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Developmental expression of immobilin in the rat epididymis.

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THE ANATOMICAL RECORD 24086-103 (1994)
Developmental Expression of Immobilin.in the Rat Epididymis
L. HERMO, K. BARIN, AND R.OK0
Department of Anatomy and Cell Biology,McGill University, Montreal, Quebec, Canada
ABSTRACT
Background: Immobilin is a protein secreted by principal
cells of the distal initial segment, intermediate zone and caput epididymidis
of adult rats, which serves to immobilize spermatozoa. In the distal cauda,
epithelial clear cells are involved in its endocytosis. The objective of this
study was to correlate the developmental events in the maturation of the
epididymis with the timing of immobilin secretion and endocytosis in order
to evaluate the testicular or epididymal factors which may influence or
regulate immobilin expression.
Methods: Our approach was to follow and compare the developmental
expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages.
Results: Coincident with the morphological maturation of the principal
cells by postnatal day 39, immobilin displayed the characteristic secretory
immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident
with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak
to non-existentin the principal cells of the caput epididymidis at day 28 and
remained so into adulthood, indicating that principal cells of this region of
the epididymis are dependent either directly or indirectly upon testicular
factors present in the lumen for immobilin expression. However, secretion
of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of
the proximal initial segment. Thus in the distal initial segment immobilin
secretion is not regulated by luminal factors originating from the testis,
while in the proximal initial segment the normal suppression of immobilin
that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49,
indicating that luminal testicular factors are not essential for stimulating
the uptake of immobilin by these cells.
Conclusions: The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is
independent of luminal testicular factors. o 1994 Wiley-Liss, Inc.
Key words: Immobilin, Development, Principal cells, Clear cells, Secretion, Endocytosis, Rat epididymis
INTRODUCTION
The ultrastructural features of the predominant epithelial cell type, i.e., the principal cell, lining the epididymis of rats ranging in age from birth to postnatal
day 90 have been studied by several investigators (Sun
and Flickinger, 1979; Francavilla et al., 1987; Hermo
et al., 1992a). Up to postnatal day 21, these cells have
a uniform appearance along the length of the epididymis and show features of structurally undifferentiated
cells; they are referred to as undifferentiated columnar
epithelial cells. By postnatal day 39, these cells, now
0
1994 WILEY-LISS,INC.
referred to as principal cells, become structurally differentiated; they present an elaborate endocytic apparatus and a well developed secretory apparatus consisting of a large Golgi apparatus, abundant cisternae of
endoplasmic reticulum and numerous 150-300 nm
electron lucent secretory vesicles present in the Golgi
and apical region of the cell (Hermo et al., 1992a). The
Received January 25, 1994;accepted March 24,1994.
Address reprint requests to Dr. L. Hermo, Department of Anatomy
and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, Canada H3A 2B2.
DEVELOPMENTAL EXPRESSION O F IMMOBILIN
differentiation of these cells occurs in spite of the absence of spermatozoa in the epididymal lumen, which
occurs by postnatal day 49 in the caput epididymidis,
but may be under the influence of androgens (Hermo et
al., 1992a), which have been shown to be high a t postnatal day 39 (Scheer and Robaire, 1980). Other factors
which may also be influential in the differentiation of
these cells include specific secretions, including proteins, by Sertoli cells (Robaire and Hermo, 1988).
Clear cells on the other hand, are highly endocytic
cells (Moore and Bedford, 1979a,b) possessing numerous coated pits and vesicles, endosomes, multivesicular
bodies and secondary lysosomes. They are active in
fluid phase and adsorptive endocytosis and in the removal of the contents of cytoplasmic droplets after the
latter detach from spermatozoa and break up in the
lumen (Hermo et al., 1988). During postnatal development, these cells become differentiated by day 39 in the
cauda region but only by day 49 in the caput and corpus
epididymidis (Hermo et al., 1992a). The factors involved in the differentiation of these cells are unknown.
Immobilin is a glycoprotein found in the caudal epididymal fluid that mechanically renders spermatozoa
in an immotile state as a result of its viscoelastic properties (Usselman and Cone, 1983; Usselman et al.,
1985). Recently, using light and electron microscope
immunocytochemistry the localization of anti-immobilin antibody was performed on adult rats. A variable
cytoplasmic reaction was noted along the entire length
of the epididymis. Principal cells of the proximal region
of the initial segment were unreactive, while those of
its distal region were intensely stained (Hermo et al.,
1992b). In the intermediate zone and proximal caput
region, principal cells showed their most intense reactivity, while in the distal caput they were less reactive.
In the corpus and cauda regions, principal cells were
unreactive. These observations extended the preliminary results obtained by Ruiz-Bravo (1988).
In the electron microscope (EM), immunogold labeling was seen over the endoplasmic reticulum, Golgi
apparatus and 150-300 nm electron lucent vesicles
present in the apical and Golgi regions of the cell. These
vesicles were considered the secretory vesicles of the
principal cell. In the lumen, immunogold labeling was
present over a fine flocculent material which appeared
sparingly in the lumen of the caput region but which
was especially abundant in the lumen of the cauda epididymidis. In the distal cauda epididymidis, epithelial
clear cells were intensely reactive. Immunogold particles were seen over the entire endocytic apparatus of
these cells. It was therefore suggested that a proportion
of immobilin in the distal cauda was taken up from the
lumen for degradation (Hermo et al., 1992b).
Using biochemical approaches to identify the presence of proteins and/or specific mRNAs during development along the entire epididymis, various investigators have noted a dramatic increase between 21 and 39
days of age (Faye et al., 1980; Scheer and Robaire,
1981; Brooks, 1987; Charest et al., 1989; Garrett et al.,
1990).However, in situ localization of these epididymal
proteins, including immobilin, during postnatal development of the epididymis is lacking.
In the present immunocytochemical investigation,
the onset and progression of immobilin expression by
87
principal cells and of endocytosis of immobilin by clear
cells along the entire rat epididymis were examined in
animals at different postnatal ages. These observations
were correlated with epididymal maturation events,
such as presence or absence of spermatozoa in the lumen, androgen levels, and the morphological state of
principal and clear cell differentiation. In order to address the question whether luminal factors secreted by
the testis influenced or regulated the normal development of immobilin secretion and endocytosis in the epididymis, bilateral ligation of the efferent ducts was
performed on 15 day old animals. The consequences of
this procedure on immobilin expression was compared
to controls by immunocytochemistry at various postnatal days thereafter. In this way it was possible to monitor the influence that the absence of luminal androgens and testicular factors including spermatozoa had
on the expression of immobilin by principal cells and on
its endocytosis by clear cells during postnatal development.
MATERIALS AND METHODS
Animals
Timed pregnant female Sprague-Dawley rats were
obtained from Charles River Canada Ltd. (St. Constant, Que.). A number of litters, out of which 58 pups
were utilized, were maintained on a 14 hour dark:lO
hour light cycle. They were provided with food and water ad libitum. After birth, their normal development
was monitored by assessing body weight gain and palpating their testes and epididymides. Only those pups
showing normal trends in development as reported by
Hermo et al. (1992a) were used. Six rats at each of the
following intervals were utilized: 7, 15, 21, 28, 39, 49
and 56 days of age. At each age, two rats were used to
obtain the weights of the paired testes and epididymides, while the other four were used to prepare
the tissue for light microscope immunocytochemical
analysis. The body weights of all animals used as well
as the testicular and epididymal weights of the two
unfixed animals were within the range obtained by
others (Scheer and Robaire, 1980; Hermo et al., 1992a).
The size and appearance of the testes and epididymides
of the four fixed animals were similar to those of the
two unfixed animals.
Light Microscope lmmunocytochemistry
Tissue preparation
Four rats at each postnatal age were anesthetized
with an intraperitoneal injection of sodium pentobarbital (Somnitol, MTC Pharmaceuticals, Hamilton,
ON), and the epididymides fixed by perfusion through
the heart (7 and 15 day old rats) or abdominal aorta
(21,28,39,49and 56 days) with Bouin’s fixative for 10
minutes.
After perfusion, the epididymides were removed and
immersed in Bouin’s fixative for another 24 hours.
Prior to immersion the epididymal tissue was cut along
its long axis in such a way that each of the major regions of the epididymis, i.e., the initial segment, intermediate zone, caput, corpus and cauda epididymidis
(diagram 1of Hermo et al., 1991),would be included for
examination. After fixation the tissue was dehydrated
and embedded in paraffin.
88
L. HERMO ET AL.
Tissue sections of 5 pm thickness were cut, mounted
on glass slides and then immersed in xylene and
graded ethanol solutions (5 minutes each) to deparaffinize and hydrate the tissue. To inactivate residual
picric acid, sections were bathed, for 5 minutes, in 70%
ethanol containing 1% lithium carbonate. They were
also immersed in a 70% ethanol solution containing 1%
vlv hydrogen peroxide to inactivate the endogenous
peroxidase activity. Once hydrated, the tissue was
washed for 5 minutes in distilled water containing 300
mM glycine in order to block the free aldehyde groups.
lmmunostaining
Prior to primary antibody incubation, the tissue sections were blocked for 15 minutes in Trizma-Base buffered saline (TBS), pH 7.4, containing 10%goat serum
in order to block any nonspecific binding. Each section
was then incubated for 1.5 hours (at 37°C) with an
affinity purified polyclonal antibody reactive against
immobilin (provided by Dr. Ruiz-Bravo)a t a dilution of
1:lO to 150. The specificity and characterization of the
antibody have been described by Ruiz-Bravo (1988).
After primary antibody incubation, sections were
washed four times in TBS containing 0.1%Tween-20,
then blocked with 10%goat serum for 15 minutes and
subsequently incubated for 1 hour with anti-rabbit IgG
conjugated to peroxidase (Sigma, St. Louis, MO) a t a
concentration of 11250 in TBS a t 37°C.
After secondary incubation, all sections were washed
thoroughly in TBS containing 0.1%Tween-20 (4 x 2
minutes each). The sections were then incubated for 10
minutes with 0.03% hydrogen peroxide and 0.05% diaminobenzidine tetrachloride in TBS containing 0.1 M
imidazole at pH 7.6 (Straus, 1982). After being washed,
the sections were counterstained with 0.1%methylene
blue for 20 to 30 minutes. Finally, the tissue was dehydrated in a graded series of ethanol (2 minutes each),
immersed in xylene (2 minutes) and mounted in Permount. Normal rabbit serum at a dilution of 1:50
served as controls.
Ligation of the Efferent Ducts of 15 Day Old Animals
For this study, twelve animals were utilized which
showed normal developmental trends. The body
weights of these animals were within the range shown
for normal untreated animals (Hermo et al., 1992a). At
postnatal day 15, both right and left efferent ducts of
these 12 pups (four per group) were ligated close to the
rete testis, to prevent the flow of testicular products
into the lumen of the epididymis. They were fixed by
perfusion with Bouin's fixative at postnatal days 28,49
and 64, i.e., 13, 34 and 49 days after ligation. Their
testes and epididymides were removed and prepared
for light microscope immunocytochemical analysis as
described above.
RESULTS
Light Microscope lmmunolocalization of lmmobilin in the
Epididymis at Different Postnatal Days
At postnatal day 7, the epithelium of the entire epididymis was cuboidal, undifferentiated and unreactive
to anti-immobilin antibody. A small empty lumen was
evident along the entire length of the duct (Fig. 1).
At postnatal day 15, the undifferentiated columnar
epithelial cells of the entire epididymis showed a weak
to moderate diffuse reaction in their apical and supranuclear regions (Figs. 2-41, with the exception of the
caput region where a few tubules presented an intense
reaction in the area above their nucleus (Fig. 3).
At postnatal day 21, in the proximal initial segment,
many undifferentiated columnar cells presented a moderate to intense spherical supranuclear reaction over
the Golgi apparatus, while some were unreactive (Figs.
5, 6). In the distal initial segment, the columnar epithelial cells displayed a similar reaction but more cells
were reactive; a weak diffuse reaction was also seen
apically (Figs. 5,7). The intermediate zone and proximal caput epididymidis showed a similar reaction to
the proximal initial segment (Figs. 5,8), while the distal caput showed weakly reactive or unreactive cells
(Fig. 9). In the corpus and cauda epididymidis, the columnar epithelial cells were weakly reactive (Figs.
10,ll). In all the regions of the epididymis, the lumen
appeared small and devoid of spermatozoa, but an intense reaction was associated with stereocilia extending into the lumen (Figs. 6-11)
At postnatal day 28, the undifferentiated columnar
epithelial cells of the initial segment, intermediate zone
and proximal caput showed a moderate to intense supranuclear Golgi reaction and a weak to moderate diffuse reaction apically (Figs. 12,13). The basal region of
these cells was weakly reactive. The principal cells of
the distal caput were weakly to moderately reactive. In
the corpus and cauda epididymidis, all undifferentiated
columnar epithelial cells were unreactive (Fig. 14). In
all regions of the epididymis, the lumen was small and
devoid of spermatozoa but a strong reaction was apparent in association with stereocilia (Figs. 12-14). In the
corpus epididymidis, an intensely immunostained
amorphous material was present in the lumen (Fig. 14).
At postnatal day 39, there was a dramatic change in
the immunostaining of the columnar epithelial cells,
now differentiated and referred to as principal cells. At
low magnification, the variability in the staining pattern in the initial segment was apparent (Fig. 15).
Principal cells of the proximal initial segment were
unreactive (Fig. 161, while those of its distal region
showed an intense supranuclear Golgi reaction and a
moderate diffuse reaction apically (Fig. 17). The same
intense staining pattern was seen in the intermediate
zone (Fig. 18) and in the proximal caput epididymidis
(Fig. 19). The basal region of principal cells of these
regions was weakly reactive (Figs. 17,18). The principal cells of the distal caput were moderately reactive.
In the corpus and cauda epididymidis, principal cells
were unreactive (Figs. 20-22). For the first time, the
epithelial clear cells of the distal cauda epididymidis
became immunostained but moderately so (Fig. 22). At
this age, the deeply immunostained amorphous material appeared in the lumen of the distal cauda epididymidis (Fig. 22). In all regions of the epididymis a lumen
was present, devoid of spermatozoa (Figs. 16-22). An
intense immunoperoxidase reaction was associated
with stereocilia of principal cells of all epididymal regions (Figs. 17-22) except the proximal initial segment
(Fig. 16).
At postnatal day 49, a variable staining pattern was
observed in the initial segment of the epididymis (Fig.
23). In the proximal initial segment, principal cells
DEVELOPMENTALEXPRESSION OF IMMOBILIN
89
Fig. 1 . Low power light micrograph of cross sections of tubules of the corpus epididymidis at postnatal
day 7 immunostained with anti-immobilin antibody. The undifferentiated epithelial (E) cells are cuboidal in appearance and unreactive. A small empty lumen (Lu) is present in all tubules. IT, intertubular
space. x335.
were unreactive (Fig. 23), while in the middle initial
segment, a moderate spherical Golgi reaction was
present supranuclearly and a weak diffuse reaction
apically (Figs. 23,24). Principal cells of the distal initial segment showed an intense supranuclear Golgi reaction and a moderate diffuse reaction apically; the
same reaction was seen in the intermediate zone (Fig.
25) and proximal caput epididymidis. In the distal
caput epididymidis principal cells were less intensely
stained (Fig. 26).
In the corpus (Figs. 27,281 and cauda (Figs. 29,301
epididymidis, principal cells were unreactive. In the
distal cauda epididymidis clear cells became intensely
reactive throughout their cytoplasm coincident with
the presence of an abundance of the deeply stained
amorphous material in the lumen (Figs. 29,30). At
postnatal day 49 no reaction was seen in the lumen of
the proximal initial segment (Fig. 23). However,
throughout the rest of the epididymis an intense reaction was seen in association with stereocilia (Figs. 2428).
At postnatal day 56, the same staining pattern was
observed along the entire epididymis as at day 49 but
a t this age spermatozoa appeared in the lumen of the
cauda epididymidis. This pattern was similar to that
seen in adult animals (Hermo et al., 1992b).
Light Microscope lmmunolocalization of lmmobilin in
Animals Ligated at Postnatal Day 75
Postnatal 28 day old animals ligated at day 15 and
immunostained with anti-immobilin antibody revealed
undifferentiated columnar epithelial cells in the initial
segment with a weak spherical Golgi reaction supranuclearly (Fig. 31). The epithelium of the rest of the
epididymis was unreactive (Fig. 32). A reaction was
seen in association with stereocilia (Figs. 31,32).
The initial segment of postnatal 49 day old animals
ligated a t day 15 presented principal cells with a moderate to intense spherical Golgi reaction in their supranuclear region with its proximal region being more intense than the distal region (Figs. 33,34). In the caput
region (Fig. 351, however, principal cells were unreactive as was the case in the corpus and cauda epididymidis (Figs. 36,371. An intense reaction was associated
with stereocilia and an amorphous material in the lumen (Figs. 35,361. In the distal cauda epididymidis a
few clear cells showed a weak reaction throughout
their cytoplasm (Fig. 37) simultaneous with the presence of only small amounts of a deeply stained amorphous material in the lumen. The lumen was devoid of
spermatozoa (Figs. 33-37).
Postnatal 64 day old animals ligated a t day 15 revealed principal cells of the initial segment with a moderate to intense spherical Golgi reaction in their supranuclear region with the proximal region being more
intense than the distal region (Fig. 38). In the caput
epididymidis, only a faint reaction was apparent supranuclearly (Fig. 39). In the corpus and cauda epididymidis, principal cells were unreactive (Figs. 40-42). The
lumen of the dislal cauda region was now filled with
large masses of a deeply stained amorphous material
(Figs. 41,42), and a t this time many clear cells became
intensely reactive (Fig. 42).
Control sections for all experiments revealed a com-
Fig. 2. Low power light micrograph of several regions of the epididp i s at postnatal day 15 immunostained with anti-immobilin antibody. The proximal (P) and distal (D) initial segment (IS), and caput
(Cap) epididymidis are indicated. The undifferentiated columnar epithelial cells of most tubules show a weak to moderate diffuse reaction
(arrows) in the area of cytoplasm above the nucleus. However, a few
tubules (curved arrows) in the caput are more intensely stained.
x 200.
Fig. 3.High power light micrograph of a portion of Figure 2 showing
several tubules of the caput epididymidis at postnatal day 15 immunostained with anti-immobilin antibody. The undifferentiated colum-
nar epithelial cells (E) present an intense reaction (curved arrows) in
the region above the nucleus. IT, intertubular space. x 335.
Fig. 4. High power light micrograph of tubules of the corpus epididymidis a t postnatal day 15 immunostained with anti-immobilin
antibody. The same staining pattern was observed in the cauda epididyrnidis. The undifferentiated columnar epithelial cells (E) reveal a
weak to moderate diffuse immunoperoxidase reaction (arrows) filling
the area of cytoplasm overlying the unstained nucleus (n). The basal
region is unreactive. Lumen (asterisk) is small and empty. IT, intertubular space. x 500.
Fig. 5. Low power light micrograph of the epididymis at postnatal
day 21 immunostained with anti-immobilin antibody. Proximal (P)
and distal (D)initial segment (IS) and caput epididymidis (Cap) are
indicated. Note variable staining pattern between the different regions. ~ 8 0 .
Fig. 6. High power light micrograph of tubules of the proximal ini-
tial segment at postnatal day 21 immunostained with anti-immobilin
antibody. Many undifferentiated columnar epithelial cells show a
moderate to intense Golgi reaction supranuclearly (arrows), while a
few are unreactive. The nucleus (n) and basal region of these cells
are unstained. Note that a reaction (arrowhead) appears in association with stereocilia (arrowhead)extending into the lumen. IT, intertubular space. x 335.
Fig. 7. High power light micrograph of tubules of the distal initial
segment a t postnatal day 21 immunostained with anti-immobilin antibody. Many undifferentiated columnar epithelial cells present a
spherical intense Golgi reaction supranuclearly (arrows) and a weak
diffuse staining apically;a few are less intensely stained. Basal region
is unstained as is the nucleus (n). A reaction is associated with stereocilia (arrowheads). IT, intertubular space. x 335.
Fig.8.High power light micrograph of tubules of the proximal caput
epididymidis at postnatal day 21 immunostained with anti-immobilin
antibody. Some columnar epithelial cells show a weak to moderate
Golgi reaction supranuclearly (arrow), while others are unreactive. A
strong reaction is associated with stereocilia (arrowhead). Nucleus (n)
and basal region of the columnar cells are unreactive. IT, intertubular
space. x375.
Fig. 9.High power light micrograph of tubules of the distal caput
epididymidis a t postnatal day 21 immunostained with anti-immobilin
antibody. A few columnar epithelial cells show a faint reaction of the
Golgi apparatus supranuclearly (arrow), while the majority are unreactive. Note the strong reaction associated with stereocilia (arrowhead). Nuclear (n) and basal region of columnar cells are unreactive.
IT, intertubular space. x 320.
Fig. 10.High power light micrograph of tubules of the corpus epididymidis at postnatal day 21 immunostained with anti-immobilin
antibody. The columnar epithelial cells (El are weakly reactive; however, a reaction is associated with stereocilia farrowhead). Nuclei (n)
and basal region are unreactive. Lumen (Lu)is small and empty. IT,
intertubular space. x 320.
Fig. 11. Low power light micrograph of tubules of the proximal
cauda epididymidis at postnatal day 21 immunostained with antiimmobilin antibody. The undifferentiated columnar epithelial cells
(E) are weakly reactive. Note a reaction is associated with stereocilia
(arrowhead). Lumen (asterisk) is small and empty. IT, intertubular
space. x210.
Fig. 12. Low power light micrograph of several regions of the epididymis a t postnatal day 28 immunostained with anti-immobilin antibody. The proximal (P)and distal (D) initial segment (IS),intermediate zone (12) and proximal caput epididymidis (Cap) are indicated.
The undifferentiated columnar epithelial cells of all these regions
appear reactive. x 80.
Fig. 13. Light micrograph a t postnatal day 28 immunostained with
anti-immobilin antibody. The columnar epithelial cells of the intermediate zone (right) show an intense Golgi reaction supranuclearly (arrows) and a moderate diffuse reaction apically. The same type
of reaction was noted in the proximal and distal initial segment. In
the caput epididymidis (left) the columnar cells (arrows) present a
similar but slightly weaker reaction. Note the strong reaction associated with stereocilia (arrowheads). x 335.
Fig. 14. High power light micrograph of tubules of the corpus epididymidis at postnatal day 28 immunostained with anti-immobilin
antibody. The epithelial cells (E)are slightly reactive. There is an
intense reaction in association with stereocilia (arrowhead) and an
amorphous material in the lumen (asterisks). IT, intertubular space.
x 335.
Fig. 15. Low power light micrograph of several regions of the epididymis a t postnatal day 39 immunostained with anti-immobilin antibody. The proximal (P)and distal (D) initial segment (IS), intermediate zone (IZ)and caput epididymidis (Cap) are indicated. Note the
large variability in the staining of epithelial principal cells from one
region to the next. ~ 8 0 .
Fig. 16. High power light micrograph of tubules of the proximal
initial segment at postnatal day 39 immunostained with anti-immobilin antibody. Principal cells (P)are unreactive. The lumen (Lu) is
devoid of spermatozoa. IT, intertubular space; arrowhead, stereocilia.
x 335.
Fig. 17. High power light micrograph of tubules of the distal initial
segment of the epididymis a t postnatal day 39 immunostained with
anti-immobilin antibody. Note the intense spherical reaction product
supranuclearly over the Golgi apparatus of principal cells (large arrows) and moderate diffuse reaction apically. A reaction is associated
with stereocilia (arrowhead). The nucleus (n) of principal cells is unstained but the basal region shows weak reactivity (small arrows). IT,
intertubular space. x 335.
Fig. 18. High power light micrograph of tubules of the intermediate
zone of the epididymis at postnatal day 39 immunostained with antiimmobilin antibody. Principal cells show a large intense Golgi reaction supranuclearly (arrows) and moderate diffuse reaction apically.
Note large vesicles (open arrows) in the apical region of principal
cells. A weak reaction is seen basally (curved arrows), while the nucleus (n) is unreactive. IT, intertubular space. X 320.
Fig. 19. Low power light micrograph of tubules of the proximal
caput epididymidis a t postnatal day 39 immunostained with antiimmobilin antibody. Principal cells (arrow) show an intense Golgi
reaction supranuclearly and, a moderate diffuse reaction apically. A
weak reaction i s seen basally (arrowheads). A strong reaction is associated with stereocilia (arrowheads). IT, intertubular space. x 210.
Fig. 20. Light micrograph of tubules of the corpus epididymidis at
postnatal day 39 immunostained with anti-immobilin antibody. Prin-
cipal cells (P) are unreactive. There is a distinct reaction over stereocilia (arrowhead). Lumen is devoid of spermatozoa, but an intense
reaction appears over an amorphous material (asterisks) in the lumen. ~210.
Fig. 21. High power light micrograph of the proximal cauda epididymidis at postnatal day 39 immunostained with anti-immobilin
antibody. Principal cells (P) are unreactive. A reaction is associated
with stereocilia (arrowhead). IT, intertubular space. x 320.
Fig. 22. Low power light micrograph of the distal cauda epididymidis at postnatal day 39 immunostained with anti-immobilin antibody.
Principal cells (P) are unreactive, whereas clear cells ( C ) are moderately stained. A reaction is associated with stereocilia (arrowhead)
and an amorphous material (curved arrow) in the lumen which is not
yet abundant. IT, intertubular space. x 125.
Fig. 23. High power light micrograph of the proximal (left) and
middle (right) regions of the initial segment of the epididymis at postnatal day 49 immunostained with anti-immobilin antibody.The two
adjacent regions show a difference in their staining pattern. Principal
cells (P) of the proximal region are unreactive, whereas in the middle
region they are moderately stained in their supranuclear region (arrow). In the middle initial segment a reaction is seen in association
with spermatozoa (asterisk) which appear in the lumen at this age.
Stereocilia (arrowhead) of the proximal initial segment are unreactive. IT, intertubular space. x 190.
Fig. 24.High power light micrograph of the middle initial segment
at postnatal day 49 immunostained with anti-immobilin antibody.
Principal cells (P) show a moderate spherical Golgi reaction supranu-
clearly (arrow) and a weak reaction apically. An intense reaction is
seen in association with stereocilia (arrowhead). x 350.
Fig.25.High power light micrograph of the intermediate zone of the
epididymis a t postnatal day 49 immunostained with anti-immobilin
antibody. The same staining pattern was seen in the distal initial
segment and proximal caput epididymidis. Principal cells show an
intense Golgi reaction in their supranculear region (large arrows) and
a moderate reaction apically. Note the large vacuoles (curved arrow)
in the apical region of principal cells. An intense reaction is seen in
association with stereocilia (arrowhead) and spermatozoa (asterisks)
which appear in the lumen a t this age. A weak staining is present in
the basal region (small arrows). x 320.
Fig. 26. High power light micrograph of a tubule of the distal caput
epididymidis at postnatal day 49 immunostained with anti-immobilin
antibody. Principal (P)cells show a weak diffuse staining apically and
a moderate staining in their supranuclear region (arrows). A distinct
reaction is evident in the lumen in association with spermatozoa (asterisks). X 320.
Fig. 27. Low power light micrograph of tubules of the proximal
corpus epididymidis at postnatal day 49 immunostained with antiimmobilin antibody. Principal cells (P) are unreactive. A distinct reaction is seen in the lumen in association with spermatozoa (arrowheads). X 130.
Fig. 28. High power light micrograph of tubules of the proximal
corpus epididymidis a t postnatal day 49 immunostained with antiimmobilin antibody. Principal cells (P)are unreactive. An amorphous
material in the lumen is intensely reactive and associated with spermatozoa (arrowhead). x 320.
Fig. 29. Low power light micrograph of the distal cauda epididymidis a t postnatal day 49 immunostained with anti-immobilin antibody.
Principal cells are unreactive; however, clear cells (arrows) show an
intense reaction throughout their cytoplasm. An amorphous material
(arrowhead) in the lumen is deeply stained and abundant a t this age
but spermatozoa are absent. IT, intertubular space. x 130.
Fig. 30. High power light micrograph of a portion of a tubule of the
distal cauda epididymidis a t day 49 immunostained with anti-immobilin antibody. Clear cells (open arrows) are intensely reactive,
whereas principal cells (P) are unreactive. Note amorphous deeply
stained material (arrowhead) in the lumen. X 320.
98
L. HERMO ET AL.
Fig. 31.High power light micrograph of tubules of the initial segment of the epididymisof a postnatal 28 day old animal ligated at day
15 and immunostained with anti-immobilin antibody. Many columnar epithelial cells show a weak Golgi reaction supranuclearly (arrows). A reaction is associated with stereocilia (arrowheads). x 335.
Fig. 32. High power light micrograph of tubules of the caput epididymidis of a postnatal 28 day old animal ligated at day 15 and
immunostained with anti-immobilin antibody. The corpus and cauda
epididymidis show the same reaction pattern. The columnar epithelial cells (E)are unreactive. There is a reaction associated with stereocilia (arrowhead).Lumen (Lu) is small and empty. IT, intertubular
space. x335.
postnatal development. At this point there appears to
be little to no regional variation in its secretion. However by day 21, a regional shift in the concentration of
immobilin secreted by the columnar epithelial cells occurs such that relatively higher levels are secreted in
the initial segment and caput than in the corpus and
DISCUSSION
cauda epididymidis. This regional shift in immobilin
Principal Cells
secretion becomes more pronounced by postnatal day
The immunocytochemical results of immobilin ex- 28 and by day 39 the pattern of immobilin secretion
pression during postnatal development of the epididy- resembles that described in adult rats (Hermo et al.,
mis in control and efferent duct ligated rats are sum- 1992b),i.e., high levels secreted by the principal cells of
marized in Table 1. The undifferentiated columnar the distal initial segment, intermediate zone and caput
epithelial cells of the epididymis first begin to secrete and negligible levels found elsewhere in the epididyrelatively low levels of immobilin around day 15 of mis. The distinguishing change from days 28 to 39 is
plete absence of staining of the epithelial cells, cells of
the intertubular space and luminal contents. These
were comparable to those already published in our previous studies (Hermo et al., 199213).
DEVELOPMENTAL EXPRESSION O F IMMOBILIN
Flg. 33.Low power light micrograph of the initial segment (IS)and
small portion of the caput (Cap)epididymidis of a postnatal 49 day old
animal ligated at day 15 and immunostained with anti-immobilin
antibody. Principal cells (arrows) of the initial segment show a moderate to intense Golgi reaction supranuclearly. An intense reaction is
associated with stereocilia (arrowheads). x 210.
Fig. 34. High power light micrograph of the proximal initial segment of the epididymisof a postnatal 49 day old animal ligated at day
15 and immunostained with anti-immobilin. Note the intense Golgi
reaction (arrows) in the supranuclear region of principal cells and a
weak reaction basally (curved arrows). The nucleus (n) is unreactive.
A reaction is associated with stereocilia (arrowhead).IT, intertubular
space. ~ 3 2 0 .
Fig. 35. High power light micrograph of tubules of the caput epididymidis of a postnatal 49 day old animal ligated at day 15 and im-
99
munostained with anti-immobilin antibody. Principal cells (P)are
unreactive, but a strong reaction is associated with stereocilia (arrowhead). x320.
Fig. 36. Photomicrograph of a tubule of the proximal cauda epididymidis of a postnatal 49 day old animal ligated at day 15 and immunostained with anti-immobilin antibody. Principal cells (P) are unreactive, while stereocilia and an amorphous material (arrowhead) in
the lumen are intensely reactive. X 320.
Fig. 37. High power light micrograph of the distal cauda epididymidis of a postnatal 49 day old animal ligated at day 15 and immunostained with anti-immobilin antibody. Principal cells (P) are unreactive, while a few clear cells (arrow) show a faint reaction. Note the
strong reaction associated with stereocilia (arrowheads). x 320.
100
L. HERMO ET AL.
directly or indirectly (by way of metabolic products)
regulate immobilin secretion in specific regions of the
Postnatal
epididymis. If specific regional effects of androgens on
PIS
DIS
PC
DC
day
immobilin secretion do indeed occur postnatally, they
7
may be explainable by the selective development of
+
+
+
+
15
androgen metabolizing enzymes, such as 5a-reductase
+
++
++
+++
21
(Viger and Robaire, 1992) and 3a-hydroxysteroid dehy+++ +++
+-+
+
28
drogenase
(Orgebin-Crist et al., 1975; Scheer and
+
+
28"
Robaire,
1980)
in specific regions of the epididymis.
+++ +++ ++
39
Clearly the present results show that immobilin ex+ + + + + +-+ + +-+
49
pression by principal cells is not dependent on sperma++++ +++
49"
tozoa, which are not present in the lumen of the epi+ + + + + + + + +-+
56
++++ +++
+
64a
didymis by day 39 (Robaire and Hermo, 1988). Our
results differ from those for proenkephalin mRNA levPIS, DIS, PC, DC, COR and CAU correspond to the proximal and
els, which increase dramatically in principal cells of
distal initial segment, proximal and distal caput, corpus and cauda
the proximal epididymis only by day 44 when spermaepididymidis, respectively.
"Results obtained in ligated rats.
tozoa appear in the lumen. Garrett et al. (1990) postuThe number of plus signs ( +) are directly proportional to the strength
of the reaction, while the minus signs (-) indicate the absence of a lated that high levels of proenkephalin were the result
of the entry of spermatozoa or a spermatozoa-like factor
reaction.
into the epididymal lumen.
The regional shift in immobilin secretion was also
accompanied by a loss in the production of immobilin
the loss in the production of immobilin by the principal by principal cells of the proximal initial segment from
postnatal days 28 to 39 (Table 1).One possible explacells of the proximal initial segment.
At the onset of immobilin secretion at postnatal day nation for such a regional specific decrease in immo15, all the yet undifferentiated columnar epithelial bilin secretion may be the progressive increase of incells express immobilin, albeit a t low levels. This sug- hibitory factors emanating from the testis. Another
gests that the factors responsible for the regional se- possibility may be the development of local inhibitory
lectivity of immobilin gene expression, which occurs factors. In order to address the question whether lumigradually until postnatal day 39, are not yet present. nal factors secreted by the testis regulate immobilin
The regional specific increases and decreases in immobilin secretion occurring in the epididymis after day 15
imply that there are both stimulatory and inhibitory
immobilin gene regulatory factors involved. What then
Fig. 38. Low power light micrograph of tubules of the initial segcould the potential factors be and from where could ment
and a portion of the proximal caput epididymidis of a postnatal
they be arising. The regional shift of immobilin secre- 64 day old animal ligated a t day 15 and immunostained with antition which begins on day 21 and becomes more pro- immobilin antibody. Principal cells (curved arrows) in the initial segnounced by day 28 and adult-like by day 39 is coinci- ment (tubules in upper half of field) show a moderate to intense Golgi
supranuclearly. In the proximal caput epididymidis (tubules
dent with several maturation events. First, the lumen reaction
in lower half of field), principal cells (arrows) present only a faint
of the seminiferous tubule becomes patent a t about day reaction in their supranuclear region. An intense reaction is associ18 and is coincident with the appearance of Sertoli cells ated with stereocilia (arrowheads). x 225.
products in the lumen of the epididymis (Vitale et al.,
Fig. 39. High power light micrograph of tubules of the proximal
1973; Tindall et al., 1975). Thus the potential exists caput
epididymidis of a postnatal 64 day old animal ligated at day 15
that one or more of these testicular products may have and immunostained with anti-immobilin antibody. Only a faint reaca regional stimulatory effect on immobilin production. tion (arrows) is present in the supranuclear region of principal cells
Second, androgen binding protein (ABP), which trans- (PI, while the basal region and nuclei (n) are unreactive. A reaction is
associated with stereocilia (arrowhead). Lumen (Lu) is small and deports and maintains high luminal concentrations of void
of spermatozoa. x 335.
testosterone, first appears in the epididymis at day 18
Fig. 40. High power light micrograph of tubules of the corpus epi(Tindall et al., 1975; Turner et al., 1985). The transported testosterone is then converted by principal cells didymidis of a postnatal 64 day old animal ligated a t day 15 and
with anti-immobilin antibody. Principal cells (P) are
into the active epididymal androgen, dihydrotestoster- immunostained
unreactive. A strong staining is associated with stereocilia (arrowone (DHT), by the enzyme 4-ene 5a-reductase (Scheer head) and an amorphous material (asterisks) in the lumen. x 335.
and Robaire, 1980; Klinefelter and Amann, 1980). This
Fig. 41. Low power light micrograph of tubules of the proximal (left)
enzyme and its mRNA undergo a steep concentration
and distal (right) cauda epididymidis of a postnatal 64 day old animal
increase in the principal cells of the initial segment ligated
at day 15 and immunostained with anti-immobilin antibody.
during postnatal days 21 to 42 (Viger and Robaire, Principal cells (P)are unreactive in the two regions, while many clear
1992). Thus it is conceivable that resulting elevated cells (arrows) in the distal cauda show an intense reaction. An intense
levels of DHT may positively regulate the secretion of reaction is associated with an amorphous material (arrowhead) in the
which is abundant at this time. No spermatozoa are present.
immobilin in the initial segment and perhaps in neigh- lumen
x 135.
bouring principal cells of the caput and more distal
regions of the epididymis. Third, circulating levels of
Fig. 42. High power light micrograph of the distal cauda epididyandrogens also begin to rise a t day 21 (Lee et al., 1975; midis of a postnatal 64 day old animal ligated at day 15 and immunostained with anti-immobilin antibody. Principal cells (P) are unreDohler and Wuttke, 1975; Scheer and Robaire, 1980; active,
while many clear cells (arrows) are intensely stained. A strong
Jean-Faucher et al., 1985). These circulating andro- reaction (arrowheads) is associated with spermatozoa and a large
gens also diffuse into the principal cells and may either amorphous mass of material in the lumen. x 335.
Table 1. Immobilin immunoreactivitv in principal cells
~
-
-
-
Figs. 38-42.
102
L. HERMO ET AL.
secretion and are involved in the regional specificity of
immobilin expression, the results of immobilin developmental expression were compared in efferent duct
ligated and control rats (Table 1).
Two striking changes in the developmental pattern
of immobilin expression can be seen in efferent duct
ligated 15 day old rats. First, a t 28 days of age and
after, immobilin secretion is absent in the principal
cells of the caput epididymidis, which is in contrast to
the normally high levels secreted by this region. This
difference suggests that factors emanating from the
testis are normally stimulatory for immobilin secretion
in this region. It is interesting to note that these factors
appear to be specifically stimulatory for immobilin in
this region, because under a similar experimental protocol (Hermo et al., 1994) the secretion of sulfated glycoprotein-2 (SGP-2) in this region was not affected by
efferent duct ligation. Secondly, high levels of immobilin, which are normally repressed by postnatal day 39,
continue to be expressed into adulthood in the principal
cells of the proximal initial segment. This difference
suggests that factors originating from the testis are
normally inhibitory for immobilin secretion in this region. Since high levels of immobilin are normally secreted by this region up to postnatal day 28, the production of these testicular inhibitory factors must take
place between days 28 and 39. Although the nature of
these inhibitory factors are unknown, it is possible that
they have an inhibitory effect on other secretory proteins of this region as well because under a similar
experimental protocol SGP-2, which is normally repressed in this region by day 39, continues to be expressed at high levels in efferent duct ligated rats
(Hermo et al., 1994).
The ligation results suggest that there are both stimulatory and inhibitory testicular factors involved in the
regional development of immobilin secretion in the epididymis. Testicular factors regulating the expression
of immobilin in the initial segment clearly differ from
those in the caput epididymidis. In addition there are
immobilin secreting regions in the epididymis, i.e., the
distal initial segment, whose secretory development is
clearly independent of luminal testicular factors. The
progressive development of immobilin secretion in the
principal cells of these regions may be either intrinsic
to the cell or regulated by “other” factors such as circulating androgens.
Clear Cells
Immunocytochemical localization revealed that clear
cells of the distal cauda epididymidis first begin to endocytose immobilin at postnatal day 39, a time coincident with the arrival of immobilin in the lumen of this
region. This time also corresponds to the age when
clear cells of the cauda have been shown to possess all
the structural features characteristic of fully differentiated cells (Hermo et al., 1992a). By postnatal day 49,
coincident with the arrival of larger amounts of an immobilin-reactive amorphous material in the lumen of
the distal cauda, the endocytosis of immobilin by clear
cells in this region becomes intense and remains so at
day 56 and i n i d u l t animals. In adults, EM immunocytochemica~analysis clearly indicated that immobilin
is endocytosed by clear cells only in the distal cauda
epididymidis (Hermo et al., 1992b). These results sug-
gest on the one hand that during development the endocytic capability of clear cells of the distal cauda is
directly proportional to the amount of immobilin secreted by the proximal portion of the epididymis. On
the other hand, it is also possible that immobilin itself
may be a factor involved in the maturation of the endocytic apparatus of these cells. This idea is substantiated in the results obtained in efferent duct ligated 15
day old rats. In these rats, the 10 day delay in the
appearance of immobilin in the lumen of the distal
cauda resulted in an equivalent delay in the maturation of endocytic apparatus of the clear cells of this
region.
In a previous study, Hermo et al. (1992b) showed
that a proportion of immobilin is endocytosed by the
clear cells of the distal cauda in adult rats. In this same
study it was also shown that clear cells in more proximal regions of the epididymis are not involved in the
endocytosis of immobilin. This observation taken together with the present observation that clear cells of
the proximal cauda only become morphologically mature by postnatal day 49 suggests that different regulatory factors are required for the differentiation of
clear cells in the various regions of the epididymis.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical
Research Council of Canada and the National Sciences
and Engineering Research Council of Canada. The
technical assistance of Sonia Bujold is gratefully appreciated. The antibody was kindly provided by Dr.
Ruiz-Bravo. The secretarial assistance of Ann Silkauskas is gratefully acknowledged.
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