/. Embryol. exp. Morph. 96, 51-63 (1986)
51
Printed in Great Britain © The Company of Biologists Limited 1986
The postnatal maturation of efferent tubules in the rat:
a light and electron microscopy study
SANDRO FRANCAVILLA, SERGIO MOSCARDELLI,
BERNARDINO BRUNO
Department of Internal Medicine, School of Medicine, University of L'Aquila,
Via S. Sisto, 67100, L'Aquila, Italy
PATRIZIO SCORZA BARCELLONA
F. Angelini Research Institute, Rome, Italy
AND CESARE DE MARTINO
Regina Elena Institute for Cancer Research, Rome, Italy
SUMMARY
The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was
investigated by light and transmission electron microscopy, from birth to 50 days of age, when
sperms were released from the seminiferous tubules and appeared in the genital duct.
At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial
cells. During the third week of life, efferent tubules developed specializations for the transport
of sperms and fluids, namely the appearance of ciliated elements interspersed among the
principal cells of the epithelium, and differentiation of myoid elements in the tubule wall.
The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear
at the end of the second week of life in the rat, along with the initial secretion of testicular fluid.
This suggested that the maturation of efferent tubules is not triggered by the passage of testicular
fluid, as surmised for the postnatal differentiation of caput epididymis.
The postnatal maturation of efferent tubules was almost complete 35 days after birth. The
appearance of sperms in the genital duct of 50-day-old animals was not associated with any
remarkable structural change.
INTRODUCTION
The postnatal maturation of the genital duct in the rat seems to precede the first
appearance of sperms (Sun & Flickinger, 1979; Francavilla et al. 1986), and it is
claimed to be influenced by testicular factors like androgens (Orgebin-Crist,
Danzo & Davies, 1975; Sun & Flickinger, 1979). The information so far available
on the maturation of the genital duct is, however, restricted to few ultrastructural
investigations in this species, dealing with epididymis (Leeson & Leeson, 1964;
Flickinger, 1969; Sun & Flickinger, 1979).
Key words: efferent tubules, epididymis, genital duct, testis, testicular fluid, rat.
52
S. FRANCAVILLA AND OTHERS
The postnatal differentiation of efferent tubules was never analysed in detail in
the rat, although efferent tubules and epididymis are embryologically (Bovy, 1929;
Brambell, 1927; Flickinger, 1969; Gier & Marion, 1970), structurally (Hamilton,
1975), and functionally (Levine & Marsh, 1971) distinct. We therefore decided to
undertake a detailed investigation, by light and transmission electron microscopy,
of the maturation of efferent tubules in the rat from birth to the time of sperm
appearance in the genital duct.
The aim of this study was to follow the development of those specializations of
the epithelium and tubule wall of efferent tubules that are related to absorption
and to the progression of luminal content.
Efferent tubules in adult animals transport fluids across the epithelium from the
lumen (Levine & Marsh, 1971), and take up endocytic markers (Hermo &
Morales, 1984). Moreover, they facilitate sperm progression through the beating
of epithelial cilia and contractility of the tubule wall (Hamilton, 1975).
MATERIALS AND METHODS
Thirty-six Long-Evans rats (Angelini breeding colony) were utilized in this investigation.
Three animals were studied on each of the postnatal days 1, 3, 5, 8,10,15, 20, 25, 30, 35, 40, 50;
the rats were anaesthetized with ether and killed by decapitation.
Gonads were carefully exposed and efferent tubules were dissected, along with the initial
segment of the caput epididymis and rete testis with the most dorsal region of the attached testis.
Tissue samples were rapidly fixed by immersion in picric acid-formaldehyde (Zamboni & De
Martino, 1967) containing 2-5% glutaraldehyde in Sorenson buffer, pH7-2, for 12 h. The
material was stored at 4°C and postfixed in 1-5% unbuffered OsO4 solution for 1-5 h, and
embedded in Epon 812. Thick sections for light microscopy were stained in buffered toluidine
blue (pH8-0), and thin sections were stained with uranyl acetate and lead hydroxide and
examined by a Siemens Elmiskop 101 electron microscope.
RESULTS
At birth, the efferent tubules appeared very coiled and easily distinct from the
epididymis because of their small cross-sectional size and thin peritubular wall
(Fig. 1). The epithelium was formed by cuboidal cylindrical cells with a basal
located nucleus and small scattered microvillar specializations on the luminal
surface (Fig. 2). Stromal cells surrounded the tubules (Fig. 2).
3-day-old animals did not show any remarkable change.
5 days after birth, the epithelial cells showed an increased number and size of
microvilli, and scattered apical invaginations of the plasma membrane seemed to
engulf an electron-dense fuzzy material (Fig. 3). In the same animals, a lumen
appeared in the neighbouring rete testis, while seminiferous cords did not show
any sign of canalization.
In 8-day-old animals, a complex endocytotic apparatus was built up in the
epithelial cells. Numerous caveolae and coated pits of the plasma membrane were
interspersed among the bases of long, packed and regularly arranged microvilli.
Hundreds of coated and uncoated vesicles crowded the apical cytoplasm, along
Efferent tubule maturation in the rat
53
with a network of dense tubules and multivesicular bodies (Fig. 4). The functional
relationship of all these specializations of the plasma membrane, during a fluid
phase, and receptor-mediated endocytosis is well documented (Ericsson & Trump,
1969; Djakiew, Byers & Dym, 1984).
One cilium was sometimes visible along the apical border of epithelial cells
(Fig. 4). The lateral plasma membranes showed numerous interdigitations along
both the apical (Fig. 5) and basal cell region (Fig. 6), as described in epithelial cells
specialized for transport of water and salts (Ericsson & Trump, 1969).
In 8-day-old animals, a wide lumen was visible in the tubules of the rete testis,
but not yet in the seminiferous cords (Fig. 7). The epithelium of tubules in the rete
was formed by cuboidal cells with a smooth luminal surface. The transition with
efferent tubules was abrupt, and featured a cylindrical epithelium provided with
packed microvilli resembling a brush border (Fig. 8).
Fig. 1. Genital duct of 1-day-old rat. Efferent tubules (t) are easily distinguished from
epididymis (ep) by the small cross-sectional diameter and thin peritubular wall. Arrow
points to the transition between the two regions of the genital duct. Toluidine blue.
X260.
Fig. 2. Efferent tubule of 1-day-old rat. The cuboidal-cylindrical epithelial cells show
scattered short microvilli and few mitochondria with transverse plate-like cristae. The
tubule is surrounded by stromal elements with a cytoplasm rich in rough endoplasmic
reticulum. X5100.
54
S. FRANCAVILLA AND OTHERS
Efferent tubule maturation in the rat
55
The epithelium of efferent tubules was seen to be formed at both the light and
electron microscope level by principal and scattered dense-stained cells. The latter
ones (Fig. 9) showed an electron-dense cytoplasm, round- and rod-shaped mitochondria, like those of principal cells, and packed microvilli. The apical cytoplasm
contained numerous vesicles and scattered multivesicular bodies, but not the
network of dense tubules visible in principal cells. The nuclei of dark cells had a
coarser chromatin than that of neighbouring principal cells. A third cell type,
resembling the wandering halo cells of adults (Hamilton, 1975) and developing
epididymis (Sun & Flickinger, 1979), was seldom present in the epithelium. The
tubules were surrounded by loosely arranged, slender stromal cells; those next
to the tubule basal lamina showed a decreased amount of rough endoplasmic
reticulum and a more electron-dense cytoplasm (Fig. 10)
No changes were recorded in 10-day-old animals.
In 15-day-old rats, efferent tubules underwent changes involving the tubule wall
and the epithelium. The stromal cells surrounding the tubules were regularly
arranged in two to four circular strips, and acquired features of myoid elements.
Numerous bundles of thin (5-6nm) filaments were visible in the cytoplasm,
anchored to dense patches along the inner plasma membrane. The cisternae of
rough endoplasmic reticulum were not so numerous as those of younger animals,
and the cells appeared surrounded by a discontinuous basement membrane. These
changes appeared first in the cells next to the epithelial basal lamina, and
progressively involved outer cells (Fig. 11).
The differentiation of a contractile wall was associated with an active ciliogenesis, involving scattered epithelial cells. Cilia appeared interspersed among
microvilli, while components of the endocytotic apparatus in the apical cytoplasm
were greatly reduced as compared with neighbouring principal cells (Fig. 12).
In 20- and30-day-old rats, the efferent tubules did not show new changes, except
for a light growth of the Golgi complex in epithelial principal cells (Fig. 13).
Microvilli and components of the endocytotic apparatus had almost disappeared in
the ciliated elements.
In 35-day-old rats, the efferent tubules appeared mature. The epithelium was
formed by a palisade of tall cylindrical principal cells and scattered ciliated
Fig. 3. Epithelium of 5-day-old rat. Coated pits of the apical plasma membrane, which
seem to engulf an electron-dense fuzzy material (arrow), are interspersed among the
bases of long, scattered microvilli. x23000.
Fig. 4. Epithelium of 8-day-old rat. Packed microvilli have differentiated along the
apical border of epithelial cells. The supranuclear cytoplasm contains numerous coated
(cv) and uncoated vesicles, and a network of short dense tubules (arrowheads), cp,
coated pits of the plasma membrane; mvb, multivesicular body; arrow, cilium.
x 19000.
Fig. 5. Epithelium of 8-day-old rat. Packed microvilli show a regular organization of a
brush border. Intercellular clefts (arrows) containing interdigitations of the lateral
plasma membrane of facing cells are visible in the apical region of the cells, x 12000.
Fig. 6. Epithelium of 8-day-old rat. The basal region of epithelial cells shows extensive
interdigitations of the lateral plasma membrane (arrows), x 15000.
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S. FRANCAVILLA AND OTHERS
Efferent tubule maturation in the rat
57
elements (Fig. 14). The latter had nuclei with coarse chromatin, often located in an
apical position, and a fairly dense-stained cytoplasm. Their apical border had
packed cilia, while microvilli and specializations related to endocytosis were
absent (Fig. 15). The wall was formed by three to four layers of circularly arranged
myoid cells.
The appearance of sperms in the duct of 50-day-old rats was not associated with
any remarkable structural change, as also reported for the epididymis maturation
in the same species (Sun & Flickinger, 1979; Francavilla et al. 1986).
DISCUSSION
This study shows that the postnatal maturation of efferent tubules in the rat
occurs in two sequential steps. In the first one, an endocytotic apparatus is
differentiated in the epithelial cells. During the second one, the apparatus for
transport of sperms and fluids is built up, and comprises the differentiation of
ciliated epithelial cells among the principal cell type, and peritubular myoid
elements.
The differentiation of an endocytotic apparatus in the epithelial cells largely
precedes the initial postnatal maturation of seminiferous cords and secretion of
testicular fluid. This, in fact, appears in the genital duct at the end of the second
week of life (Tindall, Vitale & Means, 1975), and parallels the differentiation of
the ciliated epithelium and peritubular contractile wall of efferent tubules.
The two steps of morphological maturation of this segment of the genital duct
do not overlap. The endocytotic apparatus of epithelial principal cells is fully
developed in 8-day-old animals, while ciliated cells are still absent, and the wall is
formed by poorly differentiated stromal cells. This maturation pattern of efferent
tubules is quite unique. In other regions of the genital duct, like epididymis and
intragonadal ductus deferens, the epithelium and tubule wall undergo a simultaneous differentiation (Francavilla etal. 1986).
The peculiar sequence of changes during the postnatal life seems to be related to
the embryonal ontogenesis of efferent tubules, which are the only component of
the genital excurrent pathway originated in mammals from mesonephric tubules
(Bovy, 1929; Gier & Marion, 1970; Upadhyay, Luciani & Zamboni 1981; Zamboni
& Upadhyay, 1981, 1982). In embryos, the mesonephron fulfils an excretory
function, and the tubules transiently develop a phenotype that is strictly related to
absorption, when they represent the uriniferous ducts of a functioning, though
Fig. 7. 8-day-old rat. A wide lumen is visible in the rete testis (r), while seminiferous
cords (s) are not yet canalized. Efferent tubules (t) show a cylindrical epithelium
formed by pale and dense-stained cells. Toluidine blue. X450.
Fig. 8. Ultrastructural study of efferent tubules and rete testis depicted in Fig. 7. The
epithelium of the rete testis (r) is formed by cuboidal elements, with a smooth luminal
surface. The transition with the efferent tubules features a cylindrical epithelium with
an apical brush border (arrows). Dense-stained cells are scattered among the principal
ones. Arrowhead points to a large cytosome. X1300.
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S. FRANCAVILLA AND OTHERS
Fig. 9. Epithelium of efferent tubules in a 8-day-old rat. The dense-stained epithelial
cell shows apical microvilli and numerous vesicles, while the network of dense tubules
present in the principal cells is not visible. The nucleus shows coarse chromatin.
x 10 000.
ephemeral, nephron (Leeson, 1959; De Martino & Zamboni, 1966; Krause, Cutts
& Leeson, 1979; Wettstein & Tiedeman, 1981; Zamboni & Upadhyay, 1981).
The endocytotic apparatus of the epithelium in efferent tubules, during the early
postnatal life, could be but a remnant of a phenotype this epithelium had in
embryonal life, and which is resumed as soon as a fluid appears in the tubules of
1-week-old animals. A direct demonstration of the passage of fluids in the genital
duct, at this age, is not available. The appearance of a lumen in the cords of rete
testis in 5-day-old animals could be indirect evidence of the initial transport of
fluids in the duct. A coincidence between the appearance of a lumen and the
occurrence of fluid secretion has, in fact, been demonstrated in developing
seminiferous tubules (Tindall et al. 1975). Hence the lack of a lumen in
Efferent tubule maturation in the rat
59
seminiferous cords of 5- and 8-day-old rats suggests that the canalization of rete
testis cords could be related to the initial transport of fluids across the epithelium
of the rete, as demonstrated in adult animals (Setchell & Waites, 1975).
Fig. 10. Tubule wall of efferent tubules in 8-day-old rat. Stromal cells are loosely
arranged around the tubules. They contain numerous cisternae of rough endoplasmic
reticulum. The cell next to the tubule basal lamina shows a decreased amount of rough
endoplasmic reticulum, but microfilaments are not yet visible, x 14000.
Fig. 11. Tubule wall of 15-day-old rat. Mature myoid cells are arranged in two packed
circular layers. They show bundles of thin microfilaments (arrows) anchored to dense
patches along the inner plasma membrane (arrowheads). The cells are surrounded by a
discontinuous basement membrane (double arrows), x 10000.
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S. FRANCAVILLA AND OTHERS
Efferent tubule maturation in the rat
61
In 8-day-old rats, the endocytotic apparatus of epithelial cells in efferent tubules
has reached the degree of development described in the epithelium of adult rats
(Hamilton, 1975) and other mammals (opossum, Ladman, 1967; monkey, Ramos
& Dym, 1977; bull, Goyal & Hrudka, 1980). It is worth mentioning that at this
early postnatal age (Fig. 8), the epithelium of efferent tubules shows a degree of
morphological specialization as an absorbing organ which is comparable to that of
functioning mesonephric tubules of different mammal embryos (man, De Martino
& Zamboni, 1966; opossum, Krause et al. 1979; mouse, Zamboni & Upadhyay,
1981).
During the third and following weeks of life, the efferent tubules undergo
changes which represent an adaptation of already differentiated 'uriniferous-like'
tubules, to promote transport of sperms. These are, in fact, poorly motile in the
initial segment of the adult genital duct (Bedford, 1975), and their progression in
the efferent tubules is accomplished by beating of epithelial cilia and contractility
of the tubule wall.
Stromal cells that surround the tubules of neonatal rats progressively acquire
features of myoid elements, like those that form the wall of seminiferous tubules
(Leeson & Leeson, 1963) and the initial segment of the epididymis (Francavilla
etal. 1983).
Of particular interest is the differentiation of ciliated cells. The epithelium of
efferent tubules up to 15 days after birth is formed by a single cell type provided
with packed microvilli and, rarely, one cilium, as frequently reported in developing epithelial cells not involved in the differentiation of ciliated elements (De
Martino & Zamboni, 1966). Scattered dark cells observed in the epithelium of
8-day-old rats could be precursors of ciliated cells. Both share a dense-stained
cytoplasm and nuclei with coarse chromatin. Principal and dark cells are, however, provided with micro villi and a developed endocytotic apparatus, even
though the latter does not show the apical dense tubules but numerous vesicles
only. Hence, whichever is the precursor, ciliated cells arise from fully differentiated elements, which progressively lose the features of absorbing cells and
switch their phenotype toward specializations related to movement of luminal
content.
Fig. 12. Epithelium of 15-day-old rat. The cell on the right shows numerous cilia
interspersed among microvilli. The apical cytoplasm contains few vesicles only
(compare with the endocytotic apparatus of the cell in the left corner), x 15000.
Fig. 13. Epithelium of 20-day-old rat. The principal cells show a developed Golgi
complex (G), multivesicular bodies (mvb), and numerous cytosomes (arrows),
x 12500.
Fig. 14. Survey of epithelium of efferent tubules in 35-day-old rat. The epithelium is
formed by a palisade of tall principal cells, and scattered ciliated elements with a
nucleus in an apical position showing coarse chromatin. Arrow points to the area
depicted in Fig. 15. x6000.
Fig. 15. Selected area of Fig. 14. The ciliated cell has a dense-stained cytoplasm and
the apical border has numerous cilia, while microvilli are not visible. The principal cell
on the right shows packed and regularly arranged microvilli and a rich endocytotic
apparatus, x 12000.
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S. FRANCAVILLA AND OTHERS
The second step in the maturation of efferent tubules largely coincides with the
initial differentiation of the epithelium and tubule wall of the neighbouring caput
epididymis (Francavilla et al. 1986). Both segments of the genital duct reach a full
differentiation 35 days after birth, i.e. before the arrival of sperms which appear in
the duct of 50-day-old rats. The coincidence of developing changes of efferent
tubules and caput epididymis suggests that factors like androgens, which are
claimed to induce the foetal development (Jost, 1947; Price & Pannabecker,
1956; Alexander, 1972) and postnatal maturation of the epididymis in mammals
(Orgebin-Crist et al. 1975), could also influence the maturation of efferent tubules
or, more precisely, the adaptation of survived 'mesonephric tubules' to promote
sperm progression. The occurrence during the third week of life in the rat of
increased plasma testosterone levels (Yukitaka, Nieschlag & Lipsett, 1973) and
the first appearance of the androgen-rich testicular fluid in the genital duct (Tindall
et al. 1975) seem to be more than accidental with respect to maturation changes
involving, at the same time, the epididymis and efferent tubules.
The authors gratefully acknowledge the excellent collaboration of Mrs Ingrid Adam for typing
and editing the manuscript.
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HAMILTON,
(Accepted 11 April 1986)