following administration
of -chlorhydrin in rats (Ericsson, 1970; Cooper and Jackson, 1972;
Hoffer et al., 1973). As in the case of ligation, the long term
effects of obstruction in -chlorhydrin administration include
formation of spermatoceles, sperm granuloma and fibrosis in the
ductuli, eventually leading to degeneration of the germinal epithelium
and testicular atrophy. It is not known how obstructions emerge
in the ductuli following administration of the agent; it is postulated
that -chlorhydrin causes local ischemia with the resulting epithelial
desquamation blocking the ductulus lumen (Hoffer et al., 1973).
Blockage of the flow of luminal fluids through the ductuli efferentes
also induces changes in the mouse epididymal epithelium, including
the degeneration of principal cells and the recruitment of macrophages
(Abe and Takano, 1989a,b) and the appearance of PAS+ materials
in the corpus epididymidis (Abe et al., 1982). Ligation of the
ductuli efferentes in the goat induced changes in the distal segments
that were indicative of decreases in the absorptive function of
this epithelium (Gray et al., 1983).
Occlusion of the vas deferens not only causes sperm to accumulate
in greatly stretched ductuli efferentes, but also induces long-term
auto-immune changes in the monkey (Marsh and Alexander, 1982).
Up to 7 years after vasectomy, immune complexes were found deposited
in the thickened basement membrane. Numerous macrophages and sperm
phagocytosis were observed within the ductal lumen.
There are at least two diseases associated with abnormal development
or dysfunction of the ductuli efferentes in birds. In both cases
the males have reduced fertility and the sperm exhibit functional
or morphologic abnormalities. In the turkey, the yellow semen
syndrome is associated with the abnormal accumulation of lipid
(with cholesterol clefts) in the ductal epithelium and the secretion
of excessive amounts of proteins and androgens into the semen
(Hess et al., 1982; Thurston et al., 1982; Hess and Thurston,
1984a; 1984b ; Hess et al., 1984). In the chicken, a mutated line
has been identified that is subfertile due to an abnormal maturation
of the sperm within the epididymal region. This genetic abnormality
was associated with the malformation of efferent ductules (Kirby
et al., 1990). Because the efferent ductal region has been associated
with abnormal semen production, it is reasonable to suggest that
seasonal declines in semen quality found in broiler breeders and
turkey toms (Thurston et al., 1992) may be related to ductuli
efferentes dysfunction. Certainly, the interaction of environmental
factors on hormones and/or factors that control the epididymal
function could cause these ductules to transport sperm in a fluid
milieu that is inadequate for normal sperm maturation. This mechanism
of dysfunction may help to explain some of the genetic differences
between breeds in fertilizing ability (Kirby et al., 1989).
Top
The ductuli efferentes form a unique tubular organ system that
exhibits fascinating variations in cellular morphology. These
ductules have been overlooked traditionally as part of the excurrent
duct system of the testis, primarily because of their obscure
location, either buried in thick connective tissue or a thick
layer of fat. Morphological and biochemical studies, however,
support the opinion that this part of the ductal system is physiologically
important particularly in the reabsorption of fluid and other
substances and for the maintenance of proper sperm concentrations
in the lumen of the epididymis. Although the basic morphology
of the ductules provides evidence for the role of reabsorption,
there are numerous species differences that have been noted. Therefore,
in order to improve animal models for the study of male infertility,
it is important that structural differences be considered, particularly
in reference to the blind-ending tubules and the presence of occlusions
in this part of the male reproductive tract. Considering the unique
physiology of this region and the effects of various chemicals
on the function of the efferent ductules, it may be possible to
target a chemical action specifically toward this region in an
effort to develop a male contraceptive. There is considerable
evidence that the ductuli efferentes epithelium contains several
hormone receptors; however, there is a complete lack of understanding
of hormone function in this region. Future studies, therefore,
should begin to unravel the roles that androgens, as well as estrogens
and other hormones, play in the regulation of the structure and
function of these important ductules.
ACKNOWLEDGMENTS: This publication was made possible by grant number ES-05214 from the National Institute of Environmental Health Sciences, NIH.
