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From the * Department of Morphology, Federal
University of Minas Gerais, Belo Horizonte, MG, Brazil; and the
Department of Veterinary Biosciences,
University of Illinois, Urbana, Illinois.
| Correspondence to: Dr Rex A. Hess, Department of Veterinary Biosciences, University of Illinois, 2001 S Lincoln, Urbana, IL 61802-6199 (e-mail: rexhess{at}illinois.edu). |
| Received for publication December 3, 2008; accepted for publication February 24, 2009. |
Efferent ductules of the male reproductive tract contain high
concentrations of estrogen receptors (ER), which are essential for the
regulation of fluid reabsorption and maintenance of normal epithelial
morphology. Treatments with the antiestrogen ICI 182,780 and
17β-estradiol cause a reduction in ER
expression; however, the
mechanisms governing the down-regulation are undetermined. In other tissues,
the ubiquitin-proteasome pathway appears to have a dominant role in regulating
ER turnover, although in the efferent ductules, an abundance of
epithelial lysosomes could also participate in protein turnover. To study this
activity, the expressions of proteasome, ubiquitin, and markers for the
endocytotic apparatus (early endosome antigen-1 [EEA1], clusterin, and
cathepsin D) were examined in rat efferent ductules and initial segment of
epididymis. Distinct cellular, subcellular, and regional distributions of
these proteins were observed in the epithelial cells. A gradient of
proteasome, ubiquitin, EEA1, and clusterin staining was seen in the efferent
ducts, which decreased 30%–41% from the proximal zone to the terminal
common duct. Antiestrogen treatment resulted in significant decreases in
proteasome, EEA1, and clusterin in the efferent ducts. Localization of
ubiquitin-proteasome and endocytotic pathway components suggests that
differential regulation is required for protein degradation and turnover in
efferent ductules and head of the epididymis.
Key words: Lysosome, endosome, cathepsin, ICI 182,780
(ESR1) and a wide expression of ERβ (ESR2)
throughout the tract (Hess et al,
1997,Hess et al,
1997; Nielsen et al,
2001; Nie et al,
2002; Zhou et al,
2002; Hess, 2003).
The primary function of ER in efferent ductules is the regulation of
factors involving fluid reabsorption and maintenance of nonciliated cell
morphology, particularly the endocytotic apparatus (Hess et al,
1997,1997,
2000;
Nakai et al, 2001;
Zhou et al, 2001;
Toda et al, 2008;
Yasuhara et al, 2008).
Maintenance of these ER functions is essential for male fertility
(Eddy et al, 1996;
Hess et al,
1997,Hess et al,
1997; Oliveira et al,
2001,
2002;
Cho et al, 2003).
The expression of ER in efferent ductules is down-regulated by
exogenous treatment with the antiestrogen ICI 182,780 or 17β-estradiol
(Oliveira et al, 2003,
2004). However, the mechanisms
governing this down-regulation of ER are still undetermined. A
transient increase in lysosomes and changes in their subcellular distribution
after ICI 182,780 treatment has been described for the rat (Oliveira et al,
2001,
2002); however, the
ubiquitin-proteasome pathway appears to have a dominant role in regulating
ER and ERβ turnover (El
Khissiin and Leclercq, 1999;
Lonard et al, 2000;
Wijayaratne and McDonnell,
2001; Callige and Richard-Foy,
2006; Yan et al,
2007; Picard et al,
2008). This protein degradation pathway involves covalent
attachment of multiple ubiquitin molecules to the protein substrate.
Ubiquitination involves the tagging of proteins for recognition and subsequent
degradation by the 26S proteasome complex, which is a cylindrical
multi-catalytic protease found in both cytosolic and nuclear compartments
(Palmer et al, 1996;
Ciechanover, 2005). Besides
targeting ER protein for degradation, the ubiquitination process has been
shown to regulate ER and ERβ ligand-dependent and -independent
activity and nuclear-cytoplasmic distribution
(Zhang et al, 2006;
Picard et al, 2008).
Proteasome proteolysis also participates in the entire process of estrogen
receptor–regulated transcription
(Callige and Richard-Foy, 2006;
Zhang et al, 2006;
Yan et al, 2007). These
findings indicate that function of the ubiquitin-proteasome complex might be
more important for estrogen-mediated signaling than first considered.
Information regarding the presence of the ubiquitin-proteasome complex in
the male reproductive tract is lacking. Therefore, the goals of this study
included 1) determining the expression and cellular distribution of proteasome
and ubiquitin in the epithelium of rat efferent ductules and initial segment
of the epididymis (Figure 1);
2) comparing variations in the expression of ubiquitin, proteasome, and
markers for endocytosis; and 3) determining the treatment effects of the
antiestrogen ICI 182,780 on the ubiquitin-proteasome and endocytotic pathways.
ICI 182,780 is recognized for its ability not only to block estrogen binding
but also to down-regulate the ER protein
(Dauvois et al, 1993;
Stenoien et al, 2001;
Oliveira et al, 2003;
Berry et al, 2008). The initial
segment of the epididymis was investigated for comparison, because in the rat,
this regional epithelium is ER negative and shows differential
responsiveness to estrogen and antiestrogen compared with efferent ductules
(Fisher et al, 1997; Oliveira
et al, 2003,
2004,
2005;
Hess and Carnes, 2004). Data
presented are the first to demonstrate high levels of proteasome, ubiquitin,
and early endosome antigen-1 (EEA1) proteins in rat efferent ductules and
initial segment of the epididymis, with apparent regional differences in
proteolytic activity.
|
Animals
Sprague-Dawley (Harlan Bioproducts, Indianapolis, Indiana) adult
(130–150 days old) or 30-day-old (for antiestrogen treatment) male rats
were used for this study. The rats were housed under constant conditions of
light (12 hours light, 12 hours dark) and temperature (22°C). They were
fed with commercial diet (Teklad Chow–Harlan Teklad, Madison, Wisconsin)
and received tap water ad libitum. All animal experiments were approved by the
University of Illinois, Division of Animal Resources (IACUC), and were
conducted in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council,
1996).
Tissue Preparation
The adult rats (n = 6) were anesthetized with IP injections of sodium
pentobarbital (0.1 mL/100 mg body weight) and perfused intracardially with 10%
neutral buffer formalin. After fixation, the efferent ductules plus initial
segment of the epididymis were dissected from the testis and epididymis,
embedded in paraffin, sectioned (5 µm) and mounted in electrostatically
charged glass slides for immunohistochemistry staining. The prepubertal
animals were used for antiestrogen treatment. After treatment, they were
processed following the same protocol.
Antiestrogen Treatment
Starting at 30 days of age, rats (n = 3) were treated once per week, for a
total of 73 days of treatment with subcutaneous injections of ICI 182,780
(Faslodex; provided by AstraZeneca, Macclesfield, United Kingdom) at a dosage
of 10 mg/animal in a volume of 0.2 mL of vehicle (Oliveira et al,
2001,
2002). The control group
received the same volume of castor oil. The treatment duration was chosen on
the basis of previous data that showed that after 73 days of ICI treatment,
ER was reduced to barely detectable levels
(Oliveira et al, 2003),
coincident with a maximum luminal dilation of the efferent ductules and
increased amount of lysosome-like granules in nonciliated cells. Also, at this
time point, atrophy of the ductal epithelium and seminiferous tubules of the
testis had not begun (Oliveira et al,
2002).
Immunohistochemistry
Tissues were fixed with neutral buffered formalin, embedded in paraffin,
and stained following standard methods for microwave antigen retrieval. The
antibodies used were a polyclonal rabbit anti–20S proteasome core
(Affiniti Research Products Ltd, Mamhead, United Kingdom), a polyclonal rabbit
anti-ubiquitin antiserum (Sigma, St Louis, Missouri), a monoclonal mouse
anti-human EEA1 (BD Transduction Laboratories, Lexington, Kentucky), an
affinity-purified polyclonal rabbit anti–sulfated glycoprotein-2 (SGP2,
clusterin; provided by Dr M.D. Griswold, Washington State University, Pullman,
Washington), and a polyclonal rabbit anti–cathepsin D (Dako Corporation,
Carpinteria, California). The specificity of the antibodies has been
previously determined (Sylvester et al,
1991; Igdoura et al,
1995; Greenfield et al,
1999; McDonald et al,
2003; Tengowski et al,
2005; Li et al,
2006). Sections were incubated with primary antibodies and diluted
1:1000 for proteasome and ubiquitin, 1:100 for EEA1, and 1:200 for clusterin
and cathepsin D, overnight at 4°C, except for clusterin and cathepsin D,
which were incubated for 2 hours. For negative control, the primary antibody
was replaced with phosphate-buffered saline (PBS). Liver and kidney tissues
were used as positive controls. After washing in PBS, the sections were
exposed for 1 hour to biotinylated secondary antibodies (goat anti-rabbit for
proteasome, ubiquitin, cathepsin D, and clusterin and goat anti-mouse for
EEA1—all from Dako Corporation) and diluted 1:100. The sections were
then incubated with the avidin-biotin complex (Vectastain Elite ABC kit;
Vector Laboratories, Burlingame, California) for 30 minutes, and the
immunoreaction was visualized by diaminobenzidine containing 0.01%
H2O2 in 0.05 M Tris-HCl buffer, pH 7.6. Sections were
counterstained with Mayer hematoxylin.
Scoring of Immunostaining Intensity
The intensity of immunoreaction for proteasome, ubiquitin, EEA1, and
clusterin, as well as the area occupied by the structures positive for EEA1,
clusterin, and cathepsin D, was quantified by computer-assisted image
analyses, based on previous protocols (Oliveira et al,
2002,
2007a;
Picciarelli-Lima et al, 2006).
For this procedure, pictures from five different areas of the proximal, conus
and common duct regions of the efferent ductules for each animal were taken by
using a x40 objective lens of a Nikon Eclipse E600 microscope and a
Nikon Coolpix digital camera (Nikon Co, Melville, New York). Digital images
were processed by Adobe Photoshop (Adobe Systems, Mountain View, California),
converted to grayscale mode, and inverted.
The processed images were imported by Image Tool software (University of Texas Health Sciences Center, San Antonio, Texas). For quantitative estimation of proteasome and ubiquitin immunoreaction intensity, 50 positive nuclei of epithelial nonciliated cells of the proximal, conus, and common efferent ductules were traced, and the pixel intensity was determined for the traced areas. Background intensity was determined by tracing an unlabeled area adjacent to the measured cells. Final pixel intensity was calculated by subtracting the values detected in labeled nuclei from the background.
To estimate the intensity of EEA1 and clusterin immunostaining, as well as the percent area occupied by the structures expressing EEA1, clusterin, and cathepsin D in cytoplasm of nonciliated cells of the efferent ductules, five different epithelial areas composed of four consecutive nonciliated cells with visible nuclei, for a total of 20 cells per animal, were outlined and measured by use of the Scion Image software (Frederick, Maryland). To discriminate the expression of EEA1, clusterin, and cathepsin D from surrounding background, the density-slicing mode was used. The area occupied by the highlighted positive structures was measured, then the area positive per 100 µm2 of cytoplasm was calculated. The pixel intensity for EEA1 and clusterin reaction was determined for the traced areas, subtracting the background intensity, as indicated above.
Statistical Analysis
Differences in the area and intensity of immunoreaction of the proteins
studied were analyzed statistically by multiple variance analyses (ANOVA). The
post hoc Tukey test was used for comparison between regions. Data obtained
from the ICI 182,780 experiment were analyzed by Student's t test.
Differences were considered significant at P 
.05.
Results
Proteasome
Proteasomes were strongly expressed in nuclei of nonciliated cells in the
efferent ductule epithelium (Figure 2A
through C; Table).
Ciliated cell nuclei were negative or only moderately stained; however, strong
staining was detected in basal areas of cilia. Some intermittent staining was
detected in peritubular cells and connective tissue cells. Positive staining
was common in nuclei of the connective tissue cells, whereas cytoplasmic
staining was detected in some large cells, resembling macrophages. Positive
staining was also found in endothelial and smooth muscle cell nuclei of the
blood vessels. Epithelial proteasomal staining followed a gradient, which
decreased from proximal to the common efferent ductules
(Figure 2A through C). As shown
by quantitative estimation of the immunoreaction, the level of proteasome
staining in the common efferent duct was reduced 32% compared with that found
in the proximal efferent ductules (Figure
3A). Conversely, in connective tissue cells and blood vessels,
staining intensity was similar throughout the efferent ductules. Treatment
with ICI 182,780 resulted in a slight but significant reduction (15%) in the
proteasomal immunoreaction in nuclei of proximal efferent ductule nonciliated
cells (Figures 4A through B and
5A).
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Positive staining for proteasome was also detected in the epithelium and connective tissue of the initial segment of the epididymis (Figure 2D; Table). Differing from efferent ductules, the initial segment epithelium showed positive staining for proteasome in both nuclei and cytoplasm. Nuclei of principal cells were weakly positive, but moderate cytoplasmic expression of proteasome was seen in the basal cytoplasm, surrounding the nuclei. Contrasting with the principal cells, basal cells as well as apical and narrow cells showed intense nuclear staining. Apical cells also presented moderate staining in the apical cytoplasm. Some peritubular cell nuclei were slightly stained for proteasome and both positive and negative cells were observed in connective tissue.
Ubiquitin
The pattern of ubiquitin expression was similar to that seen for proteasome
in both segments of the male reproductive tract studied, including the
gradient of nuclear staining decreasing from proximal to the common efferent
ductules, where the ubiquitin level was reduced 41% compared with proximal
ductules (Figures 2E through G
and 3B;
Table). However, there were
also some unique staining patterns in each segment. For example, differing
from proteasomal staining, ubiquitin was not detected in efferent ductule
ciliated cells. In initial segment epididymis, the intensity of ubiquitin
nuclear staining was similar among principal, basal, apical, and narrow cells
(Figure 2H). Effects of ICI
treatment on ubiquitin expression were not studied.
Early Endosome Antigen-1
In the efferent ductules, EEA1, a marker for early endosomes
(Mu et al, 1995;
Hellevik et al, 1998), was
detected in epithelial nonciliated cells, whereas ciliated cells were negative
(Figure 2I through K). In
nonciliated cells, EEA1 positivity was found along the membrane of large
vesicles occupying the apical cytoplasm. Staining intensity decreased
significantly (30%) from proximal to the common efferent ductules
(Figure 3C). The cytoplasmic
area occupied by EEA1-positive vesicles was 44% less in nonciliated epithelial
cells of the common efferent ductules, compared with the proximal efferent
ductules (Figure 3D). Few cells
in the connective tissue of efferent ductules were positive for EEA1.
Treatment with the antiestrogen ICI 182,780 resulted in a 25% reduction in
percent area of cytoplasm occupied by structures that were EEA1-positive
(Figures 4C and D and
5B).
In the initial segment of the epididymis, EEA1 was found in principal, apical, and narrow cells of the epithelium, as well as some cells in the intertubular connective tissue (Figure 2L). Immunoreaction for EEA1 in apical and narrow cells was found apically, whereas in the principal cells, the reaction was found throughout the supranuclear cytoplasm.
Clusterin (SGP2)
Clusterin, also known as SGP2 or apolipoprotein J, has been localized
throughout the endocytotic apparatus (Hermo et al,
1991,
1995). Clusterin expression
was restricted to nonciliated cells of the efferent ductule epithelium and
luminal spermatozoa (Figure 2M through
O). Epithelial immunostaining was found in vesicles of different
sizes, concentrated in the subapical and supranuclear cytoplasm of the
nonciliated cells. A gradient of staining was found for clusterin in the
epithelium; common efferent ductules showed approximately 40% less
immunoreaction than the proximal ductules
(Figure 3E). A decrease of 76%
was also observed in the cytoplasmic area occupied by the clusterin-positive
structures, from proximal to the common efferent ductules
(Figure 3F). Luminal
spermatozoa were stained less intensely when they were located in the common
efferent ductules (Figure 2O).
Compared with controls, ICI 182,780 treatment resulted in a significant
decrease (42%) in the cytoplasmic area occupied by the structures positive for
clusterin in proximal efferent ductules (Figures
4E and F and
5C). Immunoreaction for
clusterin was not detected in epithelia of the initial segment of the
epididymis (Figure 2P).
Cathepsin D
Cathepsin D, a marker for lysosomes
(Igdoura et al, 1995), was
localized in granules distributed in the supranuclear cytoplasm of nonciliated
cells and some ciliated cells of the efferent ductule epithelium
(Figure 2Q through T).
Immunoreaction for cathepsin D was heterogeneous along the efferent ductule
epithelium, and the conus region showed the strongest positive reaction,
followed by the distal part of the proximal zone. The ductules nearest rete
testis (initial zone of the proximal efferent ductules) and the common ductule
zone (near the epididymis) were negative (Figures
2Q through T and
3G). Vascular endothelial cells
and some connective cells were also positive. Effects of ICI treatment on
cathepsin D expression were not studied.
In the initial segment epididymis, positive staining for cathepsin D was primarily observed in apical and narrow cells of the epithelium (Figure 2U and V). However, an occasional basal cell was also positive, as were some connective tissue and endothelial cells.
Discussion
In this study, the expression of ubiquitin, proteasomes, and several markers of components of the endocytotic apparatus were examined by immunohistochemistry in adult rat efferent ductules and initial segment of the epididymis (Figure 1). Distinct cellular, subcellular, and regional distributions of these components were observed in epithelial cells along these regions of the male reproductive tract, strongly suggesting differences in proteolytic activity.
Proteasome and ubiquitin were found in the efferent ductules and initial
segment of the epididymis, with distribution closely resembling each other. In
efferent ductules, both proteins were located primarily in epithelial cell
nuclei. In the initial segment epididymis, nuclei of apical, narrow, and basal
cells also showed strong staining, whereas both proteins were found primarily
in the cytoplasm of principal cells. These findings add to previous evidence
that components of the ubiquitin-proteasome system are not evenly distributed
but rather show cellular compartmentalization
(Palmer et al, 1996;
Brooks et al, 2000;
Lenk and Sommer, 2000).
Nuclear predominance of the ubiquitin-proteasome pathway components in
efferent ductule epithelium might be necessary for specific activity, a view
that is consistent with the localization and function of nuclear steroid
hormone receptors (Saunders et al,
2001; Nie et al,
2002; Zhou et al,
2002; Hess and Carnes,
2004; Yamashita,
2004; Oliveira et al,
2007a), including estrogen (ER, ERβ), androgen (AR),
and vitamin D receptor (VDR). All of these nuclear receptors are targeted for
ligand-mediated down-regulation via the ubiquitin-proteasomal system
(Masuyama and MacDonald, 1998;
Alarid et al, 1999;
Nawaz et al, 1999;
Lonard et al, 2000;
Dennis et al, 2001;
Lin et al,
2002,Lin et al,
2002; Laios et al,
2005). It has already been demonstrated that ER does not
appear to degrade within the cytoplasm but rather might require degradation
within the nucleus (Alarid et al,
1999). This finding substantiates the possibility that the nuclear
localization of ubiquitin and proteasome reported here could be related to the
modulation of local steroid receptors, especially ER, whose mRNA is
found in higher concentration in efferent ductules than in uterus, a classical
target for estrogen (Hess et al,
1997,Hess et al,
1997). It is also important to consider that in addition to the
proteolytic activities, nuclear localization of ubiquitin-proteasome system
might serve other functions, such as the regulation of transcriptional
activity, protein trafficking, signal transduction, cell cycle, and apoptosis
(Tanaka and Chiba, 1998;
Salghetti et al, 2001;
Kang et al, 2002;
Lin et al,
2002,Lin et al,
2002; Ciechanover,
2005; Kwon,
2007).
Little is known about the involvement of the ubiquitin-proteasomal complex in male reproductive functions, with prior descriptions being limited primarily to spermatogenesis (Farout et al, 2000; Mochida et al, 2000; Kuster et al, 2004; Kwon et al, 2004), epididymal regulation of sperm quality (Sutovsky et al, 2001; Tengowski et al, 2005, 2007), and sperm fertilization events (Wojcik et al, 2000; Morales et al, 2003). Therefore, the present findings showing a different pattern of proteasome and ubiquitin expression in the initial segment of the epididymis raises questions regarding local functions of these specific components. The ubiquitin-proteasomal pathway has been implicated in endocytosis and turnover of several membrane receptors, transporters, and channels, targeting them for lysosome (Strous and Govers, 1999; van Kerkhof et al, 2002). Others have shown that ubiquitination is involved in the internalization of several plasma membrane proteins and that modification of proteins by ubiquitin attachment can have consequences other than direct targeting to the 26S proteasome (Urbé, 2005), leading to delayed degradation (probably in the lysosome). For several plasma membrane proteins, it has been shown that ubiquitination of the protein itself is required for endocytosis (Strous and Govers, 1999; Strous et al, 2004; van Kerkhof et al, 2007). Thus, the predominant cytoplasmic location of both proteasome and ubiquitin in principal cells might be related to their high endocytotic activity (Hermo et al, 1995, 1998). Cytoplasmic proteasome location in the apical cells is also consistent with their involvement in endocytosis (Adamali and Hermo, 1996). It is also possible that these cells could secrete ubiquitin because the surface of abnormal sperm has been shown to bind ubiquitin, which might serve as a tag for sperm elimination during epididymal passage (Sutovsky et al, 2001; Hermo and Jacks, 2002). High immunoreaction for proteasome-ubiquitin was also detected in the basal cells, which has been implicated in immune defense, phagocytosis, and protection against oxidative stress and represent cellular processes that are known to be dependent upon proteasomes for proteolysis (Veri et al, 1993; Yeung et al, 1994; Hermo and Papp, 1996; Mueller et al, 1998).
A gradient of staining was found for proteasome and ubiquitin, as well as EEA1 and clusterin, decreasing from proximal ductules to the common efferent duct, suggesting regional differences in proteolytic and endocytotic activity along these ductules. These data further support a prior observation that reabsorption of luminal fluid is greater in the proximal ductules (Clulow et al, 1994), coincident with a greater Na+/K+-ATPase activity (Ilio and Hess, 1992) and sodium-hydrogen exchanger-3 expression in the proximal ductules (Oliveira et al, 2002), both being key mediators of fluid reabsorption. On the other hand, a greater number of lysosomes has been described in nonciliated cells of the common efferent ductules (Jones and Jurd, 1987), which is in agreement with our findings that cathepsin D, a marker for lysosomes (Igdoura et al, 1995), was rarely found in the initial zone of the proximal efferent ductules (near the rete testis) and common efferent ductules but highly concentrated in the conus and distal part of the proximal region (near the conus). Thus the specific localization of cathepsin D in efferent ductules is consistent with these regions having a greater involvement in lysosomal-dependent degradation of proteins.
EEA1 and clusterin were found exclusively in the nonciliated cells of efferent ductules, supporting a strong endocytotic function ascribed to this cell type (Hermo and Morales, 1984; Hermo et al, 1988; Ilio and Hess, 1994). EEA1 appeared restricted to large vesicles, compatible with apical endosomes, contrasting with clusterin presence in components of variable size and distributed in the supranuclear cytoplasm. Clusterin has been localized along the entire endocytotic pathway, including apical tubules, endosomes, and lysosomes (Hermo et al, 1995). It is known that after secretion by Sertoli cells, clusterin binds to spermatids and then detaches from sperm within the efferent ductule lumen before being endocytosed by nonciliated cells (Igdoura et al, 1994; Hermo et al, 1995). High concentrations of the clusterin receptor (LRP-2/megalin) are found in the nonciliated cells, which would facilitate the selective endocytosis of its ligand (Hermo et al, 1999). In support of these earlier reports, clusterin staining showed a broad pattern of expression in the nonciliated cells, and there was less staining of luminal spermatozoa as they reached the common efferent duct region.
Previous studies have shown that testosterone is not a major factor
regulating the expression of clusterin protein in efferent ductules and
epididymis, suggesting that another testicular-derived luminal factor is
responsible for its regulation (Igdoura et
al, 1994). This study suggests that estrogen could be the luminal
factor that modulates clusterin expression, in that treatment with the
antiestrogen ICI 182,780 resulted in a 42% reduction in clusterin staining in
efferent ductules. This is consistent with data from the ER knockout
mice (Esr1–/– or ERKO), which also had
approximately 40% reduction in clusterin staining in the nonciliated cells
(Nakai et al, 2001). EEA1, a
marker for early endosomes, was also significantly reduced after antiestrogen
treatment, which supports a recent report of decreases in size for
"early sorting endosomes" in the
Esr1–/– efferent ductules
(Toda et al, 2008).
Collectively, these data support the hypothesis that estrogen, acting through
ER, helps to regulate endocytosis in the efferent ductule epithelium.
Reduction in the endocytotic activity would contribute to fluid accumulation
in the efferent ductule lumen after both genetic
(Hess et al,
1997,Hess et al,
1997; Nakai et al,
2001) or chemical (Oliveira et al,
2001,
2002;
Cho et al, 2003) inhibition of
ER activity.
A previous investigation using microarray analysis identified proteasome
among the genes that were down-regulated in caput epididymis of the
Esr1–/– mice
(Shayu et al, 2007). However,
on the basis of the present findings, antiestrogen action appears to inhibit
the endocytotic apparatus more than the proteasomal pathway. The decrease in
proteasome immunostaining in this study was found to be coincident with a
marked reduction in ER levels in the efferent ductule epithelium after
ICI 182,780 treatment (Oliveira et al,
2003). Although we cannot state that estrogen directly regulates
proteasome expression, together these data suggest that proteasome is reduced
as a consequence of the down-regulation of one of its main targets, ER.
This conclusion is further supported by data from the aromatase knockout male
(Cyp19–/– or ArKO), which revealed that
ER expression and morphology of efferent ductules remain normal in this
animal model, even in the absence of estrogen synthesis
(Toda et al, 2008).
The identification of components of both ubiquitin-proteasomal and endocytotic pathways in different cell types, cellular compartments, or both in efferent ductules and initial segment of the epididymis strongly suggests that differential regulation of protein degradation and turnover is important in each segment of the head of the epididymis, regions that are under the regulation of both estrogens and androgens.
Acknowledgments
We are grateful to Dr M. D. Griswold, Washington State University, Pullman, Washington, for providing the clusterin (SGP2) antibody and to André G. Oliveira for assistance in morphometry.
Footnotes
This work was partially funded by the subproject CIG-02-76 provided by CICCR, a program of CONRAD, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR. ICI 182,780 was provided by AstraZeneca in the form of Faslodex. A research fellowship by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) was provided to C.A.O. and a scholarship from Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Brazil was awarded to A.B.V.-C.
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