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Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada H3G 1Y6
Address all correspondence and requests for reprints to: Dr. Bernard Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montréal, Québec, Canada H3G 1Y6. E-mail: brobaire{at}pharma.mcgill.ca
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Introduction |
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It is well known that the epididymis is dependent on the presence of the testis for the maintenance of its structure and functions. The epididymis atrophies dramatically after orchidectomy, and treatment with an amount of androgen that mimics the circulating androgen concentration can only partially maintain epididymal tissue weight (4). This is due to the fact that about half of the epididymal weight is attributable to luminal fluid and spermatozoa (4, 5, 6). In the androgen-deficient state, tubular diameter and epithelial cell height are reduced, whereas the intertubular stroma is increased (7, 8). The lamina densa of the basement membrane underlying the epithelium is disorganized and thicker than normal, and follows the irregular outline of the basal parts of the epithelial cells (9). Ultrastructurally, orchidectomy results in greater endocytosis by principal cells, disappearance of vesicles from the cell apex, a reduction in rough endoplasmic reticulum, a drop in the volume of the Golgi cisternae, and an increase in lysosome content (10).
Orchidectomy affects not only the epididymal morphology but also its cytochemistry. Histochemical evidence shows that lipids, polysaccharide complexes, and glycogen are greatly reduced after orchidectomy, but are maintained by testosterone replacement therapy (11). The metabolic activity of the epithelium is also greatly reduced after orchidectomy (12). Orchidectomy in adult rats is followed in 30 days not only by a significant decline in epididymal weight and total protein, but also by a decrease in total RNA and DNA (13). Functionally, orchidectomy increases the amplitude of spontaneous contractions (14) and decreases sperm motility and fertilizing ability (11).
Apoptosis is a form of physiological cell death that is morphologically and biochemically distinct from necrotic cell death (15, 16). Apoptosis is characterized by chromatin condensation, cytoplasmic shrinkage, membrane blebbing, and formation of membrane-bound condensed apoptotic bodies. During apoptosis, an endogenous endonuclease is activated, which causes internucleosomal DNA fragmentation to 180- to 200-bp multiple fragments (17, 18). Apoptotic cell death can be initiated by a number of external signals, including withdrawal of some growth factors or hormones, glucocorticoids, irradiation, thermal stimuli (19), anticancer agents (20, 21), and viral infection (22).
Androgen deprivation by orchidectomy induces rapid glandular epithelial cell death in the ventral prostate via an apoptotic mechanism (17, 23, 24, 25). As many as 80% of cells are lost within 10 days after surgery (26). This type of cell death was found to be initiated by removal of the inhibitory effects of androgen on prostatic glandular cell death (26, 27). Rapid involution of the rat ventral prostate after orchidectomy provides an excellent model for studying androgen regulation of prostate functions.
Our major objective herein was to investigate whether apoptotic cell death is involved in the weight loss of the epididymis after orchidectomy and to determine the extent to which cells of the epididymis are sensitive or resistant to androgen withdrawal. We found that orchidectomy induces a wave of segment-specific apoptotic principal cell death in the rat epididymis, starting in the initial segment 18 h after surgery, moving down to the cauda epididymidis, and disappearing after a week. Androgen replacement can prevent apoptosis in all regions of the epididymis, with the exception of the initial segment. To prohibit cell death completely in the initial segment requires androgen as well as luminal components from the testis.
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Materials and Methods |
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Exp 1: bilateral orchidectomy
Animals either remained intact to serve as controls or were
bilaterally orchidectomized via the scrotal route under ether
anesthesia. Animals were anesthetized with sodium pentobarbital (0.008
ml/100 g; Somnitol, MTC Pharmaceuticals, Hamilton, Canada) administered
ip 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, or 8 days after surgery. The left
epididymis was removed, dissected free of fat, and sectioned into four
different segments: initial segment, caput, corpus, and cauda, as
previously described (1). Tissues were frozen in liquid nitrogen and
stored at -70 C before DNA extraction. The right epididymis was fixed
with Bouin’s fixative by perfusion through the abdominal aorta for 10
min, as previously described (28). Fixed tissues were then immersed in
Bouin’s fixative for at least 24 h before they were dehydrated
and embedded in paraffin.
Exp 2: bilateral orchidectomy with simultaneous androgen
replacement
Animals were either left intact to serve as controls or
bilaterally orchidectomized and sc injected with androgens in corn oil
at the time of surgery. Orchidectomized rats were randomly assigned to
each of four groups: orchidectomized and injected with 0.2 ml corn
oil/rat/day, orchidectomized and injected with 0.5 mg
testosterone/rat/day, orchidectomized and injected with 5 mg
testosterone/rat/day, orchidectomized and injected with 5 mg
5
-dihydrotestosterone (DHT)/rat/day. Doses of 0.5 and 5 mg
testosterone were chosen to mimic physiological serum testosterone
concentrations and testosterone concentrations normally found in the
epididymis, respectively. Animals were killed on days 1, 2, 3, and 7
after surgery, and the epididymides were fixed with Bouin’s fixative
by perfusion, as described above.
Exp 3: unilateral efferent duct ligation
Animals either were left intact to serve as controls or were
unilaterally efferent duct ligated. Rats were anesthetized with ether,
and the testis and epididymis on the right side were exposed through a
scrotal incision. The thin avascular attachment joining the initial
segment of the epididymis to the tunica albuginea was cut to permit
exposure of the efferent ducts coursing above and parallel to the blood
vascular supply. A silk suture was passed by needle through the thin
sheet of connective tissue between the ductules and the blood vessels,
and the efferent ducts were occluded by ligation. Great care was taken
to avoid damage to the regional blood vessels. The whole tissue was
then returned to the scrotum, and the incision was closed with wound
clips. Animals were killed 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, and 9
days after surgery. Both epididymides were fixed as described
above.
In situ detection of apoptosis
The epididymides embedded in paraffin were cut into 5-µm
sections. In situ apoptosis was detected by TUNEL
(TdT-mediated dUTP-digoxigenin nick end-labeling) staining with an
ApopTag-peroxidase kit (Oncor, Gaithersburg, MD) to identify apoptotic
cell death in situ via specific labeling of nuclear DNA
fragmentation (29). In brief, after deparaffinization and rehydration,
tissue sections were incubated with proteinase K (20 µg/ml) for 5 min
at room temperature, washed in distilled water, and then treated with
2% hydrogen peroxide in PBS for 5 min at room temperature to quench
endogenous peroxidase activity. Sections were incubated with
digoxigenin-deoxy (d)-UTP and terminal deoxynucleotidyl transferase in
a humidified chamber at 37 C for 1 h and then treated with
antidigoxigenin-peroxidase at room temperature for 30 min.
Subsequently, the sections were exposed to 0.05% substrate
(diaminobenzidine) for 20 min, washed with distilled water and PBS, and
then counterstained with 0.01% methylene blue (Sigma Chemical Co., St.
Louis, MO) for 1 min, dehydrated in 50%, 70%, 95%, and 100%
ethanol, cleared in xylene, and mounted with Permount (Fisher
Scientific, Montreal, Canada). The incidence of apoptosis was evaluated
by counting apoptotic cells and apoptosis-positive tubules (the tubules
containing at least one apoptotic cell) in each epididymal segment.
DNA fragmentation analysis
DNA from each segment of the epididymis was isolated as
previously described (30). Tissues were homogenized and digested in
digestion buffer (100 mM NaCl; 10 mM Tris-HCl;
25 mM EDTA, pH 8; and 0.5% SDS) and freshly added
proteinase K, 0.1 mg/ml] (1:1.2 mg/ml) with shaking at 50 C for
15 h. DNA was extracted with an equal volume of
phenol-chloroform-isoamyl alcohol (25:24:1) and centrifuged for 10 min
at 1700 x g. The DNA was precipitated by adding 0.5
vol 7.5 M ammonium acetate and 2 vol 100% ethanol to the
aqueous layer; samples were left overnight at -20 C, then separated by
centrifugation at 1700 x g for 5 min, rinsed with 70%
ethanol, and air-dried. The pellet was dissolved in TE buffer (10
mM Tris-HCl and 1 mM EDTA, pH 8); RNA was
degraded by incubation of the samples with 1 µg/ml
deoxyribonuclease-free ribonuclease (Boehringer Mannheim, Laval,
Canada) for 1 h at 37 C and then quantitated with
spectrophotometry at 260 nm.
An aliquot of DNA (3 µg) from each sample was labeled at the 3'-ends with 2 µl [32P]dideoxy-ATP (ddATP; 10 mCi/ml; Amersham, Aylesbury, UK) using terminal transferase (25 U/sample; Boehringer Mannheim, Indianapolis, IN) as previously described by Tilly and Hsueh (31). Labeled samples were fractionated through 2% agarose gels (50–60 V; 3.5 h). After electrophoresis, gels were dried for 2 h in a Slab-Gel Dryer without heat and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) at -70 C for 30 min.
Statistical analysis
The number of apoptotic cells per 100 tubules and the percentage
of apoptosis-positive tubules were analyzed by one-way ANOVA, followed
by Newman-Keuls test to detect significant differences. The analyses
were performed with the aid of a CSS (Complete Statistics System)
computer program (Statsoft, Tulsa, OK). P < 0.05 was
considered significant.
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Results |
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, A–D). In the caput
epididymidis, apoptosis was first found after 24 h, reached a
maximum by day 3 (49% positive tubules), and was detectable until day
5 (Fig. 1, E–H). In the corpus epididymidis, apoptosis was first seen
on day 4, peaked on day 5 (29% positive tubules), and was undetectable
by day 6 postorchidectomy (Fig. 2, A–D).
In the cauda epididymidis, apoptosis was first seen on day 5, peaked on
day 6 (25% positive tubules), and was occasionally detected on day 7
(Fig. 2, E–H).
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) and the percentage of
positive tubules (Fig. 3B) were higher in the initial segment and caput
epididymidis than in the corpus and cauda epididymidis. Thus, a wave of
apoptosis occurred along the rat epididymis after orchidectomy.
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) and from the
caput epididymidis on days 2, 3, 4, and 5 after orchidectomy (Fig. 4B).
These data demonstrated that principal cells in the rat epididymis
undergo an apoptotic form of cell death after orchidectomy.
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. In the intact rat epididymis
(controls), there were no apoptotic positive-staining tubules
throughout the tissue. In the orchidectomized rat epididymis, the low
dose of testosterone that was used to maintain accessory organ weights
had no effect on apoptosis induced by orchidectomy on the first day
after surgery (P > 0.05). However, it lowered the
number of apoptotic cells per 100 tubules on day 2 (P
< 0.05) and the percentage of apoptosis-positive tubules on day 2
(P < 0.01) and day 3 (P < 0.05). High
doses of testosterone and DHT caused a decrease in both the number of
apoptotic cells per 100 tubules and the percentage of
apoptosis-positive tubules on day 1 (P < 0.05), day 2
and 3 (P < 0.01). Neither testosterone nor DHT could
completely prevent the apoptotic cell death induced by orchidectomy in
the initial segment of the epididymis.
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Pattern of apoptosis in the unilateral efferent duct-ligated rat
epididymis
In the initial segment of the epididymis that had had its efferent
ducts ligated, the epithelial cells started to undergo apoptotic cell
death by 18 h after surgery. The number of apoptotic cells per 100
tubules and the percentage of apoptosis-positive tubules reached a
maximum on day 2 and then gradually fell to zero on day 5 (Fig. 6). In the caput epididymidis, apoptosis
was first seen on day 2, peaked on day 4, and was detectable until day
7 (Fig. 6). Compared with the orchidectomized epididymis, the pattern,
incidence, and time course of apoptosis in the initial segment after
efferent duct ligation were similar. In the caput epididymidis, the
incidence of apoptotic cells after efferent duct ligation was
significantly lower (<30 apoptotic cells/100 tubules) than that found
after orchidectomy (>500 apoptotic cells/100 tubules); the time course
of apoptosis in the caput epididymidis after efferent duct ligation was
2 days longer than that in the orchidectomized tissue. No apoptosis was
observed in the corpus or cauda epididymidis or in the contralateral
epididymis after efferent duct ligation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis occurs in the epididymis in the form of a wave that begins to appear in the initial segment 18 h after orchidectomy, moves down to the cauda epididymidis, and disappears after a week. This time course is coincident with the movement of the luminal content, including spermatozoa, through the epididymal duct. The epididymis is exposed to two sources of androgens: those that reach the tissue through the peripheral circulation and those that arrive directly in the lumen via the efferent ducts (34); it is clear that epididymal functions depend on the presence of testicular components (3). After orchidectomy, the circulating androgen level decreases rapidly, reaching a value less than 10% of the intact control level in 2 h (26). Withdrawal of luminal components from the epididymis after orchidectomy takes less than 7 days (35). The time course of apoptotic cell death in the orchidectomized epididymis is apparently caused by gradual deprivation of testicular components. This is in contrast with apoptosis in the ventral prostate, which is induced by withdrawal of circulating androgen that appears on day 1, reaches a maximum on day 3, and is still detectable 2 weeks after orchidectomy (24).
Both the number of apoptotic cells and the percentage of apoptosis-positive tubules are higher in the initial segment and caput epididymidis than in the corpus and cauda epididymidis. This region-specific responsiveness is in contrast with the ventral prostate, where apoptotic epithelial cells are not localized to a particular region but, rather, are seen throughout the glandular duct (24). However, it is in agreement with the weight loss reported by Brooks (6); in the orchidectomized rat epididymis, the tissue weight of the caput decreases more rapidly and severely than that of the cauda epididymidis. This region-specific responsiveness to orchidectomy might be explained by the regional differences in androgen concentrations in the epididymis. Using an in vivo micropuncture technique, Turner et al. (36) reported that the total androgen level in the lumen of the caput epididymidis is 6-fold higher than that in the cauda epididymidis. The tissue androgen level in the caput epididymidis was also higher than that in the cauda epididymidis (37). Furthermore, the caput epididymidis is reported to have a higher concentration of androgen receptors (38), which are prominent in the nuclei of epithelial cells (39). The caput epididymidis seems to require higher androgen concentrations to maintain its morphology and functions; thus, we propose that withdrawal of androgen by orchidectomy results in more cells undergoing apoptosis.
Throughout the rat epididymis, apoptotic cell death is seen only in the epithelium and is localized specifically to principal cells. This phenomenon is consistent with the pattern of apoptosis reported for the prostate of the orchidectomized rats. Apoptosis in the prostate is also cell type specific; only the prostatic glandular epithelial cells, and not the basal or stromal cells, are androgen dependent and thus undergo cell death after orchidectomy (17). In the epididymis, androgen receptors are located mainly in the epithelial cells (39, 40). Among different types of epithelial cells, principal cells outnumber the basal, narrow, clear, and halo cells combined by at least three to one (1). Clear and narrow cells are androgen independent; few visible changes occur in these cells after androgen ablation by orchidectomy (9, 10). For the ventral prostate, androgen receptors disappear rapidly after orchidectomy, with a 83% reduction on day 2 (40) and undetectable concentrations by day 3 (39) after removal of the testes.
In the normal rat ventral prostate, 2% of the glandular cells spontaneously undergo apoptosis every day, and this rate of cell death is balanced by an equal rate of cell proliferation (26, 41). However, in the normal epididymis, no indication of apoptosis is seen throughout the tissue, implying that the cell turnover in the epididymis is very low; this is consistent with the observations of Clermont and Flannery (42) that the population of principal and basal cells does not renew itself during adulthood in the rat. In the prostate, orchidectomy results in 70% of the glandular epithelial cells undergoing apoptosis by 7 days and 80% of the cells being lost within 10 days (17, 26). In the epididymis, because of the spermatozoa in the lumen, it is difficult to detect exactly how many cells die via apoptosis by measuring loss of DNA. However, Brooks (12) reported that in the orchidectomized rat epididymis, DNA loss is 30% more than in the efferent duct-ligated epididymis 6 weeks after surgery. The ventral prostate gland is reported to contain a mixture of clones of both androgen-dependent and androgen-independent cells (43). After orchidectomy, only the androgen-dependent cells would stop proliferating and die (23). The reason why only a fraction of the epididymal principal cells die after orchidectomy is not known, but it raises the question of whether there are different populations of principal cells existing in the epididymis. The checkerboard-like immunostaining pattern reported by many when immunostaining different regions of the epididymis (44, 45, 46) would be consistent with this suggestion.
Orchidectomy enables the epididymis to be studied in the absence of the testicular components and androgen support. Androgen replacement is used to confirm whether orchidectomy-induced changes are caused by withdrawal of androgen. Although apoptosis in the caput, corpus, and cauda epididymidis can be completely prevented by androgen replacement, the caput epididymidis requires more androgen than the rest of the tissue. Furthermore, for the initial segment, androgen administration alone, even at very high doses, could not prevent apoptotic cell death, thus further indicating the dependence of this segment on direct testicular input (3).
Efferent duct ligation prevents testicular components from entering the epididymis and therefore enables apoptosis to be detected in the circulating androgen-maintained epididymis without contribution from the testicular components. Under that circumstance, apoptosis is still seen in the initial segment, is less in the caput, and is not observed at all in the corpus and cauda epididymidis. Combining these observations lead us to suggest that apoptosis in the epididymis is segment specific with respect to androgen responsiveness. Cell death in the corpus and cauda epididymidis is prevented by the presence of circulating androgen levels; to inhibit apoptosis in the caput epididymidis, high luminal androgen levels are presumably needed. However, the maintenance of normal morphology of the initial segment of the epididymis requires not only androgen but also testicular components. These data are in agreement with a previous morphological study showing that the initial segment of rat epididymis requires intraluminal concentrations of androgens and some other constituents of testicular fluid (47). In contrast, cells lining the terminal segment of the epididymis appear to be adequately served by circulating androgen alone (48).
Although it is well established that the epididymis is dependent on the presence of the testis for the maintenance of its structure and functions (1), the mechanism by which either androgens or other testicular factors regulate epididymal functions is still poorly understood. The results presented here demonstrate that orchidectomy induces a wave of segment-specific apoptotic principal cell death throughout the epididymis that is variable and dependent on androgens and other testicular factors as well as the region being examined. These observations would provide an appropriate model to study the androgen regulation of epididymal functions.
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Footnotes |
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Received July 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4-5-reductase and 3-hydroxysteroid
dehydrogenase in the rat epididymis. Endocrinology 101:1379–1390-reductase Type 1 along the rat
epididymis during postnatal development. Endocrinology 134:2298–2306[Abstract]
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