0013-7227/03/$15.00/0
Printed in U.S.A.
Endocrinology 144(3):975–988
Copyright © 2003 by The Endocrine Society
doi: 10.1210/en.2002-220705
Gene Expression Is Differentially Regulated in the
Epididymis after Orchidectomy
NADINE EZER
AND
BERNARD ROBAIRE
Departments of Pharmacology and Therapeutics and Obstetrics and Gynecology, McGill University, Montréal, Québec
H3G 1Y6, Canada
The epididymis is the site for the transport, maturation, and
storage of spermatozoa. Regulation of epididymal structure
and function is highly dependent on the ipsilateral testis. At
the molecular level, however, few studies have been undertaken to determine which genes are expressed in the epididymis under testicular regulation. The goal of this study was to
identify genes for which expression is regulated after orchidectomy, both throughout the epididymis and in a segmentspecific manner. Microarrays spotted with 474 rat cDNAs
were used to examine gene expression changes over the first
7 d post orchidectomy in the initial segment, caput, corpus,
and cauda epididymidis of the adult Brown Norway rat. Using
k-means cluster analysis, we show that four patterns of gene
expression are activated in each epididymal segment over the
first week following orchidectomy. Transient up-regulation of
gene expression in the epididymis after orchidectomy is
described for the first time. Potential androgen-repressed
genes, including Gpx-1, show increased expression in the epididymis after orchidectomy. Several glutathione-S-transferases and calcium-binding proteins decline throughout the
epididymis after orchidectomy, indicating that these may be
novel androgen-regulated epididymal genes. Other genes coding for metabolism-associated proteins, transporters, and ␣-1
acid glycoprotein show segment-specific regulation in the epididymis after orchidectomy. Finally, we describe the expression of the previously uncharacterized heat shock proteins,
and apoptosis-associated genes in the epididymis after orchidectomy. Thus, gene expression in the epididymis is differentially affected over time after orchidectomy. These results
provide novel insight into androgen-dependent and segmentspecific epididymal function. (Endocrinology 144: 975–988,
2003)
T
HE EPIDIDYMIS, A HIGHLY convoluted tubule that
links the efferent ducts to the vas deferens, functions in
the transport, maturation, and storage of spermatozoa (1). As
early as 1926, it was established that maintenance of epididymal structure and function was dependent on an unknown
testicular factor (2), purified 5 yr later as testosterone (3).
Subsequent studies showed clearly that epididymal functions are highly dependent on the circulating androgen, testosterone; in the absence of androgens, spermatozoa become
immotile, lose the ability to fertilize, and die (4, 5).
Orchidectomy removes hormonal androgenic support arriving to the epididymis via the circulation as well as testicular factors entering the epididymal lumen directly via the
efferent ducts. Following orchidectomy, epididymal weight
is decreased to 25% of control over a 2-wk period. Testosterone replacement, even at supraphysiological levels, can
restore epididymal weight but only to 50% of control; this is
due to the large proportion (nearly half) of epididymal
weight that is attributable to spermatozoa and luminal fluid
(6, 7).
Of the four major epididymal epithelial cell types (principal cells, basal cells, clear cells, and narrow cells), principal
cells are particularly sensitive to removal of circulating an-
drogens, whereas other cell types appear unaffected (8, 9).
Following orchidectomy, altered principal cell morphology,
protein secretion, and apoptotic cell death in the caput, corpus, and cauda epididymidis can be reversed or prevented
by androgen replacement (6, 10 –12). However, testosterone
replacement is not sufficient to reverse these regressive
changes in the initial segment of the epididymis (6, 10, 13).
Efferent duct ligation studies, in which testicular contributions to the epididymis are removed but circulating androgen concentrations are maintained, have established that
testicular factors arriving through the efferent ducts are essential for maintenance of proper initial segment structure
(10), secretion (13, 14), enzyme activity (15, 16), and prevention of principal cell apoptosis (12).
At the mRNA level, several epididymal genes have been
described as androgen dependent; such genes respond by a
decline in expression after androgen withdrawal [DE/AEG/
CRISP-1 (17), glutathione peroxidase (Gpx)-5 (18), Gpx-3
(14), carbonic anhydrase (19), cyclooxygenase 2 (20), angiotensinogen (21)]. In addition, testicular factors play a role in
maintaining normal expression of proximal epididymal
genes in the initial segment and caput epididymidis [proenkephalin (22), cystatin-related epididymal specific (23), 5-␣
reductase 1 (24), ␥-glutamyl transpeptidase (GGT) (25), and
EP17 (26)]. Candidate testicular factors include high intraluminal androgens, basic fibroblast growth factor (16), and
androgen-binding protein (15). The possibility that some
genes are repressed by androgens and therefore induced, at
least in some epididymal segments, in the absence of androgens has been established [Clusterin/Trm-2/Apo-J (27),
TGF␤ (28)]. These two patterns of gene expression in the
Abbreviations: AGP, ␣-1-Acid-glycoprotein; ASCT2, sodium-dependent neutral amino acid transporter; CaBP, calcium-binding protein;
CRY␤B1, crystallin-␤ B1; DAAO, d-amino acid oxidase; Dad1, defender
against cell death protein 1; GGT, ␥-glutamyl transpeptidase; Gpx, glutathione peroxidase; grp, glucose-regulated protein; GST, glutathioneS-transferase; Hsp, heat shock protein; mAAT, mitochondrial aspartate
aminotransferase; Mcl1, myeloid cell differentiation protein 1; OCTN2,
organic cation transporter N2; ODC, ornithine decarboxylase; TNFR1,
TNF receptor 1.
975
Downloaded from endo.endojournals.org on February 28, 2005
976
Endocrinology, March 2003, 144(3):975–988
epididymis after orchidectomy suggest that regulation of
epididymal gene expression is complex.
It is not readily feasible to simultaneously measure
changes in expression of a large number of genes in the
epididymis by using conventional techniques such as differential display, Northern blot analysis, RNase protection
assays, or Q-RT-PCR. Therefore, a more comprehensive understanding of testis-dependent regulation of epididymal
gene expression remains to be attained. DNA microarrays
provide a powerful tool to analyze differential gene expression (29). We anticipated that several patterns of gene expression are activated in the epididymis after orchidectomy
and specific gene families respond differentially to the removal of androgens and/or testicular factors. To investigate
this hypothesis, we used gene expression profiling to examine the segment-specific response of 474 stress-related genes
in the rat epididymis over the first 7 d post orchidectomy.
Using k-means cluster analysis, we found four clearly distinctive expression profiles in the epididymis after orchidectomy. Novel genes that respond to orchidectomy along the
epididymis and in a segment-specific manner were identified. Finally, we have described the expression of previously
uncharacterized gene families in the epididymis after orchidectomy. By providing insight into regulation of epididymal
gene expression after orchidectomy, these results contribute
to our understanding of testis-dependent epididymal
functions.
Materials and Methods
Animals
Adult male Brown Norway rats (3 months old) were purchased from
the National Institute on Aging (Bethesda, MD) and supplied by Harlan
Sprague Dawley, Inc. (Indianapolis, IN). Rats were housed at the McIntyre Animal Resources Centre, McGill University, under controlled light
(14-h light, 10-h dark) and temperature (22 C); animals had free access
to food and water. All animal studies were conducted in accordance with
the principles and procedure outlined in the Guide to the Care and Use
of Experimental Animals prepared by the Canadian Council on Animal
Care (Animal Use Protocol no. 206).
Bilateral orchidectomy was done through the abdominal route. Efferent ducts were ligated on both sides, and testes were removed above
the ligation. Animals were killed at 0.5, 1, 2, 3, or 7 d post orchidectomy
by decapitation. Control group animals (C) were sham operated and
killed at various time points thereafter in parallel to animals that underwent orchidectomy. At the time of death, epididymides were collected; sectioned into initial segment, caput, corpus, and cauda segments; and immediately frozen in liquid nitrogen. Sections were stored
at ⫺80 C until used for RNA extraction. Blood was collected at time of
death for hormone analysis.
Serum testosterone analysis
We used a commercially available testosterone ELISA kit to establish
the time course of the decline in total serum testosterone after bilateral
orchidectomy. At the time of death, serum was obtained by centrifuging
blood for 20 min (2700 ⫻ g, 4 C); collecting the supernatant; and centrifuging again. Supernatants were collected and frozen at ⫺20 C. Total
serum testosterone was measured using ELISA (Research Diagnostics,
Flanders, NJ) according to the manufacturer’s instructions. Sensitivity of
the assay was 0.1 ng/ml, and the intraassay coefficient of variation was
less than 11%.
RNA extraction
Total RNA was extracted from each sample using guanidine thiocyanate as described previously (30). Following isolation, RNA samples
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
were DNase-treated (Atlas pure isolation kit user manual, section IV,
CLONTECH Laboratories, Inc., Palo Alto, CA), and RNA concentration
was assessed by OD determination at 260 nm (DU7 spectrophotometer,
Beckman, Montréal, Québec, Canada). To verify the quality of each
sample, 5 ␮g RNA was run on a denaturing gel containing 1% agaroseformaldehyde. Each sample consisted of single epididymal segment
obtained from an individual rat; no tissues were pooled.
cDNA arrays and hybridization
RNA samples were used to probe cDNA arrays (Atlas Rat Stress
Toxicology II array, CLONTECH Laboratories, Inc.) according to the
manufacturer’s instructions. Five arrays per epididymal segment per
time point post orchidectomy (n ⫽ 5/time/segment) were probed and
referred to as replicates, with the following exceptions: four replicates
per group were used (n ⫽ 4 group) at 0.5 d post orchidectomy for all
segments, at 3 d post orchidectomy for the initial segment and 2 d post
orchidectomy for the corpus epididymidis; three arrays (n ⫽ 3) per
group were used at 7 d post orchidectomy for the corpus epididymidis.
A total number of 112 samples were probed for this experiment. Arrays
were exposed to PhosphorImager plates (Molecular Dynamics, Inc.,
Sunnyvale, CA) 24 h before scanning with a PhosphorImager (Storm,
Molecular Dynamics, Inc.). Analysis of array images with Atlas Image
(version 2.0, CLONTECH Laboratories, Inc.) was done to quantify the
intensity of each cDNA spot, which reflects the relative abundance of
RNA in the sample. The raw data for each gene (intensity minus the
background) were imported into GeneSpring 4.0.7 (Silicon Genetics,
Redwood, CA) for further analysis. For each replicate array, a gene was
considered detected if its intensity was above threshold, with threshold
defined as two times the average background of that array. A gene was
considered expressed at any given time post orchidectomy if it was
detected in at least three replicates in that group.
To minimize experimental variation and to allow for comparison of
different time groups post orchidectomy, data were normalized with
one of two different methods (GeneSpring 4.0.7). For the standard experiment-to-experiment normalization, the median level of expression
on each array was defined as 1 and expression of each gene was normalized relative to 1; this value was averaged for all replicates in a group
to generate what is referred to as the relative intensity for a given gene.
For cluster analysis only, gene-to-gene normalization was done in addition to experiment-to-experiment normalization. Using this method,
the signal strength of each gene was normalized relative to the median
of all measurements taken for that gene in each experiment, defined as
1. This normalization allows for clearer visualization of expression profiles with all genes on the same vertical scale. All individual gene profiles
are shown using the standard experiment-to-experiment normalization.
K-means cluster analysis partitions genes into groups based on similar expression patterns. The data are divided into k different clusters of
greatest possible distinction by starting with k random clusters and
moving the genes between clusters to minimize variability within clusters and maximize variability between clusters. Before clustering by k
means, the data were normalized (gene to gene), k was defined as 4, and
the smooth correlation was selected. Clustering was done independently
on the data sets (total number of genes) for each epididymal segment.
The total number of genes represents the number of genes detected in
control groups as well as those that are detected post orchidectomy.
Changes in gene expression were considered relative to control and
when the difference in expression level was at least 2-fold in either
direction, i.e. 100% increase or 50% decrease. Genes showing transiently
increased expression were described by expression at the peak, in which
peak value was expressed relative to control. Because of the normalization procedures, standard statistical comparisons were not done.
However, sem is shown for expression of individual genes to show
variability among replicates in a given group.
We examined genes on our array that have known expression profiles
post orchidectomy; this is similar to the method we previously used to
validate gene expression profiling (30). Clusterin mRNA was detected
most abundantly in the proximal epididymis, with highest expression
in the control caput epididymidis, followed by the initial segment,
cauda, and corpus epididymidis (31); consistent with published observations, clusterin mRNA expression showed the greatest increase in the
cauda epididymidis (⬃10-fold), followed by the corpus epididymidis
(⬃2.3-fold) but was unchanged in the initial segment and caput seg-
Downloaded from endo.endojournals.org on February 28, 2005
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
ments of the epididymis after orchidectomy (27). GGT mRNA was
expressed most abundantly in the initial segment followed by the caput
epididymidis and was undetected at all time points in the corpus and
cauda epididymides. As expected, GGT mRNA declined most dramatically in the initial segment, followed by the caput epididymidis at 7 d
post orchidectomy (25). Finally, metallothionein-3 mRNA expression
was highest in the caput epididymidis, followed by the initial segment,
and its expression declined only in the caput epididymidis at 7 d post
orchidectomy (32).
Results
Serum testosterone
The average total serum testosterone in the control (shamoperated) group was 4.53 ⫾ 2.26 ng/ml (mean ⫾ sem), with
values ranging from 2.3 ng/ml to 6.0 ng/ml. By 0.5 d post
orchidectomy average serum-free testosterone fell to less
than 0.3 ng/ml and remained below this level at all subsequent times studied post orchidectomy.
Genes detected in the epididymis post orchidectomy
Of the 474 genes examined, transcripts for 39% of genes
(184) were detected in the control initial segment, 43% (203)
in the control caput epididymidis, 39% (185) in the control
corpus epididymidis, and 42% (201) in the control cauda
epididymidis. In addition, a comparison of genes expressed
in the control and 7 d postorchidectomy time point for each
Endocrinology, March 2003, 144(3):975–988 977
epididymal segment is presented in the supplemental data
for this article (see The Endocrine Society’s Journals Online
Web site at http://endo.endojournals.org). The number of
genes used for cluster analysis was 243 in the initial segment,
239 in the caput, 235 in the corpus, and 244 in the cauda
epididymidis. This number includes all genes detected in the
control group plus any additional genes that became expressed over the 7-d time course post orchidectomy.
K-means cluster analysis of gene expression in each
epididymal segment after orchidectomy
We used k-means clustering to visualize trends in gene
expression that occurred over time post orchidectomy (Fig.
1). Four distinct gene expression profiles were generated in
each epididymal segment. Decreased gene expression after
orchidectomy was classified into one of two profiles. Genes
that partitioned into the early-declining profiles (profile 1)
showed a large decline in expression before 2 d post orchidectomy, followed by smaller changes in expression between
2 d and 7 d post orchidectomy. For example, thiopurine
methyltransferase was associated with this profile in each
epididymal segment. GGT was associated with this profile in
the initial segment and caput epididymis only; this gene was
not detected in the corpus and cauda epididymidis. The
second profile (profile 2) grouped genes with expression that
FIG. 1. Profiles of gene expression obtained by
k-means cluster analysis in the epididymis after orchidectomy. 1, Early-declining gene
expression. 2, Progressively declining gene expression. 3, Transiently increased gene expression. 4, Progressively increasing gene expression. The level of gene expression is
expressed as relative intensity. Relative intensity was obtained by normalizing the signal for any given gene relative to the median
intensity of all the measurements for that
gene, defined as 1. Time post orchidectomy is
indicated on the horizontal axes. Inset, A
schematic of the profile of gene expression.
IS, Initial segment; CA, caput; CO, corpus;
CD, cauda.
Downloaded from endo.endojournals.org on February 28, 2005
978
Endocrinology, March 2003, 144(3):975–988
declined more progressively; these profiles also included
genes that showed minimal changes in expression in response to orchidectomy.
A subset of genes in each epididymal segment showed
transiently increased expression in response to orchidectomy
(profile 3). This profile was particularly evident in the caput
epididymidis. In the initial segment, this trend was less apparent; however, upon examination of these genes individually using the standard experiment-to-experiment normalization, transient up-regulation of these genes was clearly
observable. Interestingly, c-jun was assigned to this profile in
each epididymal segment (Fig. 2).
The fourth profile of gene expression identified by
k-means clustering showed a progressive increase in expression over the 7-d period after orchidectomy in each epididymal segment (profile 4). An example of one such gene is
Gpx-1 (Fig. 2).
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
Gene expression changes in the epididymis
post orchidectomy
pressed in the initial segment, caput, corpus, and cauda
epididymidis, respectively.
To identify orchidectomy-responsive gene families in the
epididymis, we compared gene expression changes 7 d post
orchidectomy across segments. Expression of five genes was
increased in all four segments of the epididymis 7 d post
orchidectomy. The segment-specific expression and increase
in expression for these genes at 7 d post orchidectomy is
presented (Table 2). Similar to Gpx-1 (Fig. 2), each of these
genes showed a progressive increase in expression with time
post orchidectomy. The expression of 27 genes was decreased throughout the epididymis at 7 days post orchidectomy; the decline in expression for these genes at 7 d post
orchidectomy is presented (Table 3). Among these were several genes belonging to the family of glutathione-S-transferases (GSTs) (Fig. 4). Expression of GST Yf, which was most
abundant in the initial segment, declined in the initial segment and caput epididymidis by 79% and 59%, respectively,
but was unchanged in the corpus and cauda epididymal
segments (Fig. 4). Expression of calcium-binding proteins
The number of genes demonstrating a minimum 2-fold
change in expression (increased or decreased) at each time
post orchidectomy relative to control was obtained for each
epididymal segment (Fig. 3). The number of genes that
showed increased expression varied little over the first week
post orchidectomy. At 7 d post orchidectomy, the proportion
of genes that remained increased represented 5%, 8%, 9%,
and 9%, respectively, of the genes expressed in the initial
segment, caput, corpus, and cauda epididymidis. In contrast,
the population of genes that showed a decline in expression
increased with time post orchidectomy. At 1 d post orchidectomy, several genes showed decreased expression in each
epididymal segment, similar to the pattern of early-declining
gene expression identified by k-means cluster analysis (Table
1). By 7 d post orchidectomy, genes that showed decreased
expression represented 30%, 42%, 51%, and 29% of genes
expressed in the initial segment, caput, corpus, and cauda
epididymidis, respectively. Expression of a large proportion
of genes remained unchanged along the epididymis at 7 d
post orchidectomy: 65%, 50%, 40%, and 62% of genes ex-
FIG. 3. Number of genes showing changes in gene expression at each
time post orchidectomy in each epididymal segment. The vertical scale
represents the total number of genes that showed at least a 2-fold
change in expression in either direction (100% increase or 50% decrease). This number was obtained independently at each time post
orchidectomy relative to control. The numbers above and below the
horizontal zero-line represent genes that show increased or decreased
expression respectively. IS, Initial segment; CA, caput; CO, corpus;
CD, cauda.
FIG. 2. Expression of c-jun and gpx-1 in the
epididymis over the first week post orchidectomy. A, C-jun. B, Gpx-1. Gene expression is presented as relative intensity,
mean ⫾ SEM for three to five replicates per
group. Time post orchidectomy is indicated
on the horizontal axis. IS, Initial segment;
CA, caput; CO, corpus; CD, cauda.
Downloaded from endo.endojournals.org on February 28, 2005
M33821
GGT
L38615
S65838
AF097593
D00753
M33821
X02904
AJ006070
Accession numbers are for the GenBank database. See abbreviations footnote for abbreviations used.
L03294
D38467
M18467
D16309
K01932
Z27118
GST Yc1
Hsp70
GST Yc1
Liver carboxylesterase 10
precursor
Lipoprotein lipase precursor
Senescence marker protein 30
mAAT
G1/S-specific cyclin D3
K01932
L46791
AB003400
AJ001933
M22413
AB003400 DAAO
AJ001933 OCTN2
M23995
GST Yb2
DAAO
OCTN2
Aldehyde dehydrogenase 1
AB003400 DAAO
AJ001933 OCTN2
J02592
Carbonic anhydrase III
AF120100
AF120100 Thiopurine methyltransferase
AF120100 Thiopurine methyltransferase
AF120100
Thiopurine methyltransferase
GGT
GST Yf
V(D)J recombination activating protein 1
N-cadherin
Contrapsin-like protease inhibitor-related protein
Glutathione synthetase
Metallothionein 3
Thiopurine methyltransferase
Accession
no.
Caput
Gene
Accession
no.
Initial segment
Gene
TABLE 1. Genes with decreased expression at 1 d post orchidectomy in each epididymal segment
Gene
Corpus
Accession
no.
Gene
Cauda
Accession
no.
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
Endocrinology, March 2003, 144(3):975–988 979
(CaBPs) also declined along the epididymis after orchidectomy (Fig. 5); expression of CaBP-2 declined by 73%, 85%,
and 66% in the caput, corpus, and cauda epididymidis, respectively, at 7 d post orchidectomy, whereas CaBP-2 expression in the initial segment remained unchanged.
Segment-specific changes in gene expression were observed in the epididymis after orchidectomy. At 7 d post
orchidectomy, several genes expressed along the epididymis
showed increased expression in various segments. The
segment-specific increase in expression for these genes at 7 d
post orchidectomy is presented (Table 4). Others, such as
vascular endothelial growth factor D, were newly detected at
7 d post orchidectomy in the caput, corpus, and cauda segments of the epididymis.
A segment-specific decline in the expression of metabolism-associated genes was observed in the epididymis after
orchidectomy (Fig. 6). Mitochondrial aspartate aminotransferase (mAAT) declined in the caput and corpus epididymal
segments by 63% and 81%, respectively, but was unchanged
after orchidectomy in the initial segment and cauda epididymidis. In contrast, the cytoplasmic aspartate aminotransferase was not affected in the epididymis after orchidectomy.
Expression of ornithine decarboxylase (ODC), which increased from the proximal to distal epididymis, declined by
63%, 74%, and 50%, respectively, in the caput, corpus, and
cauda epididymidis but was unchanged in the initial segment (not shown). Expression of d-amino acid oxidase
(DAAO), which was detected only in the caput, corpus, and
cauda epididymidis, declined to undetectable levels in these
segments by 7 d post orchidectomy. Expression of two genes
coding for transporters also declined in a segment-specific
manner after orchidectomy (Fig. 7). The transcript for organic
cation transporter N2 (OCTN2), which was most abundant
in the corpus epididymidis but not expressed in the initial
segment, declined to undetectable levels in the caput, corpus,
and cauda epididymidis by 7 d post orchidectomy. Sodiumdependent neutral amino acid transporter (ASCT2) was most
abundantly expressed in the caput epididymidis, in which it
declined by 60% at 7 d post orchidectomy. Lastly, the gene
coding for ␣-1-acid-glycoprotein (AGP), detected only in the
initial segment of the epididymis, declined by 84% at 7 d post
orchidectomy in this segment (Fig. 8).
Expression by gene family
Heat shock proteins. Members of the heat shock protein (Hsp)
family showed several patterns of gene expression in the
epididymis after orchidectomy (Fig. 9). The glucose-regulated proteins (grp)94 and grp78 (not shown) showed a
steady decline in expression in all segments of the epididymis over the first week post orchidectomy. By 7 d post
orchidectomy, grp94 expression declined by 62%, 86%, 71%,
and 78%, respectively, in the initial segment, caput, corpus,
and cauda epididymidis; expression of grp78 declined by
69%, 72%, 77%, and 82%, respectively, in the initial segment,
caput, corpus, and cauda epididymidis. Other Hsps, such as
Hsp27 and Hsp47, demonstrated a transient increase in expression post orchidectomy. At the peak of transiently increased expression, Hsp27 expression was increased by 70%,
489%, 226%, and 140%, respectively, in the initial segment,
Downloaded from endo.endojournals.org on February 28, 2005
980
Endocrinology, March 2003, 144(3):975–988
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
TABLE 2. Genes with increased expression throughout the epididymis at 7 d post orchidectomy
Name
Accession no.
Gpx-1
IGFBP-3
Timp-2
c-erb
MMP-11
X12367
M31837
L31884
M25804
U46034
Initial segment
C
PO
1.7 ⫾ 0.2
4.3 ⫾ 0.6
1.2 ⫾ 0.1
7.3 ⫾ 1.2
6.8 ⫾ 0.6 14.7 ⫾ 1.9
0.5 ⫾ 0.1
1.4 ⫾ 0.1
ND
1.0 ⫾ 0.1
Caput
%
153
508
116
180
/
C
Corpus
PO
%
2.0 ⫾ 0.5
6.4 ⫾ 1.5
1.1 ⫾ 0.1
6.2 ⫾ 0.4
7.5 ⫾ 0.8 22.8 ⫾ 3.4
0.5 ⫾ 0.1
1.3 ⫾ 0.2
0.4 ⫾ 0.1
1.2 ⫾ 0.0
C
PO
220
1.4 ⫾ 0.2
8.6 ⫾ 1.2
464
1.2 ⫾ 0.2
2.6 ⫾ 0.2
204 10.0 ⫾ 1.7 28.6 ⫾ 5.2
160
0.5 ⫾ 0.0
1.4 ⫾ 0.2
200
0.4 ⫾ 0.1
1.3 ⫾ 0.3
Cauda
%
514
117
186
180
225
C
PO
2.3 ⫾ 0.2
4.9 ⫾ 1.1
1.6 ⫾ 0.1
6.7 ⫾ 0.8
8.2 ⫾ 1.0 25.0 ⫾ 2.7
0.6 ⫾ 0.1
1.9 ⫾ 0.2
0.4 ⫾ 0.1
2.3 ⫾ 0.3
%
113
320
205
216
475
The numbers represent relative intensity ⫾ SEM for control group values (C) and values at 7 d post orchidectomy (PO). The column labeled
% shows the percent increase in expression for each gene. ND indicates that a gene was not detected, or expressed below the threshold of
detection. The backslash (/) indicates that the percent increase was not calculated.
IGFBP-3, Insulin-like growth factor binding protein 3 precursor; Timp-2, tissue inhibitor of metalloproteinase 2 precursor; MMP-11, matrix
metalloproteinase 11. Accession numbers are for the GenBank database.
caput, corpus, and cauda epididymidis; Hsp47 expression
was increased by 207%, 270%, 254%, and 81%, respectively,
in the initial segment, caput, corpus, and cauda epididymidis. Hsp90␤, the most highly expressed Hsp along the epididymis, was unaffected by orchidectomy. The small Hsp
crystallin-␤ B1 (CRY␤B1), which was expressed only in the
initial segment and caput epididymidis, showed a steady
increase in expression in the initial segment post orchidectomy; at 7 d post orchidectomy, CRY␤B1 expression was
increased by 475% in this segment.
Apoptosis. Genes coding for several apoptosis-associated proteins responded to orchidectomy throughout the epididymis
(Fig. 10). Defender against cell death protein 1 (Dad1) was
highly expressed in the proximal epididymis, particularly in
the initial segment. Dad1 mRNA expression declined after
orchidectomy by 88%, 88%, 91%, and 76%, respectively, in
the initial segment, caput, corpus, and cauda epididymidis,
respectively. Of the five bcl-2 family members [myeloid cell
differentiation protein1 (Mcl1), bcl2-associated death promoter, bcl-x, Bax-␣, and bcl2] that were expressed in the four
epididymal segments, Mcl1 was the most highly expressed
along the epididymis and, following orchidectomy, showed
transiently increased expression. At the peak of transiently
increased expression, Mcl1 expression was increased by 17%,
160%, 85%, and 76% in the initial segment, caput, corpus, and
cauda epididymidis, respectively. TNF receptor 1 (TNFR1)
was expressed at similar levels along the epididymis; TNFR1
showed a steady increase in expression before 7 d post orchidectomy when TNFR1 expression levels were increased
by 87%, 166%, 128%, and 82% in the initial segment, caput,
corpus, and cauda epididymidis, respectively.
Discussion
In this study, we examined the expression of a large number of genes simultaneously at several time points post orchidectomy in the epididymis in a segment-specific manner.
This design permitted us to visualize global trends in gene
expression in the epididymis post orchidectomy, identify
novel orchidectomy-responsive genes, and follow the expression of previously uncharacterized gene families in the
epididymis after orchidectomy.
Patterns of gene expression in the epididymis after
orchidectomy identified by k-means cluster analysis
K-means cluster analysis of gene expression is a powerful
analytical tool that can provide important information on the
responses of genes. Genes following the early-declining gene
expression profile are likely regulated by circulating factors
derived from the testis, such as androgens, because their
decline in expression parallels the decrease circulating androgenic support to the epididymis observed by 12 h post
orchidectomy. In support of this, a known androgen-dependent gene, metallothionein-3, showed an early-declining expression profile in the epididymis after orchidectomy. In the
present study, we identified some potentially novel androgen-regulated genes such as thiopurine methyltransferase,
DAAO, and OCTN2 (Table 1).
A major strength of cluster analysis is that it allowed us to
detect transiently increased gene expression in each epididymal segment within the first week after orchidectomy. Transient up-regulation of gene expression in the epididymis
after orchidectomy has not been reported previously. Transiently increased gene expression may reflect transcriptional
changes occurring in the population of principal cells that
undergoes apoptotic cell death in the epididymis after orchidectomy (12). Apoptosis is an active process that requires
protein and RNA synthesis (33). Up-regulation of the transcription factor c-jun has been characterized in numerous
models of apoptosis (34) as well as in apoptotic cells of the
regressing rat ventral prostate after cadmium treatment (35).
Alternatively, during regression of hormone-dependent tissues, such as the epididymis, expression of a number of
mRNAs that are not involved in the apoptotic process may
be induced. These genes may be induced as part of a futile
stress response in cells that die after hormone ablation, or,
alternatively, these genes may be induced in cells that resist
apoptotic cell death as part of a survival mechanism. Transiently up-regulated gene expression may, at least partially,
explain why the majority of epididymal epithelial principal
cells survive the insult of androgen withdrawal after orchidectomy, whereas more than 95% of epithelial cells in the rat
ventral prostate undergo apoptosis after orchidectomy (36).
The pattern of gene expression associated with a progressive increase in expression in the epididymis over the first
week post orchidectomy included the genes that were upregulated by at least 2-fold throughout the epididymis. At 7 d
post orchidectomy, up-regulated gene expression most likely
reflects transcriptional changes in surviving epididymal cells
because apoptotic cell death is no longer detected in the rat
epididymis at 7 d post orchidectomy (12). We propose that
some of these genes, such as Gpx-1, are novel androgenrepressed genes in the epididymis. Only clusterin and TGF␤
Downloaded from endo.endojournals.org on February 28, 2005
Downloaded from endo.endojournals.org on February 28, 2005
2.2 ⫾ 0.4
7.2 ⫾ 0.9
0.5 ⫾ 0.2
1.5 ⫾ 0.6
9.9 ⫾ 2.8
ND
11.1 ⫾ 2.7
0.7 ⫾ 0.2
3.2 ⫾ 1.3
0.4 ⫾ 0.1
1.6 ⫾ 0.3
16.6 ⫾ 1.6
0.3 ⫾ 0.2
0.7 ⫾ 0.1
2.8 ⫾ 0.8
1.4 ⫾ 0.4
1.3 ⫾ 0.1
20.8 ⫾ 2.5
2.6 ⫾ 0.8
0.9 ⫾ 0.5
0.7 ⫾ 0.2
0.7 ⫾ 0.2
0.4 ⫾ 0.2
0.5 ⫾ 0.2
1.0 ⫾ 0.3
0.5 ⫾ 0.2
ND
11.8 ⫾ 1.8
9.2 ⫾ 1.1
4.1 ⫾ 0.5
44.5 ⫾ 3.0
1.7 ⫾ 0.4
2.3 ⫾ 0.6
7.3 ⫾ 0.6
3.1 ⫾ 0.4
2.7 ⫾ 0.8
42.8 ⫾ 5.7
21.1 ⫾ 4.5
5.4 ⫾ 0.8
2.7 ⫾ 0.6
2.3 ⫾ 0.2
1.2 ⫾ 0.2
1.7 ⫾ 0.2
2.5 ⫾ 0.2
1.1 ⫾ 0.2
1.1 ⫾ 0.1
M17701
AF120100
X79328
X53363
Y00054
M14050
X15187
X71429
Z27118
D63378
Y13336
AF095740
D14014
D16309
X94351
D44495
U41853
D30616
D90038
PO
Initial segment
4.8 ⫾ 0.4
35.3 ⫾ 5.6
1.1 ⫾ 0.2
5.9 ⫾ 0.7
36.9 ⫾ 2.3
2.9 ⫾ 0.5
22.9 ⫾ 2.8
1.6 ⫾ 0.2
C
K01932
J02592
J02744
D30035
Y00404
M23995
U38379
X15958
Accession no.
88
83
74
70
67
71
60
55
/
51
/
70
62
55
52
61
63
73
/
54
80
55
75
73
/
52
56
%
9.1 ⫾ 1.9
3.7 ⫾ 1.4
1.5 ⫾ 0.3
1.8 ⫾ 0.3
1.4 ⫾ 0.4
1.2 ⫾ 0.4
2.7 ⫾ 0.6
0.9 ⫾ 0.1
0.7 ⫾ 0.1
49.4 ⫾ 5.8
0.8 ⫾ 0.3
1.8 ⫾ 0.4
6.0 ⫾ 1.6
3.2 ⫾ 0.7
7.9 ⫾ 1.6
2.5 ⫾ 0.5
44.2 ⫾ 6.0
10.9 ⫾ 2.5
5.1 ⫾ 1.0
12.6 ⫾ 1.6
42.7 ⫾ 7.9
1.1 ⫾ 0.2
3.6 ⫾ 1.4
23.0 ⫾ 6.2
2.4 ⫾ 0.5
46.0 ⫾ 8.9
1.5 ⫾ 0.2
C
PO
1.1 ⫾ 0.4
0.5 ⫾ 0.3
0.6 ⫾ 0.3
0.8 ⫾ 0.3
0.4 ⫾ 0.1
ND
ND
ND
ND
13.9 ⫾ 1.4
ND
0.5 ⫾ 0.1
0.8 ⫾ 0.1
1.0 ⫾ 0.2
1.7 ⫾ 0.2
0.8 ⫾ 0.1
9.3 ⫾ 0.5
2.7 ⫾ 1.6
ND
1.0 ⫾ 0.3
8.6 ⫾ 2.6
ND
ND
5.0 ⫾ 1.4
ND
4.4 ⫾ 2.0
0.5 ⫾ 0.1
Caput
88
86
60
55
71
/
85
/
/
72
/
72
87
69
78
68
79
75
/
92
80
/
/
78
/
90
67
%
7.1 ⫾ 0.6
2.8 ⫾ 0.7
3.7 ⫾ 0.5
2.1 ⫾ 0.5
1.2 ⫾ 0.5
0.9 ⫾ 0.1
1.6 ⫾ 0.3
0.8 ⫾ 0.1
0.8 ⫾ 0.1
32.4 ⫾ 2.2
0.8 ⫾ 0.2
1.2 ⫾ 0.3
4.1 ⫾ 0.7
4.4 ⫾ 0.4
2.9 ⫾ 0.6
2.0 ⫾ 0.4
22.8 ⫾ 2.1
6.7 ⫾ 1.5
3.1 ⫾ 0.3
16.7 ⫾ 2.1
35.4 ⫾ 4.3
0.8 ⫾ 0.1
2.5 ⫾ 0.3
28.0 ⫾ 3.4
2.2 ⫾ 0.2
3.2 ⫾ 0.6
1.2 ⫾ 0.2
C
PO
0.7 ⫾ 0.2
0.3 ⫾ 0.1
0.6 ⫾ 0.2
ND
ND
ND
ND
ND
ND
11.5 ⫾ 2.7
ND
ND
1.2 ⫾ 0.3
1.1 ⫾ 0.4
1.2 ⫾ 0.05
0.9 ⫾ 0.1
7.5 ⫾ 1.7
1.4 ⫾ 0.6
ND
1.0 ⫾ 0.3
2.5 ⫾ 0.4
ND
ND
2.5 ⫾ 0.8
ND
0.5 ⫾ 0.1
ND
Corpus
90
89
84
/
/
/
/
/
/
65
/
/
71
75
59
55
67
79
/
94
93
/
/
91
/
/
/
%
8.9 ⫾ 1.8
5.3 ⫾ 1.0
3.8 ⫾ 0.3
2.4 ⫾ 0.1
1.4 ⫾ 0.2
1.1 ⫾ 0.2
1.1 ⫾ 0.2
1.2 ⫾ 0.2
1.0 ⫾ 0.3
36.6 ⫾ 7.0
1.3 ⫾ 0.3
1.5 ⫾ 0.3
4.2 ⫾ 1.1
3.4 ⫾ 0.5
6.8 ⫾ 1.1
2.0 ⫾ 0.3
22.5 ⫾ 4.7
14.1 ⫾ 2.3
1.5 ⫾ 0.2
12.2 ⫾ 1.8
37.0 ⫾ 5.0
1.2 ⫾ 0.2
3.4 ⫾ 1.0
29.5 ⫾ 4.1
3.1 ⫾ 0.9
2.8 ⫾ 0.6
1.3 ⫾ 0.2
C
PO
1.6 ⫾ 0.6
0.6 ⫾ 0.4
1.3 ⫾ 0.4
0.8 ⫾ 0.2
0.3 ⫾ 0.1
0.3 ⫾ 0.1
0.3 ⫾ 0.1
0.3 ⫾ 0.2
ND
14.8 ⫾ 3.0
ND
0.3 ⫾ 0.1
0.9 ⫾ 0.2
1.6 ⫾ 0.6
3.2 ⫾ 0.4
0.9 ⫾ 0.2
8.3 ⫾ 1.1
4.3 ⫾ 2.6
ND
1.7 ⫾ 0.2
11.0 ⫾ 2.1
ND
0.6 ⫾ 0.3
12.1 ⫾ 4.2
ND
1.1 ⫾ 0.3
0.6 ⫾ 0.1
Cauda
82
89
77
67
/
/
/
/
/
60
/
80
79
53
53
55
63
70
/
86
70
/
83
59
/
61
54
%
The numbers represent relative intensity ⫾ SEM for control group values (C) and values at 7 d post orchidectomy (PO). ND indicates that a gene was not detected, or expressed
the threshold of detection. The column labeled % shows the percent decrease in expression for each gene. The backslash (/) indicates that the percent decrease was not calculated.
TDPX2, Thioredoxin-dependent peroxidase 2; Cu-Zn SOD1, copper-zinc superoxide dismutase 1; ALDHI, aldehyde dehydrogenase 1; Mit. Enoyl-CoA hydratase, mitochondrial CoA
hydratase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; probable protein disulfide isomerase ER60; NEDD8, ubiquitin-like protein (NEDD8); CCND1, G1/S-specific cyclin
D1; CCND3, G1/S-specific cyclin D3; CLK3, CDC-like kinase 3; AP endonuclease, apurinic/apyrimidinic endonuclease; ORP150, oxygen-regulated protein 150; PAF1, peroxisome
assembly factor 1; PMP1, peroxisomal membrane protein 1. See abbreviations footnote for other abbreviations used. Accession numbers are for the GenBank database.
Metabolism
GST Yc1
GST Yb2
GST Yb3
TDPX2
Cu-Zn
SOD1 ALDH1
GGH
Mit. Enoyl-CoA
hydratase
GAPDH
Thiopurine methyltransferase
CaBPs
CaBP-1
CaBP-3/calreticulin
Heat shock proteins
HSC70
GRP78
GRP94
HSP10
HSP70
Other
Probable protein
disulfide
isomerase ER60
DAD1
NEDD8
CCND1
CCND3
CLK3
AP endonuclease
ORP150
PAF1
PMP1
Gene
TABLE 3. Genes showing decreased expression throughout the epididymis at 7 d post orchidectomy
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
Endocrinology, March 2003, 144(3):975–988 981
982
Endocrinology, March 2003, 144(3):975–988
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
FIG. 4. Expression of GSTs in the epididymis over the first week post orchidectomy. A. GST Yb2 and GST Yc1. C.
GST Yf. Gene expression is presented
as relative intensity, mean ⫾ SEM for
three to five replicates per group. Time
post orchidectomy is indicated on the
horizontal axis. IS, Initial segment; CA,
caput; CO, corpus; CD, cauda.
FIG. 5. Expression of CaBPs in the epididymis over the first week post orchidectomy. A, CaBP-2 and CaBP-3/calreticulin. B, CaBP-1 and calnexin. Gene
expression is presented as relative intensity, mean ⫾ SEM for three to five
replicates per group. Time post orchidectomy is indicated on the horizontal
axis. IS, Initial segment; CA, caput; CO,
corpus; CD, cauda.
have been previously identified as potential testosteronerepressed genes in this tissue (27, 28). As a family, Gpx enzymes
catalyze the reduction of organic hydroperoxides and hydrogen
peroxide, using glutathione as a reducing agent, thereby protecting cells from oxidative damage caused by normal oxidative
metabolism (37). Gpx-1 is the cellular cytosolic Gpx enzyme,
whereas Gpx-3 and Gpx-5 are secretory in mammals. The latter
two have been previously localized in the epididymis and their
expression is androgen dependent (38). Consistent with our
hypothesis that Gpx-1 is androgen repressed in the epididymis,
a previous report (39) has shown that Gpx-1 mRNA expression
was unchanged after efferent duct ligation.
Gene expression changes in the epididymis
after orchidectomy
Our analysis of 2-fold changes in gene expression in each
epididymal segment revealed that there is a simultaneous
increase and decrease in the expression of several genes at
various time points over the first week after orchidectomy.
Increased expression of several genes has been noted during
regression of other androgen-dependent organs such as the
prostate (36). By comparison, decreased gene expression was
clearly the dominant response observed in the epididymis at
7 d post orchidectomy. The fact that expression of several
genes was unchanged at this time suggests that the observed
decrease in gene expression in each epididymal segment is
a consequence of testis-dependent regulation rather than an
overall decline in RNA synthesis (40).
Analysis of orchidectomy studies in the epididymis is
somewhat complex because orchidectomy removes testisderived circulating androgen support as well as direct testicular input to the epididymis. Presumably genes that respond by a decline in expression in all segments of the
epididymis by 1 wk after orchidectomy are regulated by
Downloaded from endo.endojournals.org on February 28, 2005
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
Endocrinology, March 2003, 144(3):975–988 983
TABLE 4. Genes that showed a segment-specific increase in expression at 7 d post orchidectomy in the four epididymal segments
Gene
Accession no.
Initial segment
Inhibitor of DNA binding 3
Lysyl oxidasea
11-␤-Hydroxysteroid dehydrogenase 2
Caput
RANTES
␣-2 Macroglobulin
c-jun proto-oncogenea
Tumor necrosis factor receptor 1
Apolipoprotein E precursor
Lysyl oxidasea
c-met proto-oncogene
Corpus
Thymosin ␤-10
Clusterina
RANTES
␣-2 Macroglobulin
Lysyl oxidasea
Tumor necrosis factor receptor 1
Hsp27a
Osteopontin
Matrix metalloproteinase 14
Complement component C3a
Heme oxygenase 1
Cauda
Clusterin
Thymosin ␤-10
Histone 2A
␣-2 Macroglobulin
RANTES
Apolipoprotein E precursor
c-jun proto-oncogene
c-met proto-oncogene
Control
7 d PO
% Increase
D10864
U11038
U22424
2.8 ⫾ 0.4
0.8 ⫾ 0.2
0.4 ⫾ 0.1
7.2 ⫾ 1.6
2.6 ⫾ 0.6
1.5 ⫾ 0.3
157
225
275
U06436
J02635
X17163
M63122
J02582
U11038
U65007
4.3 ⫾ 0.3
4.2 ⫾ 0.6
1.7 ⫾ 0.2
1.6 ⫾ 0.3
1.6 ⫾ 0.3
1.4 ⫾ 0.5
0.5 ⫾ 0.1
9.5 ⫾ 1.3
9.3 ⫾ 2.2
4.1 ⫾ 1.1
4.2 ⫾ 1.1
4.9 ⫾ 0.7
2.9 ⫾ 0.7
2.4 ⫾ 0.5
121
121
141
162
206
107
380
M17698
M64723
U06436
J02635
U11038
M63122
M86389
M14656
X83537
X52477
J02722
17.0 ⫾ 1.9
8.0 ⫾ 0.8
2.7 ⫾ 0.6
2.0 ⫾ 0.5
1.7 ⫾ 0.3
1.7 ⫾ 0.1
0.8 ⫾ 0.1
0.8 ⫾ 0.2
0.6 ⫾ 0.1
0.5 ⫾ 0.0
0.5 ⫾ 0.1
53.1 ⫾ 7.2
18.4 ⫾ 1.1
7.6 ⫾ 1.1
5.6 ⫾ 1.3
4.4 ⫾ 1.0
3.9 ⫾ 0.4
1.8 ⫾ 0.2
4.4 ⫾ 0.4
1.5 ⫾ 0.4
1.1 ⫾ 0.1
1.2 ⫾ 0.1
212
130
181
180
159
129
125
450
150
120
140
M64723
M17698
U95113
J02635
U06436
J02582
X17163
U65007
14.7 ⫾ 3.9
16.0 ⫾ 1.7
5.9 ⫾ 0.8
2.1 ⫾ 0.4
2.8 ⫾ 0.3
2.7 ⫾ 0.3
2.1 ⫾ 0.2
0.4 ⫾ 0.1
148.6 ⫾ 27.2
53.2 ⫾ 8.2
11.9 ⫾ 3.1
7.7 ⫾ 2.8
6.7 ⫾ 1.3
6.7 ⫾ 1.1
4.5 ⫾ 0.9
1.9 ⫾ 0.4
910
232
102
267
139
148
114
375
The numbers represent relative intensity ⫾ SEM for control group values and values at 7 d post orchidectomy (PO). Accession numbers are
for the GenBank database.
RANTES, Regulated upon activation normal T cell expressed and presumable secreted.
a
Gene expression was transiently increased prior to 7 d post orchidectomy.
FIG. 6. Expression of metabolism-associated genes in the epididymis over the
first week post orchidectomy. A, mAAT.
B, DAAO. Gene expression is presented
as relative intensity, mean ⫾ SEM for
three to five replicates per group. Time
post orchidectomy is indicated on the
horizontal axis. IS, Initial segment; CA,
caput; CO, corpus; CD, cauda.
circulating androgens and not testicular factors because the
latter have been implicated in the regulation of gene expression in the proximal epididymis but not in the distal epididymis (41). Among others, these include several glutathione GSTs and CaBPs. Interestingly, the pattern of decreased
expression for the GSTs and CaBPs is similar to that of early-
declining gene expression identified by k-means cluster analysis, which further supports androgen-dependent regulation
of these genes.
Spermatozoa produce reactive oxygen species that are essential for capacitation and chromatin condensation (42). The
GST family of enzymes function in cellular detoxification by
Downloaded from endo.endojournals.org on February 28, 2005
984
Endocrinology, March 2003, 144(3):975–988
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
FIG. 7. Expression of transporters in
the epididymis over the first week post
orchidectomy. A, OCTN2. B, ASCT2.
Gene expression is presented as relative intensity, mean ⫾ SEM for three to
five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput;
CO, corpus; CD, cauda.
FIG. 8. Expression of AGP in the initial segment of the epididymis
over the first week post orchidectomy. Gene expression is presented
as relative intensity, mean ⫾ SEM for three to five replicates per group.
Time post orchidectomy is indicated on the horizontal axis.
acting as molecular scavengers that conjugate harmful electrophiles with reduced glutathione (43). In the epididymis,
GSTs are expressed at high levels in principal and basal cells
throughout the epididymal epithelium (44, 45) and are
thought to form part of the epididymal antioxidant system
that protects luminal spermatozoa and the epididymal epithelium from oxidative damage (46). The decline in epididymal expression of several GST transcripts after orchidectomy suggests that androgens regulate epididymal
antioxidant functions. In support of this concept, our observation that the mRNA for GST Yf declined after orchidectomy in the proximal epididymis is in accordance with histochemical observations that GST Yf protein declines in these
regions after androgen withdrawal by orchidectomy, but not
after efferent duct ligation (47). The lack of an effect of orchidectomy on the expression of GST Yf in the corpus and
cauda epididymidis is consistent with the observation that
this subunit is selectively located in basal cells in these segments (46) and these cells seem to be minimally affected by
orchidectomy (8). Moreover, the GST Yf promoter in the
mouse contains several androgen-response elements (48),
indicating that GST Yf expression in the epididymis may be
regulated by androgens at the transcriptional level. Androgens have also been implicated in regulating epididymal
GST enzyme activity (49).
In contrast, expression and regulation of CaBPs has not
been previously described for the epididymis. CaBPs, originally isolated from rabbit endoplasmic reticulum (50), play
an important role in maintaining cellular Ca2⫹ homeostasis.
Our observations that several CaBPs are highly expressed
throughout the epididymis and CaBP expression declines
throughout the epididymis after orchidectomy suggest that
circulating androgens are involved in the regulation of intracellular Ca2⫹ in the epididymal epithelium. In the prostate, expression of CaBP-3/calreticulin mRNA is androgen
dependent (51); down-regulation of this gene increases sensitivity to apoptosis induced by the calcium ionophore
A23187 in the androgen-sensitive prostate cell line LNCaP
(52). This evidence suggests that the decreased expression of
CaBP-3/calreticulin after androgen withdrawal by orchidectomy may be linked to androgen-dependent apoptosis of
principal cells in the epididymis after orchidectomy (12).
Alternatively, a recent study (53) has shown that differing
expression patterns of high molecular weight CaBPs are observed in spermatozoa collected from the caput and cauda
epididymal segments, indicating that CaBPs may be secreted
from the epididymal epithelium during sperm maturation.
The precise function of CaBPs in the epididymis and the
significance of their regulation after orchidectomy remain to
be elucidated.
Segment-specific up-regulation of genes in the epididymis
has not been previously described. In contrast, segmentspecific down-regulation of gene expression after the withdrawal of androgens or testicular factors is a hallmark characteristic of the epididymis (54). ODC is involved in the
biosynthesis of polyamines, and ODC enzyme activity in the
epididymis is regulated by androgens (55). Similarly, expression of mAAT, which we describe for the first time in the
epididymis, is likely to be regulated by androgens. In the
prostate, citrate production is an androgen-dependent pro-
Downloaded from endo.endojournals.org on February 28, 2005
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
Endocrinology, March 2003, 144(3):975–988 985
FIG. 9. Expression of Hsp in the epididymis over the first week post orchidectomy. A, Grp94. B, Hsp47 and
Hsp27. Gene expression is presented as
relative intensity, mean ⫾ SEM for three
to five replicates per group. IS, Initial
segment; CA, caput; CO, corpus; CD,
cauda.
FIG. 10. Expression of apoptosis-associated genes in the epididymis over the
first week post orchidectomy. A, Dad1.
B, Mcl1. C, TNFR1. Gene expression is
presented as relative intensity, mean ⫾
SEM for three to five replicates per
group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial
segment; CA, caput; CO, corpus; CD,
cauda.
cess that requires mAAT (56); mAAT expression and stability
in that tissue are regulated by androgens (57), likely through
an androgen response element in its promoter (58). DAAO
is a ubiquitous flavoenzyme that catalyzes the oxidative
deamination of d-amino acids in many species including
mammals (59). Although the existence of in vivo substrates
for DAAO was not known for many years, recent evidence
has demonstrated modulation of N-methyl-d-aspartate neurotransmission in the brain by d-ser, with a role for DAAO
in brain function by regulating the level of these compounds
(60). The significance of DAAO expression and regulation in
the epididymis remains to be resolved.
Our observation that several transporters are expressed
and differentially regulated along the epididymis is of in-
Downloaded from endo.endojournals.org on February 28, 2005
986
Endocrinology, March 2003, 144(3):975–988
terest in light of the changing microenvironment bathing
spermatozoa in the epididymal lumen (61). OCTN2 has recently been characterized in the epididymis (62); OCTN2 is
thought to function to transport carnitine into the epididymal
lumen. ASCT2 has been proposed to be involved in transport
of amino acids across the brush-border membrane of the
intestine and kidney (63). Transport of amino acids has been
characterized in the epididymis, particularly in the caput
region (64), although specific transporters have yet to be
identified. Our observation that ASCT2 is expressed and
regulated in the epididymis after orchidectomy suggests that
this particular transporter may be involved in androgendependent transport of amino acids across the epididymal
epithelium.
Finally, we describe for the first time the expression and
regulation of AGP mRNA specifically in the initial segment
of the epididymidis. AGP belongs to the family of acute
phase proteins synthesized by the liver and released into the
circulation in response to tissue injury, inflammation, or
infection. Drug binding to AGP, including retinoic acid and
the endogenous steroid cortisol, has also been reported (65).
It is possible that AGP is involved in transport of androgens
across the epididymal epithelium in a manner similar to
the well-characterized epididymal androgen-binding
protein (66).
Expression by gene family
Hsps. The expression of Hsps has not been described previously in the epididymis. Hsps are highly conserved proteins
that act as molecular chaperones within the cell. In addition
to their housekeeping functions, which include stabilization
against protein aggregation, protein folding, intraorganellar
transport, and refolding of denatured proteins (67), the expression of several family members can be modulated by
cellular stress. Inducible Hsps, including Hsp27 and Hsp70,
can accumulate in cells under a variety of stressful stimuli
such as heat or oxidative stress (68). The family of grp, which
include grp94 and grp78, can be regulated by perturbations
in function such as Ca2⫹ depletion (69). Hsp47 is a collagenspecific molecular chaperone essential for collagen synthesis
in the endoplasmic reticulum (70). We have observed that
throughout the epididymis, progressively decreased expression of grp94 coincides with transient up-regulation of Hsp47
and Hsp27 over the first week post orchidectomy. Interestingly, transiently increased expression of TGF␤1 has been
noted also in the epididymis after orchidectomy (28), and
TGF␤1 has been implicated in the transcription of Hsp47 in
mouse osteoblast cells (71). Transient up-regulation of Hsp47
after orchidectomy suggests that epididymal collagen production is altered in the epididymis after androgen withdrawal.
A transient increase in the expression of Hsp27 has been
reported in epithelial cells during involution of the rat ventral prostate after castration/orchidectomy (72); Hsp27 has
been shown recently to have antiapoptotic functions (73),
suggesting that it may be directly involved in the survival of
epididymal principal cells after orchidectomy. It is interesting to note that increased expression of the transcription
factor c-jun coincided with the rise in Hsp27 and Hsp47
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
expression along the epididymis, with the greatest increase
for all three genes observed in the caput epididymidis. This
suggests that c-jun may be involved in the transcription of
these genes in the epididymis after orchidectomy.
Expression of Hsp90␤, which regulates androgen receptor
activation (74), was unchanged in the epididymis after orchidectomy. This observation was surprising in light of reports that have shown a decline in androgen receptor expression in the epididymis after androgen withdrawal (75,
76). Similar to clusterin, the small Hsp CRY␤B1 showed
increased expression in the initial segment of the epididymis
after orchidectomy. CRY␤B1 may be an androgen-repressed
gene in the initial segment of the epididymis in a manner
analogous to the androgen-repressed status of clusterin in
the corpus and cauda epididymidis (27).
Apoptosis-associated proteins. Mechanisms underlying apoptotic cell death in the epididymis after androgen withdrawal by
orchidectomy (12) or efferent duct ligation (12, 77) are largely
unknown. The involvement of the FAS pathway in epididymal apoptotic cell death after orchidectomy is controversial;
an initial study indicated that FAS signaling was involved in
apoptosis of male reproductive organs after orchidectomy
(78), although a subsequent report on the FASR and FAS
ligand null mutants failed to show prevention of apoptotic
cell death in male reproductive organs (79), indicating that
the FAS-FAS ligand pathway is not essential in mediating
apoptosis after orchidectomy in these tissues.
In the present study, we show differential regulation of
three apoptosis-associated transcripts after orchidectomy in
the epididymis, Dad1, the bcl-2 family member Mcl1, and
TNFR1. Dad1 has largely been implicated in N-linked glycosylation (80). In cell culture, inhibition of N-linked protein
glycosylation in cell lines that undergo apoptosis after loss of
Dad1 function suggests that loss of N-linked glycoproteins is
associated with onset of apoptotic cell death (81). In the
mouse, the null mutation for Dad1 is lethal and causes abnormal N-linked glycoproteins and increased embryonic apoptosis (82). High levels of Dad1 expression, particularly in
the initial segment of the epididymis, indicate that there is a
differential requirement for N-linked protein glycosylation
along the epididymis. This decline in the expression of Dad1
along the epididymis suggests that androgens modulate
Dad1 expression and protein glycosylation, although the link
with apoptotic cell death in the epididymis after orchidectomy remains unclear. Interestingly, Dad1 has been shown
to bind Mcl1 (83), which we have shown to be transiently
increased along the epididymis after orchidectomy. Mcl1 is
an antiapoptotic member of the bcl-2 family of proteins (84),
which form a key group of intracellular factors that regulate
apoptosis by binding to each other to form heterodimers (85).
Transient up-regulation of Mcl1 expression may reflect an
antiapoptotic mechanism by which the majority of principal
cells are able to survive the apoptotic insult of orchidectomy.
Studies in cell lines have shown that Mcl1 is capable of
suppressing cell death induced by various stimuli (86).
TNFR1 is a transmembrane glycoprotein receptor that
binds intracellular proteins to control signaling within the
target cell. TNFR1-dependent signaling can be either pro- or
antiapoptotic (87). At present, the significance of increased
Downloaded from endo.endojournals.org on February 28, 2005
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
TNFR1 expression in the epididymis after orchidectomy is
unclear, although studies on caspase-8, a downstream target
of TNFR1 signaling, should help clarify this observation.
In summary, our study of gene expression in the epididymis after orchidectomy has provided a clearer understanding of patterns of mRNA expression underlying epididymal
regression after orchidectomy. The identification of novel
genes that are regulated in the epididymis after orchidectomy, both throughout the epididymis and in a segmentspecific manner, enhances understanding of androgendependent and segment-specific epididymal functions.
Acknowledgments
Received July 10, 2002. Accepted November 14, 2002.
Address all correspondence and requests for reprints to: B. Robaire,
Department of Pharmacology and Therapeutics, McGill University, 3655
Promenade Sir-William-Osler, Montréal, Québec H3G 1Y6, Canada. Email: [email protected].
This work was supported by a program project grant from the National Institute on Aging, NIH (Grant AG08321).
References
1. Ezer N, Robaire B 2002 Androgen regulation of epididymal structure and
function. In: Robaire B, Hinton BT, eds. The epididymis from molecules to
clinical practice. New York: Kluwer Academic/Plenum Publishers; 297-316
2. Benoit MJ 1926 Recherches anatomiques, cytologiques et histophysiologiques
sur les voies excrétrices du testicule, chez les mammifères. Arch Anat Hist
Embryo 5:173– 412
3. Butenandt A 1931 Uber die chemish untersuchungder der sexualhormone. Z
Angnew Chem 44:905–908
4. White WE 1932 The effect of hypophysectomy on the survival of spermatozoa
in the male rat. Anat Rec 54:253–273
5. Dyson AL, Orgebin-Crist MC 1973 Effect of hypophysectomy, castration and
androgen replacement upon the fertilizing ability of rat epididymal spermatozoa. Endocrinology 93:391– 402
6. Robaire B, Ewing LL, Zirkin BR, Irby DC 1977 Steroid ⌬4 –5␣-reductase and
3␣-hydroxysteroid dehydrogenase in the rat epididymis. Endocrinology 101:
29 – 40
7. Brooks DE 1979 Influence of androgens on the weights of the male accessory
reproductive organs and on the activities of mitochondrial enzymes in the
epididymis of the rat. J Endocrinol 82:293–303
8. Moore HD, Bedford JM 1979 Short-term effects of androgen withdrawal on
the structure of different epithelial cells in the rat epididymis. Anat Rec 193:
293–311
9. Orgebin-Crist MC, Davies J 1974 Functional and morphological effects of
hypophysectomy and androgen replacement in the rabbit epididymis. Cell
Tissue Res 148:183–201
10. Fawcett DW, Hoffer AP 1979 Failure of exogenous androgen to prevent
regression of the initial segments of the rat epididymis after efferent duct
ligation or orchidectomy. Biol Reprod 20:162–181
11. Ruiz-Bravo N 1988 Tissue and cell specificity of immobilin biosynthesis. Biol
Reprod 39:901–911
12. Fan X, Robaire B 1998 Orchidectomy induces a wave of apoptotic cell death
in the epididymis. Endocrinology 139:2128 –2136
13. Holland MK, Vreeburg JT, Orgebin-Crist MC 1992 Testicular regulation of
epididymal protein secretion. J Androl 13:266 –273
14. Schwaab V, Faure J, Dufaure JP, Drevet JR 1998 GPx3: the plasma-type
glutathione peroxidase is expressed under androgenic control in the mouse
epididymis and vas deferens. Mol Reprod Dev 51:362–372
15. Robaire B, Zirkin BR 1981 Hypophysectomy and simultaneous testosterone
replacement: effects on male rat reproductive tract and epididymal ⌬4 –5␣reductase and 3␣-hydroxysteroid dehydrogenase. Endocrinology 109:1225–
1233
16. Lan Z-J, Labus JC, Hinton BT 1998 Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat
epididymis by testicular factors: role of basic fibroblast growth factor. Biol
Reprod 58:197–206
17. Haendler B, Habenicht UF, Schwidetzky U, Schuttke I, Schleuning WD 1997
Differential androgen regulation of the murine genes for cysteine-rich secretory proteins (CRISP). Eur J Biochem 250:440 – 446
18. Rigaudiere N, Ghyselinck NB, Faure J, Dufaure JP 1992 Regulation of the
epididymal glutathione peroxidase-like protein in the mouse: dependence
upon androgens and testicular factors. Mol Cell Endocrinol 89:67–77
19. Kaunisto K, Fleming RE, Kneer J, Sly WS, Rajaniemi H 1999 Regional
Endocrinology, March 2003, 144(3):975–988 987
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
expression and androgen regulation of carbonic anhydrase IV and II in the
adult rat epididymis. Biol Reprod 61:1521–1526
Cheuk BL, Leung PS, Lo AC, Wong PY 2000 Androgen control of cyclooxygenase expression in the rat epididymis. Biol Reprod 63:775–780
Leung PS, Wong TP, Lam SY, Chan HC, Wong PY 2000 Testicular hormonal
regulation of the renin-angiotensin system in the rat epididymis. Life Sci
66:1317–1324
Garrett JE, Garrett SH, Douglass J 1990 A spermatozoa-associated factor
regulates proenkephalin gene expression in the rat epididymis. Mol Endocrinol 4:108 –118
Cornwall GA, Orgebin-Crist MC, Hann SR 1992 The CRES gene: a unique
testis-regulated gene related to the cystatin family is highly restricted in its
expression to the proximal region of the mouse epididymis. Mol Endocrinol
6:1653–1664
Viger RS, Robaire B 1996 The mRNAs for the steroid 5␣-reductase isozymes,
types 1 and 2, are differentially regulated in the rat epididymis. J Androl
17:27–34
Palladino MA, Hinton BT 1994 Expression of multiple ␥-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is
differentially regulated by androgens and testicular factors in a region-specific
manner. Endocrinology 135:1146 –1156
Lareyre JJ, Winfrey VP, Kasper S, Ong DE, Matusik RJ, Olson GE, OrgebinCrist MC 2001 Gene duplication gives rise to a new 17-kilodalton lipocalin that
shows epididymal region-specific expression and testicular factor(s) regulation. Endocrinology 142:1296 –1308
Cyr DG, Robaire B 1992 Regulation of sulfated glycoprotein-2 (clusterin)
messenger ribonucleic acid in the rat epididymis. Endocrinology 130:2160 –
2166
Desai KV, Kondaiah P 2000 Androgen ablation results in differential regulation of transforming growth factor-␤ isoforms in rat male accessory sex
organs and epididymis. J Mol Endocrinol 24:253–260
Van Hal NL, Vorst O, van Houwelingen AM, Kok EJ, Peijnenburg A, Aharoni A, van Tunen AJ, Keijer J 2000 The application of DNA microarrays in
gene expression analysis. J Biotechnol 78:271–280
Jervis KM, Robaire B 2001 Dynamic changes in gene expression along the rat
epididymis. Biol Reprod 65:696 –703
Cyr DG, Hermo L, Blaschuk OW, Robaire B 1992 Distribution and regulation
of epithelial cadherin messenger ribonucleic acid and immunocytochemical
localization of epithelial cadherin in the rat epididymis. Endocrinology 130:
353–363
Cyr DG, Dufresne J, Pillet S, Alfieri TJ, Hermo L 2001 Expression and
regulation of metallothioneins in the rat epididymis. J Androl 22:124 –135
Oppenheim RW, Prevette D, Tytell M, Homma S 1990 Naturally occurring
and induced neuronal death in the chick embryo in vivo requires protein and
RNA synthesis: evidence for the role of cell death genes. Dev Biol 138:104 –113
Dragunow M, Preston K 1995 The role of inducible transcription factors in
apoptotic nerve cell death. Brain Res Brain Res Rev 21:1–28
Achanzar WE, Achanzar KB, Lewis JG, Webber MM, Waalkes MP 2000
Cadmium induces c-myc, p53, and c-jun expression in normal human prostate
epithelial cells as a prelude to apoptosis. Toxicol Appl Pharmacol 164:291–300
Nickerson T, Pollak M, Huynh H 1998 Castration-induced apoptosis in the
rat ventral prostate is associated with increased expression of genes encoding
insulin-like growth factor binding proteins 2, 3, 4 and 5. Endocrinology 139:
807– 810
Sunde RA, Hoekstra WG 1980 Structure, synthesis and function of glutathione
peroxidase. Nutr Rev 38:265–273
Ghyselinck NB, Dufaure I, Lareyre JJ, Rigaudiere N, Mattei MG, Dufaure
JP 1993 Structural organization and regulation of the gene for the androgendependent glutathione peroxidase-like protein specific to the mouse epididymis. Mol Endocrinol 7:258 –272
Zini A, Schlegel PN 1997 Identification and characterization of antioxidant
enzyme mRNAs in the rat epididymis. Int J Androl 20:86 –91
Brooks DE 1977 The androgenic control of the composition of the rat epididymis determined by efferent duct ligation or castration. J Reprod Fertil 49:
383–385
Hinton BT, Lan ZJ, Rudolph DB, Labus JC, Lye RJ 1998 Testicular regulation
of epididymal gene expression. J Reprod Fertil Suppl 53:47–57
Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC 1989 Analysis of
the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 10:214 –220
Strange RC, Spiteri MA, Ramachandran S, Fryer AA 2001 Glutathione-Stransferase family of enzymes. Mutat Res 482:21–26
Hales BF, Hachey C, Robaire B 1980 The presence and longitudinal distribution of the glutathione S-transferases in rat epididymis and vas deferens.
Biochem J 189:135–142
Papp S, Robaire B, Hermo L 1995 Immunocytochemical localization of the Ya,
Yc, Yb1, and Yb2 subunits of glutathione S-transferases in the testis and
epididymis of adult rats. Microsc Res Tech 30:1–23
Veri JP, Hermo L, Robaire B 1993 Immunocytochemical localization of the Yf
subunit of glutathione S-transferase P shows regional variation in the staining
of epithelial cells of the testis, efferent ducts, and epididymis of the male rat.
J Androl 14:23– 44
Downloaded from endo.endojournals.org on February 28, 2005
988 Endocrinology, March 2003, 144(3):975–988
47. Hermo L, Papp S 1996 Effects of ligation, orchidectomy, and hypophysectomy
on expression of the Yf subunit of GST-P in principal and basal cells of the adult
rat epididymis and on basal cell shape and overall arrangement. Anat Rec
244:59 – 69
48. Ikeda H, Serria MS, Kakizaki I, Hatayama I, Satoh K, Tsuchida S, Muramatsu M, Nishi S, Sakai M 2002 Activation of mouse Pi-class glutathione
S-transferase gene by Nrf2(NF-E2-related factor 2) and androgen. Biochem J
364:563–570
49. Robaire B, Hales BF 1982 Regulation of epididymal glutathione S-transferases:
effects of orchidectomy and androgen replacement. Biol Reprod 26:559 –565
50. Ostwald TJ, MacLennan DH 1974 Isolation of a high affinity calcium-binding
protein from sarcoplasmic reticulum. J Biol Chem 249:974 –979
51. Zhu N, Pewitt EB, Cai X, Cohn EB, Lang S, Chen R, Wang Z 1998 Calreticulin:
an intracellular Ca2⫹-binding protein abundantly expressed and regulated by
androgen in prostatic epithelial cells. Endocrinology 139:4337– 4344
52. Zhu N, Wang Z 1999 Calreticulin expression is associated with androgen
regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP
prostate cancer cells. Cancer Res 59:1896 –1902
53. Gye MC, Park S, Kim YS, Ahn HS 2001 Mobility shift assay of calciumbinding proteins of mouse epididymal spermatozoa. Andrologia 33:193–198
54. Cornwall GA 2002 Gene expression and epididymal function. In: Robaire B,
Hinton BT, eds. The epididymis: from molecules to clinical practice. New York:
Kluwer Academic/Plenum; 169 –199
55. Las Heras MA, Calandra RS 1987 Androgen-dependence of ornithine decarboxylase in the rat epididymis. J Reprod Fertil 79:9 –14
56. Franklin RB, Kukoyi BI, Akuffo V, Costello LC 1987 Testosterone stimulation
of mitochondrial aspartate aminotransferase levels and biosynthesis in rat
ventral prostate. J Steroid Biochem 28:247–256
57. Qian K, Franklin RB, Costello LC 1993 Testosterone regulates mitochondrial
aspartate aminotransferase gene expression and mRNA stability in prostate.
J Steroid Biochem Mol Biol 44:13–19
58. Juang HH, Costello LC, Franklin RB 1995 Androgen modulation of multiple
transcription start sites of the mitochondrial aspartate aminotransferase gene
in rat prostate. J Biol Chem 270:12629 –12634
59. Weimar WR, Neims AH 1977 The development of D-amino acid oxidase in rat
cerebellum. J Neurochem 29:649 – 656
60. Pilone MS 2000 D-Amino acid oxidase: new findings. Cell Mol Life Sci 57:
1732–1747
61. Turner TT 1991 Spermatozoa are exposed to a complex microenvironment as
they traverse the epididymis. Ann NY Acad Sci 637:364 –383
62. Rodriguez CM, Labus JC, Hinton BT 2002 Organic cation/carnitine transporter, OCTN2, is differentially expressed in the adult rat epididymis. Biol
Reprod 67:314 –319
63. Avissar NE, Ryan CK, Ganapathy V, Sax HC 2001 Na(⫹)-dependent neutral
amino acid transporter ATB(0) is a rabbit epithelial cell brush-border protein.
Am J Physiol Cell Physiol 281:C963–C971
64. Hinton BT, Howards SS 1982 Rat testis and epididymis can transport [3H]
3-O-methyl-d-glucose, [3H] inositol and [3H] ␣-aminoisobutyric acid across its
epithelia in vivo. Biol Reprod 27:1181–1189
65. Fournier T, Medjoubi N, Porquet D 2000 ␣-1-Acid glycoprotein. Biochim
Biophys Acta 1482:157–171
66. Feldman M, Lea OA, Petrusz P, Tres LL, Kierszenbaum AL, French FS 1981
Androgen-binding protein. Purification from rat epididymis, characterization,
and immunocytochemical localization. J Biol Chem 256:5170 –5175
67. Bukau B, Horwich AL 1998 The Hsp70 and Hsp60 chaperone machines. Cell
92:351–366
Ezer and Robaire • Epididymal Gene Expression and Orchidectomy
68. Kregel KC 2002 Heat shock proteins: modifying factors in physiological stress
responses and acquired thermotolerance. J Appl Physiol 92:2177–2186
69. Lee AS 2001 The glucose-regulated proteins: stress induction and clinical
applications. Trends Biochem Sci 26:504 –510
70. Hendershot LM, Bulleid NJ 2000 Protein-specific chaperones: the role of hsp47
begins to gel. Curr Biol 10:R912–R915
71. Yamamura I, Hirata H, Hosokawa N, Nagata K 1998 Transcriptional activation of the mouse HSP47 gene in mouse osteoblast MC3T3–E1 cells by TGF-␤
1. Biochem Biophys Res Commun 244:68 –74
72. Guenette RS, Daehlin L, Mooibroek M, Wong K, Tenniswood M 1994
Thanatogen expression during involution of the rat ventral prostate after
castration. J Androl 15:200 –211
73. Garrido C, Gurbuxani S, Ravagnan L, Kroemer G 2001 Heat shock proteins:
endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286:433– 442
74. Fang Y, Fliss AE, Robins DM, Caplan AJ 1996 Hsp90 regulates androgen
receptor hormone binding affinity in vivo. J Biol Chem 271:28697–28702
75. Zhu LJ, Hardy MP, Inigo IV, Huhtaniemi I, Bardin CW, Moo-Young AJ 2000
Effects of androgen on androgen receptor expression in rat testicular and
epididymal cells: a quantitative immunohistochemical study. Biol Reprod
63:368 –376
76. Goyal HO, Bartol FF, Wiley AA, Khalil MK, Williams CS, Vig MM 1998
Regulation of androgen and estrogen receptors in male excurrent ducts of the
goat: an immunohistochemical study. Anat Rec 250:164 –171
77. Turner TT, Riley TA 1999 p53 independent, region-specific epithelial apoptosis is induced in the rat epididymis by deprivation of luminal factors. Mol
Reprod Dev 53:188 –197
78. Suzuki A, Matsuzawa A, Iguchi T 1996 Down regulation of Bcl-2 is the first
step on Fas-mediated apoptosis of male reproductive tract. Oncogene 13:31–37
79. Sugihara A, Yamada N, Tsujimura T, Iwasaki T, Yamashita K, Takagi Y,
Tsuji M, Terada N 2001 Castration induces apoptosis in the male accessory sex
organs of Fas-deficient lpr and Fas ligand-deficient gld mutant mice. In Vivo
15:385–390
80. Makishima T, Nakashima T, Nagata-Kuno K, Fukushima K, Iida H, Sakaguchi M, Ikehara Y, Komiyama S, Nishimoto T 1997 The highly conserved
DAD1 protein involved in apoptosis is required for N-linked glycosylation.
Genes Cells 2:129 –141
81. Yoshimi M, Sekiguchi T, Hara N, Nishimoto T 2000 Inhibition of N-linked
glycosylation causes apoptosis in hamster BHK21 cells. Biochem Biophys Res
Commun 276:965–969
82. Brewster JL, Martin SL, Toms J, Goss D, Wang K, Zachrone K, Davis A,
Carlson G, Hood L, Coffin JD 2000 Deletion of Dad1 in mice induces an
apoptosis-associated embryonic death. Genesis 26:271–278
83. Makishima T, Yoshimi M, Komiyama S, Hara N, Nishimoto T 2000 A subunit
of the mammalian oligosaccharyltransferase, DAD1, interacts with Mcl-1, one
of the bcl-2 protein family. J Biochem (Tokyo) 128:399 – 405
84. Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW 1993 MCL1, a gene
expressed in programmed myeloid cell differentiation, has sequence similarity
to BCL2. Proc Natl Acad Sci USA 90:3516 –3520
85. Adams JM, Cory S 1998 The Bcl-2 protein family: arbiters of cell survival.
Science 281:1322–1326
86. Reynolds JE, Li J, Craig RW, Eastman A 1996 BCL-2 and MCL-1 expression
in Chinese hamster ovary cells inhibits intracellular acidification and apoptosis
induced by staurosporine. Exp Cell Res 225:430 – 436
87. Baud V, Karin M 2001 Signal transduction by tumor necrosis factor and its
relatives. Trends Cell Biol 11:372–377
Downloaded from endo.endojournals.org on February 28, 2005