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Center for Biomedical Research, The Population Council, New York, New York 10021
Address all correspondence and requests for reprints to: Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: hardy{at}popcbr.rockefeller.edu
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Abstract |
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-methyl-19-nortestosterone, 0.7 mg/kg BW·day). AR protein was
visualized by immunohistochemistry and measured by computer-assisted
image analysis in Leydig and Sertoli cells using frozen sections of
testes. After NalGlu treatment, AR levels in Leydig cells declined
sharply to 42% and 31% of vehicle control (P <
0.01) in the 21 and 35 days postpartum age groups, respectively, but in
90-day-old rats there was no change. AR levels were partially
maintained by exogenous LH, and completely maintained by exogenous
androgen treatments in Leydig cells from 21- and 35-day-old rats,
whereas in Leydig cells from 90-day-old rats, AR levels were unaffected
in all treatment groups. In contrast, after NalGlu treatment, the AR
concentration in Sertoli cells from 90-day-old rats were reduced to
32% of control (P < 0.01). Moreover, in Sertoli
cells from 90-day-old rats, AR levels were partially maintained by LH
and completely maintained by androgens. A similar trend was observed on
day 35. On day 21, however, AR levels in immature Sertoli cells were
unaffected in all treatment groups. These results indicate that
androgen maximally stimulates AR levels in immature Leydig cells but is
without significant effect in adult Leydig cells. In contrast, AR
levels in Sertoli cells are more sensitive to androgen regulation in
adult compared with immature animals. These findings indicate that
there are distinct mechanisms controlling AR concentrations in Leydig
and Sertoli cells during the development of the testis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The postnatal differentiation of Leydig cells is a continuous process that may be subdivided into three stages (progenitor, immature, adult) based on the structural and functional properties of differentiating Leydig cells (5). In the present study, androgen receptor (AR) levels were measured in Leydig cells of these three developmental stages: on day 21 postpartum, when Leydig cells exist as mesenchymal-like progenitors (6); on day 35 when they are still immature, producing low amounts of testosterone (7, 8, 9, 10); on day 90 when they are fully functional in the sexually mature rat (11, 12). LH is required throughout Leydig cell differentiation but is not the only stimulus involved in the conversion of Leydig cell progenitors into immature Leydig cells. Our previous studies showed that androgen in combination with LH stimulated testosterone production by Leydig cell progenitors in vitro (3, 13), and androgen stimulates messenger RNA (mRNA) levels for both AR and LH receptors within these cells in vivo (14). This suggests that differentiating Leydig cells possess AR and are sensitive to androgen action, a hypothesis supported by studies showing deficient steroidogenesis in Leydig cell from rats (15) and mice (16) with testicular feminization (androgen resistance).
Androgens are believed to exert their effects on spermatogenesis indirectly (17), primarily through Sertoli cells and peritubular myoid cells (18). Immunohistochemical and in situ hybridization studies have shown that AR is present in Sertoli cells, peritubular myoid cells (19, 20, 21, 22, 23) in seminiferous tubules, and possibly in germ cells (22).
In an earlier study of androgen action, testosterone replacement maintained levels of androgen receptor mRNA, assessed by Northern blot in Leydig cell progenitors purified from 21-day-old rats that were treated with an LHRH antagonist to suppress gonadal function (14). The aim of the present study was to examine the effects of androgen on AR levels in Leydig and Sertoli cells during puberty and adulthood, to determine whether sensitivity to androgen action is developmentally regulated. Using an antiserum that was raised against an AR synthetic peptide, AR protein was detected in the nuclei of testicular cells that are known to express AR but was not detected in germ cells. The findings suggest that Leydig and Sertoli cells maintain AR levels differently, which is consistent with contrasting roles for androgen in the regulation of these two cell types: facilitation of Leydig cell differentiation prepubertally and support of Sertoli cell function post-pubertally.
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Materials and Methods |
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-methyl-19-nortestosterone (MENT) was custom
synthesized. T or MENT was dissolved in cottonseed oil containing 5%
ethanol. Doses of T were selected to maintain testis weight in NalGlu
treated animals, based on the results of a previous study (14).
Animals and hormonal treatments
Male Sprague-Dawley rats, purchased from Charles River
Laboratories (Wilmington, MA), were randomly distributed into five
groups with n = 3 per group.
Group 1, control. Animals were treated with vehicle, receiving a daily ip injection of 8% mannitol and an sc daily injection of cottonseed oil containing 5% ethanol.
Group 2, LHRH antagonist. NalGlu was administrated to animals by daily ip injection at a dose of 0.3 mg/kg BW (24, 25) and also received the sc vehicle.
Group 3, NalGlu + LH. Animals received a daily ip injection of NalGlu and 0.2 mg LH/kg BW plus the sc vehicle. This dose of LH has been shown to fully stimulate Leydig cells in vivo (14).
Group 4, NalGlu + T. Animals received a daily NalGlu injection plus T (7.5 mg/kg BW) by daily sc injection.
Group 5, NalGlu + MENT. Animals received a daily injection of NalGlu plus MENT (0.7 mg/kg BW) by sc injection.
The treatments began on days 17, 31, and 86 postpartum, and the animals were killed by asphyxiation with CO2 on days 21, 35, and 90, respectively. The animal procedure was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (no. 91200R1).
Antibody against AR peptide
The rabbit polyclonal antibody against a synthetic AR N-terminal
peptide (residues 14–32) and preimmune serum were described previously
(26). The anti-AR peptide antibody recognizes the 110-kDa AR protein
from isolated Leydig cells, testis, and other tissues after
denaturation in SDS (4). This antibody also recognizes the insoluble,
denatured AR produced by AR complementary DNA transfected Sf9 insect
cells after solubilization in SDS (26), and the denatured AR in
formalin fixed tissue sections.
In the present study, a series of preliminary experiments were performed to examine the sensitivity and specificity of the antiserum in AR immunohistochemical detection. The dilution of antiserum was examined over a range from 1:200 to 1:2000; 1:500 was selected as the optimal concentration for AR immunohistochemical detection in that the background level in unlabeled cytoplasmic areas was low in proportion to specific localization in nuclei. Specificity of the antiserum was demonstrated by showing that preadsorption of the antibody against a 10-fold excess of the AR peptide reduced the staining to undetectable levels. The preimmune serum also did not produce a positive signal.
Immunohistochemical staining and image analysis
Tissues were immediately frozen in liquid nitrogen and stored at
-70 C. Eight micron-thick frozen sections were cut in a cryostat
(Hacker Instruments Inc., Fairfield, NJ). To avoid possible
interexperimental variation in staining intensity, the testicular
sections from all of the treatment groups at each age were mounted on
one slide for simultaneous immunohistochemical detection. The sections
were fixed in 10% neutral buffered formalin for 12 min at 25 C,
followed by washes in PBS (pH 7.4), and endogenous peroxidase activity
was quenched by incubation in 3% hydrogen peroxide in methanol for 10
min. After overnight incubation at 4 C with the primary antibody,
followed by 30 min in solutions of biotinylated secondary antibody and
horseradish peroxidase-streptavidin, respectively, the colored product
was developed by staining in a chromogen solution that contained
3-amino-9-ethyl-carbazole using a kit (Histostain-SP, catalog no. AEC
95–6143, Zymed Laboratories Inc., South San Francisco, CA). Sections
of spleen, prostate, and testis from a 35-day-old rat were mounted on
one slide and monitored in the microscope during incubation with the
chromogen, and a nuclear signal was apparent in prostate and testis
after 2 min but did not increase further after 6 min even in prostate
epithelium, which contained the highest nuclear AR concentrations.
Therefore, the incubation was stopped at 4 min.
Stained testicular section areas were first recorded at 100 times magnification using a Nikon Optiphot-2 microscope (Nikon, Inc., Melville, NY) equipped with a Dage MTI video camera (CCD 72, Michigan City, IN). The video images were then digitized using a frame grabber (Quick Capture, Data Translation, Inc., Marlboro, MA) and displayed on a Sun IPC work station (Mountain View, CA). The stained nuclear areas of labeled Leydig and Sertoli cells were traced. The integrated pixel intensity was determined for the traced areas using image analysis software (Image-Pro, Media Cybernetics, Silver Spring, MD). The intensities were normalized for possible changes in nuclear size by dividing the integrated pixel intensity by the nuclear area (which equaled the average number of pixels per unit of traced nuclear area). Intensities of the background were determined for each group by tracing an unlabeled area adjacent to the labeled cells. The background was subtracted from the values obtained for the labeled cells, and the adjusted values are referred to as the relative signal intensities (RSI) providing a measure of nuclear AR concentration.
Statistics
The treatment protocol was repeated three times: three
replications x three rats per group x five groups equalled
a total of 45 rats in each of the three age groups for the study. One
testis from one rat per group was randomly selected from each of the
three replications, and used for image analysis (on 15 rats per age
group). For each cell type, four cells were measured in a randomly
observed field and the four measurements were then averaged. This was
performed three times, resulting in three averaged measures per testis.
Three such averaged measures were obtained for each of three testes
from all five groups, to calculate the final mean ±
SE, with n = 9 RSI observations/group. The data were
analyzed by the Kruskal-Wallis analysis of variance (27). Significant
differences between groups were identified by the Tukey test using the
rank-transformed data (28). Differences were regarded as significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), in which AR is known to be
abundant (29). Testicular cells were identified on the basis on nuclear
shape and histological location a described in a previous study (5). In
the testicular interstitial areas, AR protein was detected in the
nuclei of spindle-shaped Leydig cell progenitors on day 21 (Fig. 1B),
immature Leydig cells on day 35 (Fig. 1C), and adult Leydig cells on
day 90 (Fig. 1D). The nuclear AR was also detected in pericytes
(vascular smooth muscle cells, 21, 22, 30), peritubular myoid cells
(Fig. 1C), and Sertoli cells but was not observed in germ cells (Fig. 1D). Spleen (Fig. 1F) had a negligible signal of AR, consistent with
its lack of AR protein (20, 31). No signal was detected in control
sections that were incubated with preimmune serum (prostate, Fig. 1E;
testis, Fig. 1G), or with preabsorbed antiserum (testis, Fig. 1H).
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. The frequency distributions of AR RSI were plotted
for 36 cells from each tissue. The RSI of prostate epithelial cells was
1.5 times higher than immature Leydig cells (mean ±
SE: 20.9 ± 1.0 vs. 13.9 ± 0.7,
respectively). In contrast, the RSI of spleen was 1.2 ± 0.1, a
value not different from 1, which would be expected if the nuclear
signal did not differ from the signal seen in the adjacent unlabeled
cytoplasm.
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, B–D), the AR signal intensities were measured
by image analysis after simultaneous immunohistochemical assay on a
single slide and the results are summarized in Table 1.
The RSI of AR was intermediate in Leydig cell progenitors on day 21 at
10.1 ± 0.3, highest in immature Leydig cells on day 35 at
15.4 ± 0.8, and lowest in adult Leydig cells on day 90 at
8.0 ± 0.7. The reverse trend was seen in Sertoli cells, where the
RSI of AR was lowest on day 21 at 7.8 ± 0.5, intermediate on day
35 at 8.9 ± 0.3, and highest on day 90 at 14.3 ± 1.1. The
above results indicate that, in Leydig cells, AR is highest during
puberty, whereas AR is highest in adult Sertoli cells at stages VII and
VIII on day 90.
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and Table 2 indicated that the nuclear AR signal was first evident
in Sertoli cells at stages IV and V at 8.5 ± 0.5, increased
progressively and reached its greatest intensity at 14.3 ± 1.1
during stages VII and VIII, then declined with barely detectable at
1.6 ± 0.2 during stages X and XI, and was negligible (0.8 ±
0.1) at stage XII. The AR staining was predominantly localized in the
basal region of the seminiferous epithelium at stages VII and VIII, the
staining was localized to the nuclei of cells that bordered the basal
lamina and had nuclear profiles extending between the large pachytene
spermatocytes (Fig. 3), consistent with localization in Sertoli
cells.
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) and
summarized in Table 3. On days 21 and 35, NalGlu
treatment reduced RSI of AR levels in progenitor (Fig. 4, A2) and
immature Leydig cells (Fig. 4, B2) to 42% and 31% of control,
respectively. LH (Fig. 4, A3 and B3) partially maintained AR, whereas
T- (Fig. 4, A4 and B4) and MENT- (Fig. 4, A5 and B5) treated rats had
complete maintenance in both 21 and 35 day age groups. On day 90,
however, the AR RSI in adult Leydig cells was not significantly
influenced by any of the hormonal treatments (Fig. 4, C1–C5).
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), and are summarized in
Table 4. Hormonal treatment had no effect on RSI of AR
levels in immature Sertoli cells on day 21. In contrast, on days 35
(Fig. 4, B2) and 90 (Fig. 4, C2), NalGlu treatment decreased AR RSI in
Sertoli cells to 41%, and 32% of control, respectively. LH (Fig. 4, B3, and C3) partially maintained AR RSI in both 35-and 90-day-old
animals, whereas, in T- and MENT-treated groups, the maintenance of AR
was complete (Fig. 4, B4 and C4; B5 and C5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional activity in Sertoli cells varies depending on the stage of the seminiferous epithelial cycle (35, 36). AR expression in Sertoli cells from 90-day-old rats peaked at stages VII and VIII, which mirrored the pattern of AR mRNA level examined by in situ hybridization (23), consistent with the highest AR protein concentration in isolated seminiferous tubules of stages VII and VIII (37). Interestingly, the stage-dependent expression of Sertoli cell AR occurs at a time when endogenous testosterone is thought to reach its maximal concentration in stages VII and VIII tubules (35). This suggests that the requirement for androgen action is maximal during stages VII and VIII (21), when androgen stimulation of protein secretion by seminiferous tubules peaks (38).
The hypothesis that Leydig and Sertoli cells respond differently to androgen regulation during development was tested by measuring the effects of androgen on AR levels in Leydig cell progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90 postpartum, with Sertoli cells comparatively analyzed at each of these ages. The LHRH antagonist, NalGlu, was used to suppress endogenous secretion of both LH and androgen (39, 40). Administration of LHRH antagonists reduces seminiferous tubule diameter in rats (41), and decreases cytoplasmic and nuclear areas of Leydig cells. These effects are caused by suppression of the circulating levels of LH to 2% of control, which in turn suppresses T production to less than 6% of control (24, 25). NalGlu treatment lowered AR levels in progenitor and immature Leydig cells, and in adult Sertoli cells. Treatment with NalGlu plus LH maintained AR levels in these cells, probably by stimulating endogenous biosynthesis of testosterone (42, 43).
The present results showed that treatment with NalGlu plus T or MENT
completely prevented the suppression of AR levels seen in rats treated
with NalGlu alone. Considering the difference in dose, MENT was more
effective than T, consistent with the higher potency of MENT in
restoring ventral prostate mass and maintaining sex behavior in
castrated rats and other species (44, 45). The affinity of MENT for AR
is higher compared with T (46), explaining its biological potency in
influencing Leydig and Sertoli cells at doses that are 10-fold lower
than testosterone (Tables 3 and 4). These results suggests that MENT
may act on testicular cells without 5-reduction as it does on
muscle, where this androgen also has a 10-fold greater effect with
respect to T (47).
Leydig cells, in contrast to Sertoli cells, were most sensitive to
androgen regulation in pubertal animals. Previous studies have shown
that Leydig cells are a target for the steroids they produce (3).
Androgen in combination with LH has been shown to increase testosterone
production by Leydig cell progenitors after 3 days in vitro
(3), and treatment with androgen in vivo increases
steady-state mRNA levels for AR and LH receptor in Leydig cell
progenitors (14). The higher levels of AR in immature Leydig cells and
the heightened sensitivity of these cells to androgen regulation
suggests that low level prepubertal production of androgen by Leydig
cell progenitors acts within these cells, stimulating their
differentiation into immature Leydig cells. This was consistent with
earlier studies showing that: AR levels were low in adult Leydig cells
(4) and were not influenced by androgen treatment. The results of the
present study indicate that the significance of androgen action for
Leydig cell function probably changes during development. In contrast
to progenitor and immature Leydig cells, adult Leydig cells are targets
of testosterone negative feedback inhibition of testosterone
biosynthesis, which is exerted primarily at the level of 3ß-HSD
activities (48) and P450–17 (2).
The highest RSI of AR immunoreactivity in adult Sertoli cells at stages VII and VIII reflects the critical role of androgen in spermatogenesis. According to a recent report (49), both T and DHT increase adult testis size and can completely maintain fertility in hpg mice (which are genetically deficient in LHRH), probably by maintaining intracellular calcium in Sertoli cells (50). The role of androgen by itself in maintaining testicular function may be unique to the adult rodent because FSH, in addition to androgen, is necessary for spermatogenesis in immature rodents and adult humans (51). The lower AR RSI and relative insensitivity to androgen regulation of immature compared with adult Sertoli cells imply that the former are less sensitive to androgen than the latter (52). AR expression was unchanged by androgen treatment of immature Sertoli cells and peritubular myoid cells isolated from 15-day-old rats (53), whereas, in adult rats, the total testicular amount of AR was reduced after deprivation of T caused by chemical destruction of Leydig cells (54).
In summary, the developmental trends for androgen regulation of AR levels in Leydig and Sertoli cells were defined. In Leydig cells on day 35, AR levels were highest and most stimulated by androgen. These findings support the hypothesis that androgen has an important role in Leydig cell differentiation. In Sertoli cells, androgen stimulated AR levels most effectively on day 90 which probably reflects the requirement for androgen in spermatogenesis.
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Acknowledgments |
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Footnotes |
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Received October 3, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstane-3,17ß-diol and
5-androstane-3ß,17ß-diol by rat testicular cells in
vitro. J Reprod Fertil 57:143–147-hydroxylase activity. J Endocrinol 131:443–449-methyl-19-nortestosterone, an androgen that
does not undergo 5-reduction. Biol Reprod 49:577–581[Abstract]
-methyl-19-nortestosterone by rat liver, prostate, and epididymis.
Endocrinology 123:2187–2193-methyl-19-nortestosterone
is not amplified in male reproductive tract as is that of testosterone.
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x
). The three distributions were
significantly different from one another at P <
0.001.
