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Department of Developmental Anatomy, University of California School of Medicine, San Francisco, California 94143-0738
Address all correspondence to: Dr. Barbara A. Foster, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: grcunha{at}itsa.ucsf.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-methyl-19-nortestosterone and methyltrienolone (R1881), which
cannot be 5-reduced to DHT, also induced extensive ductal branching
and elicited responses less than those to T and not statistically
different from those to DHT. This suggests that although DHT is
sufficient for prostatic development, it is not necessary for postnatal
ductal branching morphogenesis and growth of the prostate.
5-Androstan-3,17ß-diol was particularly potent in inducing
ductal branching, eliciting a response greater than or comparable to
those of T and DHT. Androsterone, androstanedione,
5-androstan-3ß,17ß-diol and 5ß-androstan-3,17ß-diol
induced ductal branching, but to a lesser extent than either T or DHT.
These studies challenge the assumption that DHT is essential for
prostatic development, specifically during ductal branching
morphogenesis of the neonatal rat prostate. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostatic development involves a coordinated sequence of events beginning with the undifferentiated embryonic urogenital sinus (UGS) and culminating in the attainment of full adult function. The embryonic UGS under the influence of androgens becomes committed to form prostate. The first morphological change indicative of prostatic development is the formation of prostatic buds at 17.5 days gestation in the mouse and 19 days gestation in the rat (1). Under the continued influence of androgens the prostatic buds elongate and undergo ductal branching morphogenesis and extensive growth (2, 3, 4). During pubertal growth the epithelium undergoes functional cytodifferentiation and expresses prostate-specific secretory proteins as growth and branching morphogenesis continue.
Testosterone (T) is the principle circulating androgen produced
by the testes and is readily taken up by the prostate. T is metabolized
in accessory sex tissues (5) including the prostate, where it is
converted to several androgenic metabolites, including the potent
androgen dihydrotestosterone (DHT) (6, 7, 8, 9, 10). DHT can be
metabolized by three enzymes into two pathways. One leads to a circular
pathway in which the products can be interconverted to reform DHT. All
of the reactions of this pathway are reversible, and all of the
intermediates have been reported to have androgenic properties. The
interconversion of DHT to 5-androstan-3,17ß-diol (3-diol) is
favored in the direction of DHT (11). It is believed that the
metabolites of this pathway exert their androgenic effect by conversion
to DHT (8, 12). This pathway is thought to be involved in the tight
local regulation of the availability of DHT in the prostate (12, 13).
Alternatively, DHT can be reduced to form
5-androstan-3ß,17ß-diol (3ß-diol), which is rapidly and
irreversibly hydroxylated at either the 6 or 7 position to form
6-triol or 7-triol (14, 15). The triol metabolites do not have
androgenic activity when tested by classical bioassays in rats (14).
This pathway is believed to be responsible for clearing androgenic
metabolites from the prostate.
It has been hypothesized that the effects of T are not due to the
activity of T alone, but to the summation of the effects of all of its
metabolites. The action of T is elicited through the local formation of
androgenic metabolites in target cells (16, 17). T is
metabolized to several compounds in the prostate, and thus, T is
thought to act as a prohormone in this tissue (17, 18). There has been
extensive research into the androgenic potency of T metabolites in the
prostate, with an emphasis on DHT, 3-diol, and 3ß-diol.
It is believed that formation of DHT is essential for the development
of the prostate, and that DHT is the active androgen in the developing
prostate and external genitalia (19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Prostatic development is
partially or completely inhibited when T is unable to be metabolized to
DHT, such as when the 5-reductase enzyme is defective or when an
inhibitor of 5-reductase is administered prenatally (21, 22, 23, 25, 27, 28, 29, 30). Human males with 5-reductase deficiency have a defect in
type II 5-reductase, such that T is inefficiently converted to DHT.
Affected males have feminized external genitalia and small or absent
prostates. Such findings have lead to the conclusion that
masculinization of the external genitalia and development of the
prostate are dependent on DHT (22, 26, 27, 31, 32). Supporting these
conclusions are the findings that treatment of pregnant rats with
5-reductase inhibitors elicits feminization of the external
genitalia and inhibits prostatic development (21, 25). However, such
prenatally treated male rats have normal reproductive capacity and
normal reproductive tract morphology in adulthood (33), suggesting that
acute inhibition of prostatic development by 5-reductase inhibitors
early in development can be overcome given a sufficient period of
recovery. Additionally, DHT restores prostatic development when given
to pregnant rats in combination with a 5-reductase inhibitor (21).
Further supporting evidence that DHT is the active androgen in the
prostate comes from studies that 5-reductase activity and DHT
formation in the UGS are detectable both before and during prostatic
development (19, 20).
Synthetic analogs of T that cannot be 5-reduced to form DHT have
been developed, e.g.
17ß-hydroxy-17-methyl-estra-4,9,11-trien-3-one (R1881 or
methyltrienolone) and 7-methyl-19-nortestosterone (MENT) (34, 35, 36).
Both bind to the androgen receptor (AR) with high affinity and elicit
androgenic responses in target organs (34, 35, 36, 37, 38, 39). MENT increases the
weights of the ventral prostate and seminal vesicle in castrated rats.
The activity of MENT is not blocked by 5-reductase inhibitors, but
is inhibited by antiandrogens that block binding to the AR (35, 37).
The androgenicity of non-5-reducible T analogs is thought to be due
to their binding to the AR. Androgenicity of synthetic androgens in the
prostate correlates with their binding affinity for the AR, indicating
that androgens do not necessarily have to be 5-reduced to be active
(40). The effects of these synthetic androgens on the developing
prostate are unknown. The experiments performed in this study examine
the effects of various androgens (natural and synthetic) on postnatal
prostatic development, as assayed by quantification of ductal branching
morphogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstan-3ß,
17-ß diol (3ß-diol) and 4-androstene-3,17-dione
(androstenedione) were obtained from Sigma Chemical Co.
(culture grade; St. Louis, MO). 5-Androstan-17ß-ol-3-one (DHT),
5-androstan-3, 17ß-diol (3-diol),
5ß-androstan-3,17ß-diol (5ß3-diol),
5-androstan-3,17-dione (androstanedione), and
5-androstan-3-ol-17-one (androsterone) were obtained from
Steraloids (Wilton, NH). Methyltrienolone (R1881, metribolone) was
obtained from Roussel-UCLAF (Romainville, France).
7-Methyl-19-nortestosterone (MENT) was obtained from The Population
Council (New York, NY).
Organ culture
Male Fisher 344 rat pups less than 12 h old (Simonsen Laboratories, Inc., Gilroy, CA) were used for organ culture
experiments. The rats were killed by decapitation. The reproductive
tracts with attached bladder were removed. Further dissection of the
tract was performed under a dissecting microscope in a watchmaker’s
depression slide using no. 5 biological forceps and a 27.5-gauge needle
as a scalpel. All dissections were performed in 50% DMEM H-16–50%
Ham’s F-12 medium. To isolate the AP, the seminal vesicle,
dorsolateral prostate, and AP complex was cut into right and left
halves through the midline. The vas deferens and dorsolateral prostate
were removed, and the AP was dissected from the seminal vesicle. Care
was taken during the dissection so that the APs placed in culture were
intact and undamaged. The AP cultures used for morphometric analysis
were unbranched at the start of culture.
Glands were cultured on floating Millicell-CM filters (Millipore Corp., Bedford, MA) in 1 ml culture media in four-well culture dishes. The culture medium contained 50% DMEM H-16–50% Ham’s F-12 medium with 1.401 g/liter glucose, 0.11 g/liter sodium pyruvate, 0.365 g/liter L-glutamine, linoleic acid, and 1.2 g/liter NaHCO3 (University of California-San Francisco Cell Culture Facility) and was supplemented with 50 µg/ml gentamicin, 10 µg/ml insulin (University of California-San Francisco Cell Culture Facility), and 10 µg/ml human holo-transferrin (Sigma Chemical Co.). Glands were transferred to floating filters (Millicell-CM 0.4-µm Culture Plate Insert) using forceps in a drop of medium. The drop of medium was approximately 4 times larger than the gland. The cultures were grown at 37 C in an atmosphere of 5% CO2 and 95% air. The medium was changed every 2–3 days. Under these conditions the glands grew two-dimensionally rather than three-dimensionally.
Androgens were dissolved in 100% ethanol, and 10-4 M stocks were stored at -20 C in the dark. Stocks were diluted in medium to the working concentration. Ethanol concentrations were never greater than 1% in the culture medium. Ethanol at 1% was tested and did not have an effect on the development of the prostate in culture.
Image analysis
Image analysis was performed on the glands using the procedures
of Alarid et al. (41). Whole-mount images of the glands were
captured and digitized using a Dage-MT1 CCD-72 TV camera interfaced
with a Macintosh Quadra 800 computer and processed with PRISM VIEW
software (Dapple, Sunnyvale, CA). PRISM VIEW software calculated
epithelial area and perimeter from computer-generated binary images of
individual glands. The morphological complexity of epithelial shape was
assessed using the node number parameter and the form factor parameter
of PRISM VIEW. Node number is a topological measurement whose value is
proportional to the complexity of ductal branching and indicates the
number of branch points. Form factor is an indication of the complexity
of the shape. A circle has a form factor of 1. As shape becomes more
complex, its form factor decreases. The form factor is calculated based
on the ratio of area to perimeter (form factor = 4
area/perimeter2). The inverse of the form factor was
calculated so that as morphological complexity of ductal branching
increased so did the values for inverse form factor.
Statistical analysis
Statistical analysis of the dose-response experiments was
performed using ANOVA, with differences determined using Fisher’s
protected least significance differences at a 95% confidence interval
using Abacus Concepts StatView 4.02 software (Abacus Concepts, Inc., Berkeley, CA). Each experiment contained at least three
glands per treatment, and all experiments were performed at least three
times.
Metabolism studies
The metabolism studies were essentially performed according to
the methods described by Tsuji et al. (42, 43). Briefly,
glands were cultured for 24 or 48 h with [3H]T,
[3H]DHT, or [3H]3-diol. Steroids were
extracted from the culture media with 1 ml ether-ethylacetate (9:1),
followed by 1 min of vortexing and 10 min of centrifugation at 1500
rpm. The supernate containing the steroids was evaporated using
nitrogen. The steroids were then brought up in 100 µl chloroform and
used for TLC. A carrier mixture of reference steroids was added to each
sample, such that 5 µg of each carrier were loaded per sample. Next,
the sample was spotted on silica gel plastic plates and developed in a
double ascent system of chloroform-methanol (98:2) at 4 C, followed by
benzene-ethyl acetate (13:1) at room temperature. The plates were
air-dried, sprayed with a glacial acetic acid-sulfuric
acid-anisaldehyde mixture (25:1:1), and heated to 80-90 C for 2–5
min. The steroids were marked, cut from the plate, and extracted with
100% ethanol, and radioactivity was counted using a scintillation
counter. The percent conversion was based on the total radioactivity
added to the cultures. Control cultures lacking glands were included to
determine the breakdown of androgens in the absence of tissue.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Between birth and 6 days of age, the
AP undergoes striking morphogenetic changes in situ. A
highly branched ductal tree developed after 6 days of culture with
androgens, mimicking the development of the gland in vivo.
As expected, epithelial ductal branching morphogenesis in
vitro was androgen dependent. APs cultured for 6 days in the
absence of androgens failed to undergo epithelial ductal branching
(Fig. 1B). T, DHT, and 3-diol were potent inducers of epithelial
branching in culture (Fig. 1, C–E). The synthetic androgens R1881 and
MENT induced extensive ductal branching (Fig. 1, F and G).
However, androsterone, androstanedione, 3ß-diol, and 5ß3-diol
were less effective at inducing ductal branching (Fig. 1, H–K). Thus,
dramatic morphogenetic changes can be elicited by androgens in newborn
rat APs during a 6-day culture period.
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Morphometric analysis
APs cultured in the absence of androgens. APs cultured for 6
days without androgens survived, but the epithelium remained as a
single unbranched duct (Fig. 1B
). Those glands that were completely
unbranched at the start of culture remained unbranched after 6 days of
culture in the absence of androgen (Fig. 1, A and B). However, glands
that were beginning to branch at the start of culture (one or two
branches) continued to branch for the first 24 h of culture even
when cultured without androgen. This is probably due to "playing
out" of the androgenic stimulus the gland received in
vivo. The more branching present at the start of culture, the more
ductal branching achieved in glands cultured without androgens. For
this reason, only unbranched APs were used for all of the experiments
reported here. Newborn rat APs had 0.186 ± 0.394 nodes/gland and
an inverse form factor of 2.6 ± 1.3. The number of nodes per
explant, inverse form factor, and epithelial perimeter of glands grown
in the absence of T were not different from those of glands freshly
dissected from 0-day-old rats. However, after 6 days of culture in the
absence of androgens, the epithelial area was 173% of that at the
start of culture. This increase in neonatal prostate growth in the
absence of androgens in vitro is in agreement with in
vivo studies demonstrating that the prostate continues to grow
even when neonates are castrated and treated with antiandrogens (2).
The increase in epithelial area in the absence of androgen was very
modest, increasing only 58,074 µm2. The increase
in epithelial area in culture in the absence of androgens could be due
in part to flattening and spreading of the gland in culture. Thus,
after 6 days of culture in the absence of the androgens, APs did not
undergo branching morphogenesis, but may have grown slightly. However,
the increase in epithelial area in culture in the absence of androgen
was limited compared with that in APs grown in the presence of
androgens either in vivo or in vitro.
APs cultured with T. APs cultured 6 days in the presence of T
underwent extensive ductal branching morphogenesis (Fig. 1C). Based
upon the analysis of 118 APs in 8 replicate experiments, an increase in
ductal branching, relative to that of glands grown in the absence of
androgen, was detectable as an increase in both node number and inverse
form factor. APs cultured for 6 days with T concentrations as low
as 10-10 M had 3.6 ± 2.9 nodes/explant
and an inverse form factor of 7.7 ± 5.1. The maximal response to
T was observed at 10-8 M with 8.9 ± 3.1
nodes/explant and an inverse form factor of 16.2 ± 8.0 (Fig. 2). Thus, T was a potent inducer of
prostatic development.
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) similar in extent to that induced by
T (Fig. 2). Based upon the analysis of 264 APs in 17 replicate
experiments, branching was initially detectable as an increase in both
node number and inverse form factor at DHT concentrations as low as
10-10 M, with 3.5 ± 2.3 nodes/explant
and an inverse form factor of 7.1 ± 3.3. The maximal response was
achieved with DHT at 10-8 M, with 7.6 ±
2.5 nodes/explant and an inverse form factor of 12.5 ± 4.9 (Fig. 2). The degrees of epithelial branching induced by T and DHT were
comparable for nodes per explant, epithelial area, and epithelial
perimeter (Fig. 2). Surprisingly, at the dose of DHT that elicited the
maximal response (10-8 M), the inverse form
factor for DHT was significantly less than that elicited by T at the
same concentration (P = 0.02). DHT induced 77.2% of
the response elicited by T as measured by inverse form factor. However,
DHT induced 85.4% of the response elicited by T as measured by node
number. Thus, T at 10-8 M induced greater
complexity of the epithelial shape as measured by the inverse of form
factor and a higher degree of ductal branching than DHT. Although DHT
is a potent androgen, the effect of T was equal to or greater than that
of DHT at all concentrations.
APs cultured with 3-diol. 3-Diol induced extensive
ductal branching morphogenesis (Fig. 1E) similar in extent to that
induced by DHT and T (Fig. 2). Based upon the analysis of 346 APs in 21
replicate experiments, branching was initially detectable by both node
number and inverse form factor at concentrations as low as
10-11 M (P = 0.0058 and
P = 0.0009, respectively), with 1.5 ± 1.6
nodes/explant and an inverse form factor of 4.9 ± 2.9. Maximal
ductal branching was induced at 10-7 M, with
8.6 ± 3.4 nodes/explant and an inverse form factor of 13.0
± 5.0 (Fig. 2). At the dose eliciting a maximal response, the nodes
per explant was 96.6% of that obtained using T at 10-8
M, and the inverse form factor was 80.2% of that obtained
using T at 10-8 M.
The degrees of ductal branching morphogenesis induced by T and
3-diol were comparable for node number, epithelial area, and
epithelial perimeter (Fig. 2). However, the number of nodes per explant
was slightly decreased with 3-diol at 10-8
M compared with T at the same concentration
(P = 0.009; Fig. 2A). Similarly, in cultures treated
with 3-diol (10-8 M), the inverse form
factor (P = 0.002; Fig. 2B) and epithelial perimeter
(P = 0.009; Fig. 2C) were consistently less than those
in explants treated with T (10-8 M).
Additionally, the inverse form factor of explants cultured with
3-diol (10-9 M) was slightly decreased
compared to that of explants cultured with T at the same concentration
(P = 0.023; Fig. 2B). T at 10-8 and
10-9 M was more effective than 3-diol at
the same concentrations. T induced a greater complexity of the
epithelial shape as measured by inverse form factor, indicating an
increase in ductal branching morphogenesis. However, the amount of
epithelial area induced by 3-diol was comparable to that induced by
T (Fig. 2D). Although 3-diol is a potent androgen, at higher
concentrations 3-diol did not elicit the same degree of prostatic
development as that elicited by T.
APs cultured with synthetic androgens. R1881 and MENT are both
synthetic androgens capable of binding to the AR, but cannot be
5-reduced to form DHT (34, 35, 36). Both R1881 and MENT induced
extensive ductal branching morphogenesis (Fig. 1, F and G). Stimulation
of ductal branching was initially detectable by both node number and
inverse form factor using R1881 at concentrations as low as
10-9 M, with 5.3 ± 1.9 nodes/explants
and an inverse form factor of 8.3 ± 2.2 (Fig. 3, A and B). The maximal response to
R1881 was observed at 10-8 M, with 6.3 ±
0.5 nodes/explants and an inverse form factor of 10.9 ± 4.4 (Fig. 3, A and B). At the dose eliciting maximal response, the number of
nodes per explant was 77.8% of that using T at 10-8
M and 82.9% of that using DHT at 10-8
M, whereas the inverse form factor was 67.3% of that using
T at 10-8 M and 87.2% of that using DHT at
10-8 M. Thus, at the dose eliciting
a maximal response, R1881 induced significantly fewer nodes per explant
than T (P = 0.032), but the result was not statistically
different from that obtained using DHT, and the inverse form factor
induced by R1881 was not statistically different from that induced by
either T or DHT.
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, A and B).
Maximal ductal branching was induced by MENT at a concentration of
10-9 M, with 6.143 ± 1.069 nodes/explant
and an inverse form factor of 10.409 ± 1.577 (Fig. 3, A and B).
At the dose eliciting a maximal response, the number of nodes per
explant was 68.5% of that obtained using T and 80.3% of that obtained
using DHT, and the inverse form factor was 64.2% of that obtained
using T and 83.2% of that obtained using DHT. Thus, at the dose
eliciting a maximal response, MENT induced significantly fewer nodes
per explant than T (P = 0.003), but was not
statistically different from DHT. Furthermore, at the dose eliciting a
maximal response, the inverse form factor induced by MENT was
significantly less than that induced by T (P = 0.049),
but was not statistically different from that induced by DHT. The
biological response, as measured by ductal branching, elicited by
either of these synthetic androgens was not statistically different
from that elicited by DHT and was slightly less than that elicited by
T.
APs cultured with androsterone, androstanedione, 3ß-diol, or
5ß3-diol. Androsterone, androstanedione, 3ß-diol, and
5ß3-diol were relatively ineffective in inducing ductal branching
morphogenesis compared with T (Fig. 1, H–K). At optimal
doses (10-7 M) the maximal number of nodes
per explant induced by these androgens was between
35.9–70.8% of the nodes per explant induced by T. With
androsterone at concentrations as low as 10-9
M, stimulation of ductal branching was initially
detectable, with 2.9 ± 1.2 nodes/explant and an inverse form
factor of 6.0 ± 1.4 (Fig. 4, A and
B). Maximal ductal branching was induced by androsterone at
10-7 M, with 6.3 ± 1.3 nodes/explant and
an inverse form factor of 10.1 ± 2.8 (Fig. 4, A and B). At the
dose eliciting a maximal response, the number of nodes per explant was
70.8% of that obtained using T at 10-8 M, and
the inverse form factor was 62.3% of that obtained using T at
10-8 M. At all concentrations,
androsterone was less effective than T. Androstanedione induced ductal
branching at concentrations as low as 10-8 M,
with 4.3± 2.6 nodes/explant and an inverse form factor of 6.4 ±
2.4 (Fig. 4, A and B). The maximal response of androstanedione
was achieved at 10-8 M. At the dose
eliciting a maximal response, the number of nodes per explant was
48.3% of that obtained using T at 10-8 M and
the inverse form factor was 39.5% of that obtained using T at
10-8 M. 3ß-Diol stimulated ductal
branching at concentrations as low as 10-9 M,
with 2.1 ± 2.0 nodes/explant and an inverse form factor of
5.2 ± 3.0 (Fig. 4, A and B). Maximal ductal branching was induced
by 3ß-diol at 10-8 M, with
3.9 ± 1.7 nodes/explant and an inverse form factor of 8.0 ±
3.2 (Fig. 4, A and B). At the dose eliciting a maximal response, the
number of nodes per explant was 43.8% of that obtained with T at
10-8 M, and the inverse form factor was 49.4%
of that obtained with T at 10-8 M.
5ß3-Diol was the least effective androgen tested (Fig. 1K). Ductal
branching was initially detectable using 5ß3-diol at a
concentration of 10-7 M, with 2.9 ± 1.8
nodes/explant and an inverse form factor of 6.2 ± 1.7 (Fig. 4, A
and B). 5ß3-Diol at 10-7 M was also the
concentration at which the maximal response was induced. At the dose
eliciting a maximal response, the number of nodes per explant was
32.6% of that obtained using T at 10-8 M, and
the inverse form factor was 38.3% of that obtained using T at
10-8 M. Thus, at all of the concentrations
tested, androstanedione, androsterone, 3ß-diol, and 5ß3-diol
were less effective than T at inducing ductal branching
morphogenesis.
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-diol in culture-diol to
other metabolites during culture. After culture of APs with
[3H]T at 10-8 M for 48 h,
34.9% of T remained unmetabolized, whereas 52.6% was metabolized to
DHT, 5.7% was metabolized to androstanedione, and 3.5% was
metabolized to 3-diol. Thus, T was extensively metabolized to DHT by
the developing rat AP during culture. After 24 h of culture with
[3H]DHT (10-7, 10-9, and
10-11 M), 82.5–89.0% of the DHT remained
unmetabolized, whereas 6.7–11.7% was metabolized to 3-diol,
1.7–2.3% was metabolized to 3ß-diol, 1.0–2.4% was metabolized to
androstanedione, and 1.2–1.8% was metabolized to androsterone. Thus,
DHT was only slightly metabolized to other androgens by the developing
rat AP during culture. After 24 h of culture with
[3H]3-diol (10-7, 10-9, and
10-11 M), 63.4–70.8% remained unmetabolized,
whereas 18.9–25.1% was metabolized to DHT, 4.1–10.5% was
metabolized to 3ß-diol, 1.2–1.7% was metabolized to androsterone,
and 0.2–1.5% was metabolized to androstanedione. Thus, only about
20% of 3-diol is metabolized back to DHT in the developing rat AP
during culture. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on 25 separate dose-response experiments used in the current study with 294 APs, DHT was clearly no more effective than T in inducing ductal branching morphogenesis in the developing prostate, as quantitated by 4 parameters of ductal growth and branching morphogenesis. The effectiveness of T and DHT in inducing ductal branching was determined using a serum-free organ culture system in which systemic effects were eliminated. In organ culture the 0 day prostate recapitulates the normal androgen-dependent process of development as observed in vivo. In the presence of androgens the epithelium grows into and fills the mesenchymal compartment during the 6 days of growth either in vivo or in vitro.
Epithelial area, epithelial perimeter, node number, and inverse form factor were equivalent when T or DHT was used at 10-10 M, a suboptimal concentration for both hormones. At higher concentrations (10-9–10-7 M) T was more effective than DHT in inducing prostatic ductal branching, as indicated by an increased number of nodes and inverse form factor. The increased maximal response observed with T vs. DHT may be explained by the ability of T (but not DHT) to be metabolized to estradiol. In utero exposure to extremely low levels of estradiol induced an increase in the number of buds and prostatic enlargement postnatally, but did not change the epithelial area (44). This is in agreement with our in vitro observations that at high concentrations T (10-8 M) induced more nodes and a greater inverse form factor, but did not increase the epithelial area. Therefore, the greater response observed with T may be due in part to the ability of T to be metabolized to estradiol.
Our results appear to contradict the conclusions of studies by George
and Peterson (21) and Iguchi et al. (33),
which demonstrated that treatment of pregnant rats with
5-reductase inhibitors impaired prostatic development, thus
implying that the elevated circulating levels of T were not sufficient
to induce prostatic development. It is important to recognize that
these former studies initiated during embryonic periods deal with a
multitude of phenomena not applicable to our study (determination of
prostatic development, induction of prostate buds, elongation of
prostate buds, epithelial growth, and ductal branching morphogenesis).
Our study focuses exclusively on epithelial growth and ductal
branching, and thus is not strictly comparable to these earlier
reports. It is possible that DHT may activate a different set of genes
than T in the undifferentiated embryonic UGS, which is responsible for
initiation of prostatic bud development. The presence or absence of DHT
or T at the initiation of embryonic prostatic development may
profoundly affect all subsequent steps in prostate development.
DHT is believed to be the active androgen in prostatic development and
has been suggested to act by amplifying the activity of T by as much as
10-fold, because DHT has a higher relative affinity for and forms a
more stable complex with the AR (45, 46, 47). However, in the studies
presented here DHT was less effective than T in inducing ductal
branching. Similarities in the dose-response curves for DHT and T
indicate that the effect of T was probably not due simply to the
conversion of T to DHT. Given the presumed 10-fold higher efficacy of
DHT over T, the dose-response curves for T and DHT, particularly at the
lower concentrations, should have been different, yet the dose-response
curves of T and DHT were virtually superimposable (Fig. 2). Thus, the
degree of ductal branching morphogenesis induced by T cannot be
accounted for simply by the conversion of T to DHT.
The supremacy of DHT in prostate development is further questioned by
the ability of synthetic androgens, MENT and R1881, to support ductal
branching morphogenesis of the 0-day-old rat prostate. MENT and R1881
cannot be 5-reduced to DHT (34, 35, 36). However, both of these
compounds were potent inducers of ductal branching in the developing
prostate and elicited a response similar to that elicited by T and not
statistically different from that elicited by DHT. This is the first
set of experiments to show that these synthetic androgens are able to
induce androgen-dependent prostatic development. Thus, although T can
be metabolized to form potent inducers of prostatic development, such
as DHT and 3-diol, the experiments with MENT and R1881 indicate that
DHT is not required for ductal branching morphogenesis or growth of the
rat neonatal prostate.
The biological system used in this study to assay the potencies of the androgens (epithelial growth and ductal branching morphogenesis) is very complex and has a certain degree of variability in its end point. It is possible that the variation within the system could mask slight differences in the potencies of the androgens. Thus, slight differences between T and DHT would not have been detected. However, the expected 7- to 10-fold difference in activity between T and DHT (46, 47, 48) would have been detectable. The data presented here, based upon the largest dataset examined to date, indicate that T and T-like molecules are just as effective as DHT at inducing ductal branching morphogenesis in the developing rat AP.
With regard to the relative potencies of T vs. DHT,
Lasnitzki and Mizuno (49) cultured rat urogenital sinuses (14.5–19.5
days gestation) with either T or DHT in serum-containing medium. The
researchers did not perform dose-response experiments comparing the
effectiveness of T vs. DHT. However, the effectiveness of T
vs. DHT to induce prostatic bud formation in urogenital
sinuses from 14.5- to 19.5-day-old fetal rats was compared at the same
pharmacological concentration (1.5 µg/ml = 5 x
10-6 M). DHT was more effective than T at the
earliest time points. At 14.5 days gestation, DHT induced 2.9 prostatic
buds/UGS, whereas T was unable to induce prostatic buds. Furthermore,
at 15.5 and 16.5 days gestation, DHT was more effective than T in
inducing prostatic buds. However, at 17.5 days gestation, T and DHT
were equivalent in their ability to induce prostatic buds in the UGS.
The researchers interpreted the difference between T and DHT in the
15.5- and 16.5-day-old UGS as indicating that DHT is the active
androgen that masculinizes the UGS. The authors further suggest that at
14.5 days of development T was not able to elicit prostatic bud
formation because of the low content or absence of 5-reductase at
this stage and suggest the 5-reductase activity is acquired in the
period between 14 and 15 days gestation (49). The exact timing of
5-reductase activity in the developing rat UGS has not been
determined. The studies by Lasnitzki and Mizuno suggest that DHT may be
the active androgen in initial prostatic bud formation. By 17.5 days
gestation, at which time the UGS is known to have 5-reductase
activity (19, 50), T and DHT are equally effective at inducing
prostatic bud formation. Further studies are needed to determine the
dose-response effectiveness of T and DHT both in the commitment of the
UGS into the prostatic pathway of differentiation and in initial
prostatic bud formation of the UGS, especially as the dose used by
Lasnitzki and Mizuno was in the pharmacological to toxic range.
After birth, circulating levels of androgens rapidly decline to their
postnatal nadirs. Androgen levels rise at puberty and remain high
through adulthood (51). It has been hypothesized that 5-reductase
activity is required for prostatic development because the low
circulating levels of T at birth are inadequate to drive prostatic
development (21, 46, 48, 52). Local formation of DHT may serve to
amplify the androgenic signal by enhancing the interaction between
hormone and AR. DHT may be required to induce prostatic development in
the embryonic UGS because of the low circulating levels of T present
during the time of prostatic bud formation and subsequent ductal
branching morphogenesis (45, 46). However, the circulating level of T
in the plasma of newborn rats is about 0.3 ng/ml, which is
approximately 10-9 M T, and increases to about
3 ng/ml in adulthood (10-8 M T) (51). In the
studies presented here the dose-response curve for T-induced ductal
branching of the 0 day rat AP indicates that the circulating level of T
in the newborn rat is more than sufficient to induce ductal branching
morphogenesis in the developing prostate. Furthermore, at low
concentrations, T was just as effective in inducing branching
morphogenesis as DHT. Thus, the data presented here do not support the
hypothesis that formation of DHT is required to enhance the activity of
T during neonatal prostatic development. However, in vivo
the release of hormones, including androgens, is pulsatile. In the
studies presented here the developing prostate was grown in the
continuous presence of androgens. Therefore, we cannot rule out the
possibility that in vivo the pulsatile release of androgens
may influence the efficacies of the various androgens.
In the studies presented here 3-diol was also a potent inducer of
prostatic ductal branching morphogenesis in vitro. This is
in agreement with others who have reported that 3-diol is a potent
androgen. After castration, 3-diol restores male sexual behavior and
increases ventral prostate and seminal vesicle weights (5, 53).
3-diol is a potent inducer of benign prostatic hyperplasia in
dogs (5, 54, 55, 56) and is believed to work by backconversion to DHT (13, 57). If 3-diol was eliciting its effect solely through
backconversion to DHT, an equivalent efficacy of DHT and 3-diol
would require a nearly 100% backconversion of 3-diol to DHT. Our
metabolism studies indicate that approximately 22% of 3-diol was
converted to DHT, and the dose-response curves for 3-diol and DHT
were almost identical. The data presented herein are not consistent
with the hypothesis that the androgenic activity of 3-diol is due
solely to the backconversion of 3-diol to DHT.
Androsterone, androstanedione, and 3ß-diol are also able to be
converted to DHT (5). In the studies presented here these androgens
were able to induce ductal branching morphogenesis, but to a lesser
degree than either T or DHT. This may be due to the lower affinity of
these compounds for the AR (5, 40). Androsterone, androstanedione, and
3ß-diol were not as potent at inducing ductal branching as 3-diol,
which also has a low affinity for the AR. This was probably due to the
fact that androsterone, androstanedione, and 3ß-diol are not as
rapidly converted to DHT as 3-diol (14, 15). 5ß,3-Diol was the
least effective in eliciting ductal branching in the developing
prostate, and this is consistent with 5ß,3-diol’s low affinity
for the AR as well as its inability to be converted to the more potent
androgen, DHT (5).
The studies presented herein quantitated ductal branching morphogenesis
in the AP of the newborn rat. To date, this is the most extensive and
detailed study of the effectiveness of various natural and synthetic
androgens on postnatal development of the prostate. Nine androgens were
tested using 808 newborn rat APs in 68 dose-response experiments. Based
on these studies, T is just as effective as DHT in supporting ductal
branching in the developing neonatal rat AP. Furthermore, the activity
of T could not be accounted for simply by metabolism of T to DHT. The
synthetic androgens, MENT and R1881, which cannot be 5-reduced to
DHT, also induced extensive ductal branching and elicited responses
similar to those to T and not statistically different from those to
DHT. This suggests that although DHT is sufficient for prostatic
development, it is not necessary for postnatal branching morphogenesis
and growth of the prostate. 3-Diol was particularly potent in
inducing ductal branching morphogenesis, eliciting a response
comparable to those to T and DHT. Androsterone, androstanedione,
3-diol, and 5ß3-diol induced ductal branching, but to a lesser
extent than either T or DHT. These studies challenge the assumption
that DHT is necessary for prostatic growth and ductal branching
morphogenesis in the neonatal prostate.
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Acknowledgments |
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Footnotes |
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1 This work was supported by NIH Grants DK-45861, DK-52708, CA-64872,
and CA-59831. 
Received March 17, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), DHT (
), and 3
) at 10-x
M concentrations and in the absence of androgen (
; w/o;
Table 1
), androstanedione (X), 3ß-diol (