Nongenomic effect of testosterone on chloride secretion in
cultured rat efferent duct epithelia
G. P. H.
Leung,
S. B.
Cheng-Chew, and
P. Y. D.
Wong
Department of Physiology, Faculty of Medicine, The Chinese
University of Hong Kong, Shatin, New Territories, Hong Kong
 |
ABSTRACT |
Short-circuit current
(Isc) technique was used to investigate the role
of testosterone in the regulation of chloride secretion in cultured rat
efferent duct epithelia. Among the steroids tested, only testosterone,
and to a lesser extent, 5
-dihydrotestosterone (5
-DHT), reduced
the basal and forskolin-induced Isc in cultured rat efferent duct epithelia when added to the apical bathing solution. Indomethacin, a 3
-hydroxysteroid dehydrogenase, did not affect the
inhibitory effect of 5
-DHT. The effect of testosterone occurred within 10-20 s upon application and was dose dependent with
apparent IC50 value of 1 µM. The effect was abolished by
removal of Cl
but not HCO
from the
normal Krebs-Henseleit solution, suggesting that testosterone mainly
inhibited Cl
secretion. The efferent duct was found to be
most sensitive to testosterone, while the caput and the cauda
epididymidis were only mildly sensitive. Cyproterone acetate, a
steroidal antiandrogen, or flutamide, a nonsteroidal antiandrogen, did
not block the effect of testosterone on the forskolin-induced
Isc, nor did protein synthesis inhibitors,
cycloheximide, or actinomycin D. However, pertussis toxin, a
Gi protein inhibitor, attenuated the inhibition of
forskolin-induced Isc by testosterone.
Testosterone caused a dose-dependent inhibition of forskolin-induced
rise in cAMP in efferent duct cells. It is suggested that the rapid
effect of testosterone was mediated through a membrane receptor that is
negatively coupled to adenylate cyclase via Gi protein. The role of nongenomic action of testosterone in the regulation of electrolyte and fluid transport in the efferent duct is discussed.
testosterone; chloride secretion; efferent duct
 |
INTRODUCTION |
IT CAN HARDLY BE
OVEREMPHASIZED that the male posttesticular duct system plays an
indispensable role in male reproduction. The efferent duct, or ductuli
efferentes, forms the initial portion of the duct where the bulk of the
testicular fluid is reabsorbed (7). Water reabsorption
results in an increase in the concentration of spermatozoa and also of
the constituents that are essential for the maintenance of
epididymal integrity and functions. Although the efferent duct is
engaged in net water reabsorption (7, 16), electrolyte
secretion is also known to take place (6, 23). Secretion
of anions may act as a counterbalance to absorption, thereby exerting a
fine control over net water movement across the ductules. It is
generally held that in the epididymis, unlike absorption which is held
at a tonic rate, secretion is subject to short-term neurohumoral
regulation (39, 40). Rapid changes in secretion will have
impact on the fluidity of the epididymal microenvironment in which
maturing spermatozoa are bathed (40).
Androgens are essential for the normal growth and differentiation of
the male reproductive tract during sexual development (12, 18,
36). The acquisition of fertilizing capacity of spermatozoa in
epididymis also depends on androgens (4, 25, 28). It is
generally accepted that androgens bind to intracellular androgen
receptors resulting in mRNA and protein synthesis (32). Nevertheless, rapid responses to androgens have been observed in many
nonepithelial tissues that cannot be explained by involvement of mRNA
and protein synthesis (2, 5, 8, 13, 30). These rapid,
nongenomic effects are also seen for other steroid hormones (for
review, see Ref. 31). Recently, unconventional membrane
receptors for testosterone have been visualized under confocal
microscopy (1).
It has been shown that fluid reabsorption in efferent duct and
epididymis is androgen dependent (15, 41). Methods for the
isolation and culture of epithelial cells from efferent duct have
already been described (34). Epithelia so-derived display apical/basolateral polarity and possess morphological features and
secretory functions of the intact efferent duct in vivo. In the present
study, we have elucidated the nongenomic effect of testosterone on
chloride secretion in cultured rat efferent duct epithelia. The
implications of this effect in relation to male reproduction are discussed.
 |
MATERIALS AND METHODS |
Tissue culture technique.
All experiments were carried out according to the guidelines laid down
by the Laboratory Animal Services Centre of the Chinese University of
Hong Kong. The procedures of primary cultures of rat efferent duct
epithelial cells were modified from Rozewicka et al. (34).
Immature male Sprague-Dawley rats weighing 150 g were used
as a source of efferent duct. Adult rats were not used, as it has been
shown previously that the presence of spermatozoa prevents the plating
of epithelial cells, rendering formation of monolayers difficult. Rats
were killed by asphyxiation with a rising concentration of
CO2. The lower abdomens were opened, and the efferent ducts
were isolated and microdissected under sterile conditions to remove fat
and connective tissue. The ductules were cut into several small
segments, transferred to Hanks' balanced salt solution (HBSS)
containing 0.1% (wt/vol) trypsin and 0.2% (wt/vol) collagenase I, and
incubated in a water bath at 32°C for 1 h with vigorous shaking
(150 strokes/min). Then, the tissue was separated by low-speed
centrifugation (800 g, 5 min). The supernatant was
discarded, and the pellet resuspended in HBSS containing 0.2% (wt/vol)
collagenase I for 30 min at 32°C with vigorous shaking. After
centrifugation at 800 g for 5 min, cell plaques were
resuspended in HBSS containing 0.2% (wt/vol) collagenase I and
subjected to repeated pipetting for 15 min. The cells were centrifuged
at 800 g for 5 min and resuspended in Eagle's minimum essential medium (MEM) containing non-essential amino acids (0.1 mM),
sodium pyruvate (1 mM), glutamine (4 mM), 5
-dihydrotestosterone (5
-DHT, 1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and
streptomycin (100 µg/ml). The cell suspension was decanted and seeded
into the wells of Matrigel-coated Millipore filter assemblies with a
diameter of 0.2 cm2 (cell concentration 5 × 104 cell plaques/ml, plating density 2.5 × 104 cell plaques/cm2 filter) floating on 15 ml
of culture medium. Cultures were incubated for 5 days at 32°C in 5%
CO2. Thereafter, the monolayers reached confluence and were
ready for the measurement of short-circuit current
(Isc). Sample cultures were stained with
toluidine blue and the morphological examination was made under light microscopy.
The procedures of primary cultures of rat caput and cauda epididymidis
have been described previously (9, 39). Briefly, the
tissues were dissected out, finely chopped with scissors, and then
digested with 0.25% (wt/vol) trypsin followed by 0.1% (wt/vol)
collagenase I. Epithelial cells were seeded into the wells of Millipore
filter assemblies floating on 15 ml of culture medium. Cultures were
incubated for 3 days at 32°C in 5% CO2.
Isc measurement.
Confluent monolayers were washed three times with Normal
Krebs-Henseleit solution to get rid of any sex hormone-binding globulin that may be present in culture medium. They were then clamped between
two halves of Ussing chambers with a 0.6-cm2 window. The
tissue was short-circuited by the use of a voltage-clamp amplifier
(model DVC 1000; World Precision Instruments, New Haven, CT). The
Isc was displayed on a pen recorder.
Transepithelial resistance was obtained from Ohm's law by clamping the
tissue intermittently at a voltage at 0.1 mV displaced from zero. The two channels of the amplifier were mostly used simultaneously on
parallel monolayers so that studies could be made under control and
experimental conditions. In most situations, monolayers were bathed on
both sides with Krebs-Henseleit solution, gassed with 95%
O2-5% CO2, and warmed to 32°C.
Measurement of cAMP.
Efferent duct monolayers from rats were grown on 24-well plates
(Costar, Cambridge, MA). After reaching confluence, they were washed twice with Krebs-Henseleit solution and then incubated in 0.5 ml
of the same buffer containing isobutylmethylxanthine (IBMX) (1 mM) for
10 min at 32°C. Forskolin and testosterone were added to the wells
and incubated for a further 10 min. The reaction was terminated by
adding 10 µl of 60% (wt/vol) perchloric acid to each well. The
content of each well was mixed thoroughly and transferred to a 1.5-ml
microcentrifuge tube and was then centrifuged at 10,000 g
for 5 s. Supernatant (300 µl) was neutralized by KOH (1 M). The
mixture (100 µl) was taken and assayed for cAMP by an immunoassay
kit. The principle of the assay is based on the competition between
cAMP in the sample and a fixed amount of alkaline phosphatase-labeled
cAMP for binding to a specific antibody. The concentration of cAMP in
the sample is inversely proportional to the alkaline phosphatase activity.
Solutions.
Krebs-Henseleit solution had the following composition (in mM): 117 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4 · 7H2O, 2.56 CaCl2 · 2H2O, 24.8 NaHCO3,
and 11.1 glucose. This solution had a pH of 7.4 when bubbled with 95%
O2-5% CO2. In Cl
-free solution,
NaCl, KCl, and CaCl2 were replaced by sodium gluconate, potassium gluconate, and calcium gluconate, respectively. When HCO
-free solution was used, NaHCO3 was
replaced with NaCl, and the solution was buffered with 10 mM HEPES
at pH 7.4, gassed with 100% O2.
Materials.
MEM, fetal bovine serum, and nonessential amino acids were purchased
from GIBCO. Penicillin/streptomycin, HBSS, sodium pyruvate, trypsin,
collagenase I, forskolin, chlorophenylthio-cAMP,
aldosterone, estrogen, cyproterone acetate, flutamide,
cycloheximide, actinomycin D, pertussis toxin, IBMX, and indomethacin
were from Sigma. Dexamethasone was from Research Biochemicals
International. Testosterone and 5
-DHT were from Fluka. Matrigel was
purchased from Collaborative Biochemical (Bedford, MA). The immunoassay
kit for cAMP measurement was bought from R & B Systems
(Minneapolis, MN). Steroids were dissolved in 95% ethanol. It was
found that solvent alone did not affect the Isc.
Statistical analysis.
Results are expressed as means ± SE. Comparisons between groups
of data were made by the Student's unpaired t-test.
P < 0.05 was considered statistically significant.
 |
RESULTS |
Formation of polarized epithelia on permeable supports.
Light microscopic study showed that efferent duct epithelial cells
grown on Millipore filters formed confluent monolayers 5 days in
culture (Fig. 1). Epithelial polarity was
established with stereocilia appearing on the apical side of the cells.

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Fig. 1.
Light microscopic photograph of a confluent rat efferent
duct epithelial monolayer grown on Millipore filter 5 days in culture.
Arrows, cells with large nuclei and stereocilia at the apical border;
f, Millipore filter.
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|
Basal and forskolin-stimulated Isc in rat efferent duct
epithelia.
When bathed in normal Krebs-Henseleit solution, cultured efferent duct
epithelia exhibited a transepithelial potential difference of 0.7 ± 0.03 mV (n = 189 epithelia), a basal
Isc of 7.10 ± 0.4 µA/cm2
(n = 189), and a transepithelial resistance of
157.4 ± 6.3
· cm2 (n = 189), when calculated from transient current changes elicited by intermittent voltage pulses.
Forskolin (10 µM), an adenylate cyclase activator, added to the
basolateral side caused a rise in Isc that
reached a peak level after 3 min, +Isc = 6 ± 0.3 µA/cm2 (n = 147 epithelia).
The current then stabilized at a lower level, +Isc = 3.5 ± 0.2 µA/cm2
(n = 147) after about 15 min (Fig.
2). Experiments with ion transport inhibitors were carried out to investigate the ionic basis of the
forskolin-induced Isc. At the plateau phase of
the response, addition to the apical side of amiloride and benzamil,
both of which are inhibitors of apical epithelial Na+
channels, and phloridzin, an inhibitor of the
Na+-glucose cotransporter, had no effect on
Isc. In contrast, diphenylamine-2-carboxylate (DPC), an anion channel blocker, resulted in dose-dependent
inhibition of Isc (Fig. 2A). The
results suggested that the major part of forskolin-induced
Isc was due to anion secretion. Figure
2B shows basolateral addition of bumetanide, an inhibitor of
Na+-K+-2Cl
symport, produced a
dose-dependent inhibition of the Isc. Addition of acetazolamide, a carbonic anhydrase inhibitor, SITS, an inhibitor of
the Cl
/HCO
exchanger, or amiloride, an
inhibitor of the Na+/H+ exchanger, produced
only a negligible inhibition of the Isc when compared with bumetanide.

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Fig. 2.
Effects of ion channel blockers and transport inhibitors
on the forskolin-induced short-circuit current
(Isc) in efferent duct epithelia: area = 0.2 cm2. A: results show % inhibition of
forskolin-induced Isc by different
concentrations of diphenylamine-2-carboxylate (DPC, ),
amiloride ( ), benzamil ( ), or
phloridzin ( ) added apically. B: results
show % inhibition of forskolin-induced Isc by
different concentrations of bumetanide ( ), amiloride
( ), acetazolamide ( ), or SITS
( ) added basolaterally. Each record is representative
of 4 different experiments.
|
|
Effect of steroid hormones on basal and forskolin-induced
Isc.
Different steroid hormones were screened for their effects on anion
secretion by the efferent duct epithelial cells. In each case,
Isc was stimulated by forskolin (10 µM), which
increases intracellular cAMP. At the plateau phase of the response,
addition of testosterone to the apical side inhibited
Isc within 10-20 s. The inhibition was dose
dependent, with the IC50 value at ~1 µM and maximal
inhibition at 10 µM testosterone (Fig.
3, inset). In contrast, only
20% of forskolin-induced Isc could be reduced by 10 µM of 5
-DHT (Fig.
4D). Testosterone and 5
-DHT
also inhibited the basal Isc by 37% and 11%,
respectively (results not shown). Aldosterone, dexamethasone, estrogen,
and dehydroisoandrosterone (a weak androgenic steroid) did not
affect the basal Isc (results not shown) nor the
forskolin-induced Isc (Fig. 4,
A-C and E). Basolateral
application of testosterone or 5
-DHT had no effect on
forskolin-induced Isc (results not shown).

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Fig. 3.
Effect of testosterone on forskolin-induced
Isc. Forskolin (10 µM) was added to the
basolateral side (bl) followed by testosterone (0.1, 1, and 10 µM)
added sequentially to the apical side (ap). The record is
representative of 5 different experiments. Horizontal line indicates
zero Isc. Inset:
concentration-inhibition curves for testosterone on forskolin-induced
Isc in rat efferent duct epithelia
( ), caput epididymal epithelia ( ), and
cauda epididymal epithelia ( ). Each point shows the
mean ± SE of 4 experiments.
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Fig. 4.
Effect of different steroids on forskolin-induced
Isc. In each case, forskolin (10 µM) was added
to the basolateral side to stimulate Isc
followed by estrogen (A), dexamethasone (B),
aldosterone (C), 5 -dihydrotestosterone (5 -DHT,
D), or dehydroisoandrosterone (E) added to the
apical bathing solution in increasing concentrations: 0.1, 1, 10, and
100 µM. Each record is representative of 4 different experiments.
Horizontal lines indicate zero Isc.
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|
Figure 3, inset, shows the degree of inhibition by
testosterone of the forskolin-induced anion secretion was dependent on the regions from which the epithelia were derived. In the efferent duct, the forskolin-induced Isc could be
completely abolished by apical application of 10 µM testosterone.
However, in epithelia derived from the rat caput or cauda epididymidis,
the same concentration of testosterone inhibited the
forskolin-induced Isc by only 20%.
To elucidate whether the low efficacy of 5
-DHT was due to the rapid
metabolism by 3
-hydroxysteroid dehydrogenase (3
-HSD), the effect
of indomethacin, a potent 3
-HSD inhibitor (17, 29), was
studied. As shown in Fig. 5, indomethacin
(100 µM) did not affect the inhibitory effect of 5
-DHT.

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Fig. 5.
Effect of a 3 -hydroxysteroid dehydrogenase (3 -HSD) inhibitor
on the inhibition of Isc by 5 -DHT. Monolayer
was pretreated with 100 µM indomethacin. After 15-20 min,
forskolin (10 µM) was added to the basolateral side to stimulate
Isc followed by 5 -DHT (0.1, 1, and 10 µM)
added apically (B). Control response to 5 -DHT is shown in
A. Each record is representative of 4 different experiments.
Horizontal lines indicate zero Isc.
|
|
Effect of testosterone on forskolin-induced Isc in
Cl
-free or HCO
-free solution.
Tissues were bathed in Cl
-free solution and stimulated
with forskolin. At the steady state of the response, testosterone added apically had no effect on the current (Fig.
6B). However, in the absence
of HCO
, apical addition of testosterone (10 µM)
completely inhibited the forskolin-induced Isc
(Fig. 6C).

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Fig. 6.
Effects of testosterone in normal and in modified
Krebs-Henseleit solution. In each case, forskolin was added to the
basolateral side to stimulate Isc, followed by
sequential addition of testosterone in increasing concentrations: 0.1, 1, and 10 µM to the apical bathing solution. Tissue was bathed in
normal Krebs-Henseleit solution (KHS, A),
Cl -free solution (B), and
HCO -free solution (C). Each record is
representative of 4 different experiments. Horizontal lines indicate
zero Isc.
|
|
Effects of testosterone receptor antagonists, protein synthesis
inhibitors, and pertussin toxin.
To verify whether the inhibitory effect of testosterone was mediated
through the classic testosterone receptors, the effect of
anti-androgens were studied. Cyproterone acetate (100 µM), a
steroidal anti-androgen, or flutamide, a nonsteroidal anti-androgen (100 µM), did not affect the inhibition of Isc
by testosterone (Fig. 7, A and
B). Figure 8, A and
B, shows that pretreatment for 2 h with actinomycin D
(10 µg/ml) and cycloheximide (100 µg/ml), inhibitors of DNA
transcription and mRNA translation, respectively, were unable to
prevent the inhibitory effect of testosterone. To determine the
involvement of G protein in testosterone action, epithelia were
pretreated with pertussis toxin (100 ng/ml) for 6 h before
addition of testosterone. Figure 9 shows
that the concentration-inhibition curve for testosterone was shifted to
the right, with the apparent IC50 value shifted from 1 to
40 µM after pretreatment with pertussis toxin.

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Fig. 7.
Effects of anti-androgens on the inhibition of
Isc by testosterone. Results show % inhibition
of forskolin-induced Isc by different
concentrations of testosterone with ( ) or without
( ) preincubation with cyproterone acetate (100 µM,
A) and flutamide (100 µM, B). Each column shows
the mean ± SE of 4 experiments.
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Fig. 8.
Effects of protein synthesis inhibitors on the inhibition
of Isc by testosterone. Results show % inhibition of forskolin-induced Isc by different
concentrations of testosterone with ( ) or without
( ) pretreatment with actinomycin D (10 µM,
A) and cycloheximide (100 µM, B). Each column
shows the mean ± SE of 4 experiments.
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Fig. 9.
Effect of pertussis toxin on the inhibition of
Isc by testosterone: concentration-inhibition
curves for testosterone on forskolin-induced Isc
with ( ) or without ( ) pretreatment with
pertussis toxin (100 ng/ml). Each point shows the mean ± SE of 4 experiments.
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|
Effect of testosterone on intracellular cAMP.
Immunoassays were performed to study the effect of testosterone on
intracellular cAMP level in the efferent duct epithelium. Under basal
condition, the intracellular cAMP content was 45.8 ± 17.2 pmol/well (mean ± SE, n = 7). Stimulating the
tissues with forskolin (10 µM) led to a rise in intracellular cAMP
content to 140.7 ± 11.7 pmol/well (mean ± SE,
n = 4) (P < 0.01). Testosterone (10 µM) did not affect the basal intracellular cAMP but significantly inhibited the forskolin-induced rise of cAMP in a
concentration-dependent manner (Fig.
10).

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Fig. 10.
Effect of testosterone on intracellular cAMP content in
efferent duct epithelial cells in the absence or presence of forskolin
(10 µM). Each column shows the mean ± SE of 4-7
experiments. *P < 0.05 compared with
forskolin-stimulated control.
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|
 |
DISCUSSION |
The primary function of the efferent duct in vivo is to reabsorb a
major portion of fluid flowing down from the testis (7). However, previous work in our laboratory has shown that efferent duct
epithelial cells are also capable of secreting chloride
electrogenically in the presence of humoral agents that increase
intracellular cAMP (6, 23). The present studies with
forskolin, ion removal (Fig. 6), and transport inhibitors (Fig. 2)
confirm these results. Several steroid hormones were investigated for
their effects on anion secretion by the efferent duct. Among the
steroids tested, only testosterone and 5
-DHT inhibited the basal
(results not shown) and forskolin-stimulated Isc
(Figs. 3 and 4D). This inhibitory effect was dose dependent
(Fig. 3, inset) and highly specific for testosterone, as
dehydroisoandrosterone, a weak androgenic steroid, and other
nonandrogenic but structurally related steroids could not mimic the
effect of testosterone (Fig. 4). Although the effect of testosterone
was mostly seen when the agent was added to the apical side of the
epithelium, a basolateral action of the hormone could not be excluded.
Testosterone is highly lipophilic, and it is possible that the
Matrigel-coated Millipore filters may have prevented testosterone from
reaching the basolateral membranes of the cells. Ion replacement
experiments indicated that the fall in Isc after
testosterone was due to a decrease in chloride secretion, since the
inhibition was prevented by removal of chloride from the bathing
solution (Fig. 6).
In the epididymis, testosterone is converted by 5
-reductase to
5
-DHT, which has a greater affinity for the nuclear androgen receptors (14). However, in the present work, 5
-DHT was
found to be less effective than testosterone in inhibiting the
forskolin-induced anion secretion (Fig. 4D). The inability
of indomethacin, a 3
-HSD inhibitor, to augment the inhibition by
5
-DHT (Fig. 5) indicates that the low efficacy of 5
-DHT was not
due to rapid metabolism of 5
-DHT by 3
-HSD. It seems that a
testosterone-binding site other than the classic nuclear androgen
receptor is involved in the inhibition of secretion by testosterone. It
is of interest that testosterone has a greater effect on the efferent
duct than on the caput and cauda epididymidis (Fig. 3,
inset). This regional difference can be ascribed to a higher
5
-reductase activity in the epididymis to metabolize testosterone
(32, 33), or alternatively, it could be due to a regional
difference in the distribution of the nonclassic testosterone-binding site.
The effect of testosterone occurred within 10-20 s, too rapid to
have been attributed to the genomic action of steroid hormones. This
notion was supported by the lack of effect of actinomycin D, a DNA
transcription inhibitor, or cycloheximide, a mRNA translation inhibitor
(Fig. 8). Moreover, the inability of the classic nuclear testosterone
receptor antagonists, flutamide and cyproterone acetate, to block the
effect of testosterone (Fig. 7) further indicated that testosterone may
act on receptors that are distinct from the classic nuclear androgen
receptors. Preincubation of the efferent duct epithelium with pertussis
toxin, a Gi protein inhibitor, attenuated the effect of
testosterone (Fig. 9). Lieberherr and Grosse (24) found in
osteoblasts the rapid increase of intracellular calcium by androgens
was inhibited by pertussis toxin, suggesting that the effects of
androgens were mediated through a Gi protein-coupled membrane receptor. In the efferent duct, testosterone was found to
inhibit the forskolin-induced rise of intracellular cAMP (Fig. 10),
prompting speculation that testosterone acted on membrane receptors
negatively linked to adenylate cyclase via Gi protein, as
first proposed by Ravindra and Aronstam (30).
There is strong evidence that sex steroids are of central importance in
regulating fluid reabsorption in the efferent duct (15,
19). Using in vivo microperfusion technique, Hansen et al.
(15) demonstrated that in the rat, estrogen reduced fluid reabsorption substantially, causing a 2.5-fold increase in fluid flow
through the efferent duct. However, when fluid reabsorption was
measured in closed segments of the efferent duct isolated from the
estrogen receptor knockout mice (ERKO) in vitro, Hess et al.
(19) found no fluid reabsorption by the ducts. In
addition, in normal mice, the anti-estrogen, ICI-182,780, was found to
greatly reduce fluid reabsorption. Further investigations are required to resolve the controversy over the control of fluid reabsorption by
estrogen. In our experiments, we did not find any effect of estrogen on
chloride secretion by cultured rat efferent duct epithelia (Fig. 4).
Systemic administration of testosterone in the rat has been reported to
cause a small increase in fluid reabsorption in the efferent duct
(15). This effect could be due to an increase in sodium
reabsorption, as the Na+-K+-ATPase activity of
the efferent duct is known to be regulated by androgens
(21). In the renal proximal tubule, a tissue that resembles the efferent duct, expression of the brush-border
Na+/H+ exchanger was found to be increased by
testosterone (26). In addition to its effect on sodium
reabsorption, we propose that testosterone decreases electrogenic
chloride secretion via a rapid nongenomic effect. Little is known about
the physiological role of the local effect of testosterone in efferent
duct. Vreeburg (38) and Turner et al. (37)
reported that the rat seminiferous tubular fluid has a total
testosterone concentration of 82 and 62 nmol, respectively, falling
short of 100 nM free testosterone required to inhibit chloride
secretion in the efferent duct (Figs. 3 and 6A). It is
therefore not known whether the effect observed is of physiological
relevance. In certain diseases such as Leydig cell tumor, production of
testosterone may be greatly increased (10, 35). Most of
the men with such tumors have difficulty to father children because of
poor semen quality (low sperm count and poor sperm quality)
(3). In addition to the impairment of spermatogenesis
(20), high testicular testosterone might also inhibit
electrolyte transport in the efferent duct, and this could have
contributed to infertility in this case. The effect of
testosterone on efferent duct electrolyte transport may therefore have
a pathological role to play. It would be of interest to speculate a
synergism between the genomic and nongenomic actions of testosterone in
regulating fluid transport. In the efferent duct, we found testosterone
decreases intracellular cAMP, which, in the renal tubule, downregulates
the Na+/H+ exchanger (22) known to
be regulated by androgens (26). There is increasing
evidence that cAMP is able to reduce (11) or enhance (27) the expression of some androgen-dependent genes.
These findings therefore support a possible interaction between the genomic and nongenomic actions of testosterone and open up new areas of
investigation into the hormonal regulation of the male reproductive system.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Research Grants Council of Hong
Kong, the Chinese University of Hong Kong, and the International Consortium on Male Contraception, Population Council, New York.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. Y. D. Wong, Dept. of Physiology, Chinese Univ. of Hong Kong,
Shatin, NT, Hong Kong (E-mail: patrickwong{at}cuhk.edu.hk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 May 2000; accepted in final form 18 December 2000.
 |
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