BIOLOGY OF REPRODUCTION 48, 248-251 (1993)
DNA Synthesis Occurs throughout the Rat Ventral Prostate during
Its Postnatal Development'
PARTHA P. BANERJEE, 2 SUBHADRA BANERJEE, and BARRY R. ZIRKIN
Division of Reproductive Biology, Department of Population Dynamics, The Johns Hopkins University
School of Hygiene and Public Health, Baltimore, Maryland 21205
ABSTRACT
We previously used autoradiographic methods to show that cells proliferate throughout the rat ventral prostate during its
postnatal development (Biol Reprod 1991; 45:773-782). The objective of the present study was to quantify the extent to which
DNA synthesis, and thus growth, occur in the distal and proximal segments of the rat ventral prostate. To this end, rats were
administered 3H-thymidine in vivo. Subsequently, 3H-thymidine incorporation per prostatic segment and per g DNA was determined, as was the number of labeled cells in the proximal and distal segments. We report that 'H-thymidine was incorporated
into both the distal and proximal segments of the ventral prostate of rats 10, 20, 45, and 60 days of age, with significantly greater
amounts (6-21 times) incorporated into the distal segment at each age. Consistent with this, the distal segment contained 520 times more labeled cells than the proximal segment. However, when 3 H-thymidine incorporation was expressed per FLg DNA,
only relatively small, though significant, differences were seen between the distal and proximal segments of Days 10 and 20; and
no differences were seen at Days 45 and 60. Taken together, these results strongly support the contention that DNA synthesis
occurs throughout the rat ventral prostate, and suggests therefore that prostatic growth is not limited to the distal tips of the
gland.
INTRODUCTION
two segments to determine the extent to which growth occurs throughout the ventral prostate.
Recent studies have shown that there is regional variation in cell type [1-3], responsiveness to androgens [4, 5],
gene expression [6], protein synthesis [7, 8], and DNA synthetic activity [2, 9, 10] in the rodent ventral prostate. With
respect to the latter, a number of publications have reported that prostatic ducts of the mouse and rat ventral
prostate grow primarily, if not exclusively, at their distal
tips [9, 10]. This important conclusion was based largely upon
the results of whole-mount autoradiographic studies [9, 10]
of microdissected prostatic ducts that had been incubated
with 14C-thymidine, and upon analyses of mitotic figures [8]
in sections of prostatic ducts.
In contrast, we recently reported that cells synthesizing
DNA occur throughout the ventral prostate of rats of 1060 days of age [11]. In our study, 3 H-thymidine was administered in vivo, and autoradiographic analyses of sections
of the distal, intermediate, and proximal segments of the
ventral prostate were performed. We reported that at all
ages studied, both epithelial and stromal cells were labeled
in all segments of the prostate.
In the present study, biochemical methods were used to
quantify 3 H-thymidine incorporation into the distal and
proximal segments of the ventral prostate of rats 10-60 days
of age. Our objective was to quantify DNA synthesis in the
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats of ages 8, 18, 43, and 58 days
were purchased. They were housed for 2 days in large plastic cages (4 rats per cage), in an air-conditioned room, with
lights-on from 0700 to 1900 h.
Quantification of DNA Synthesis in Ventral
ProstateSegments
Rats (n = 3-6) from each age group were injected i.p.
(1 Ci/g body weight) with 3 H-thymidine (sp. act. 80 Ci/
mmol; New England Nuclear, Boston, MA). After 1 h, rats
were killed, and the prostate gland was removed and microdissected into distal and proximal segments [11]. The
segments were separately homogenized in cold TE buffer
(10 mM Tris, 0.1 mM EDTA, pH 7.5). Ten-microliter aliquots
of tissue homogenate were spotted onto Whatman No. 1
filter paper (Whatman, Clifton, NJ) saturated with 0.5 M dithiothreitol (DTrF) and dried. The filters were then placed
in 10% trichloroacetic acid (TCA) for 10 min at room temperature, and washed three times (10 min per wash) with
3% TCA/1% inorganic pyrophosphate. During the last wash,
the solution was brought to boiling and then cooled. The
filters were further washed in absolute ethanol, air-dried,
and counted in a Beckman scintillation counter (Beckman
Instr., Palo Alto, CA). The results were expressed as counts
per minute (cpm) per segment or per ,ug DNA per segment. To obtain Rg DNA per segment, 10 pul of tissue ho-
Accepted September 7, 1992.
Received May 27, 1992.
'This research was supported by NIH grant DK19300 and by Hopkins Population
Center grant HD06268.
2
Correspondence: Dr. Partha P. Banerjee, Department of Population Dynamics,
Division of Reproductive Biology, The Johns Hopkins University, School of Hygiene
and Public Health, 615 North Wolfe Street, Baltimore, MD 21205. FAX: (410) 9550792.
248
249
DNA SYNTHESIS IN RAT VENTRAL PROSTATE
mogenate was diluted with DNA assay buffer (0.05 M NaPO 4,
0.002 M EDTA, 2 M NaCI, pH 7.4), and the DNA content was
measured by fluorometry according to the method of Labarca and Paigen [12].
Quantification of Cell Number in Ventral
ProstateSegments
Rats 10, 20, 45, and 60 days of age (5-10 rats per group)
were anesthetized with ether and then killed by cervical
dislocation. After the ventral lobe of the prostate was carefully dissected out with minimal damage to the stromal tissue, the distal and proximal segments were separated from
each other with microdissection tools [10], weighed, and
frozen at -70 0C until use. Frozen segments subsequently
were thawed and homogenized with chilled DNA assay buffer
through use of a Polytron homogenizer (Polysciences, Inc.,
Warrington, PA) at medium speed. Fifty microliters of tissue
homogenate was diluted 10-fold with DNA assay buffer and
the DNA content was measured by a fluorometric method
[12], using a DNA mini-fluorometer (TKO-100; Hoefer Scientific Instruments, San Francisco, CA) with Hoechst 33258
dye and calf thymus DNA as the standard. The total amount
of DNA per segment was determined from the total volume
of tissue homogenate and the dilution factor.
The total number of cells was determined by dividing
DNA per segment by the amount of DNA per cell. To quantify DNA per cell, ventral prostate cells were dissociated by
a modification of the method of Montpetit and Tenniswood
[13]. In brief, the ventral prostate was finely minced (1-2
mm 3) and rinsed 3-5 times with ice-cold Hanks' balanced
salt solution (HBSS; Gibco, Grand Island, NY). The tissue
was then dissociated in 10 ml of 1% collagenase (Gibco)
and 1% trypsin (Gibco) in Ca2+- and Mg2 +-free HBSS at 37 °C
for 30 min, using a water bath with continuous shaking at
120 cycles per min. After removal of supernatant, 10 ml of
1% collagenase solution containing 1% normal horse serum
was added to the tissue pellets, and incubations were continued for 30 min. Cells were then collected by centrifugation at 600 x g for 10 min. Cell pellets were diluted with
1 ml of HBSS, and 100 R1l of 0.4% DNase-I (BoehringerMannheim, Mannheim, Germany) was added to eliminate
fragmented DNA. After incubation for 5 min with DNase-I
at 4°C, the cells were washed, collected at 1000 x g, suspended in 1 ml of HBSS, and counted with a hemocytometer. To quantify DNA per cell, 500 p.l of cell suspension
was homogenized via a Polytron homogenizer at medium
speed for 1 min. Cell homogenates were diluted with DNA
assay buffer (see above), and the DNA content was measured directly by fluorometry [12]. The amount of DNA
present per prostatic cell was determined by dividing the
total amount of DNA by the total number of cells in a 1-ml
cell suspension. The amount of DNA per cell was found to
be 8.21 ± 1.43 pg, a value similar to a previously published
value of 8.7 + 1.2 pg/cell [14].
30000
*
o0
25000
oS
o
0
20000
Distal
* Proximal
E
rr.
15000
oa
10000
O.
a5000
0
10
20
45
60
Age (Days)
3
FIG. 1. H-Thymidine incorporation (cpm/prostatic segment) in the distal
and proximal segments of 10-60-day-old rat ventral prostate. Bars represent mean
SEM.
Statistical Analyses
All group data are presented as the mean - standard
error of the mean. Student's t-test was used to detect significant differences between the groups [13]. The level of
significance selected was p < 0.05.
RESULTS
The incorporation of 3H-thymidine into newly synthesized DNA of the distal and proximal segments of the ventral prostate of 10-60-day-old rats is shown in Figure 1. At
all ages, DNA synthesis occurred in both segments of the
prostate. Figure 1 shows that incorporation of 3 H-thymidine
into DNA was significantly higher in the distal than in the
proximal segment of prostates of 10-60-day-old rats;
3
H-thymidine incorporation was 6 to 21 times greater in the
distal than in the proximal segment, depending upon the
age of the rat.
Table 1 shows the total number of cells (epithelial plus
stromal) in the distal and proximal segments of the ventral
prostate of rats 10-60 days of age. At each age, significantly
greater numbers of cells were seen in the distal than in the
proximal segments, ranging from 3.5-fold greater at Day 10
to 18.5-fold greater at Day 60.
Table 2 shows the percent labeled cells (epithelial plus
stromal) in the segments of the ventral prostate of 10-60TABLE 1. Number of cells in 10-60 day-old rat ventral prostatic
segments.
Number of cells at different ages (x 10-6)
Prostatic segment
Distal
Proxima
10
20
45
60
8.1 - 0.7
12.3 ± 0.4
25.0 - 1.5
4.3 ± 0.3
100.0 -+4.6
7.5 - 1.3
150.0 +±21.0
8.1
0.4
250
BANERJEE ET AL.
TABLE 2. Percent labeled cells in 10-60-day-old rat ventral prostatic
segments. a
Age
(days)
500
-
Prostate segments
10
20
45
60
Distal
Proximal
12.2
0.8
4.0 0.6
3.0 - .3
2.6 0.3
8.1 + 0.9
2.7
0.4
4.6 0.6
2.0
0.3
*
E
400 -
Distal
Proximal
z
300 -
aThis table was derived from Banerjee et al., Fig 13 [11].
a
200-
o
day-old rats. These data were derived from our previous
quantitative analyses of autoradiographs of ventral prostate
sections after in vivo administration of 3 H-thymidine [11].
In rats 10 days of age, the percentage of labeled cells in
the distal segment of the prostate was significantly higher
than in the proximal segment. On each of Days 20, 45, and
60, the percentages of labeled cells in the distal and proximal segments were similar. It should be noted that at each
age, labeling was seen in both segments of the prostate.
Figure 2 shows the total number of labeled cells in the
distal and proximal segments of Day 10-60 rats. The values
depicted in this figure were derived by multiplying the
number of cells per segment (Table 1) by the percentage
of labeled cells per segment (Table 2). At Day 10, the number of labeled cells in the distal segment (10 x 105) was 5
times greater than the number in the proximal segment
(1.9 x 105). At Day 20, labeled cells in the distal (9.9 x
105) segment were 9 times greater in number than in the
proximal segment (1.1 x 105). At Day 45, the number of
labeled cells (32 x 105) was 10 times greater in the distal
segment; and at Day 60, the number of labeled cells in the
distal segment (40 x 105) was 21 times greater than that in
the proximal.
50 -
*
·
Io
X
Distal
Proximal
T
40 -
0
o
30 -
0
J0
.0
20-
o
10-
E
z
o-
I-
10
L
20
45
Age (Days)
FIG. 2. Numbers of labeled cells (epithelial plus stromal) in the distal
and proximal segments of 10-60-day-old rat ventral prostate. Bars represent mean + SEM.
100 -
010
20
45
60
Age (Days)
FIG. 3. DNA synthetic activity (cpm/Rg DNA) in the distal and proximal segments of 10-60-day-old rat ventral prostate. Bars represent mean
+ SEM.
Figure 3 shows 3 H-thymidine incorporation into DNA of
the distal and proximal segments, expressed as cpm/[tg DNA.
Incorporation of 3 H-thymidine, though significantly different in the distal than in the proximal segments of Day 10
and 20 prostates, was only 1.5-fold greater at both ages. At
Days 45 and 60, the incorporation of 3 H-thymidine was not
significantly different between the two segments.
DISCUSSION
We show herein that 3 H-thymidine, when administered
in vivo to rats 10-60 days of age, was incorporated into
both the distal and proximal segments of the ventral prostate at each age. Expression of DNA synthesis per [1g DNA
per segment revealed only relatively small differences between the distal and proximal segments of Days 10 and 20
rats, and no differences at Days 45 and 60. These analyses
clearly show that DNA synthesis occurs throughout rat ventral prostate, supporting the contention that growth occurs
throughout the prostate rather than only at the distal tips.
These conclusions, derived from biochemical analyses, are
consistent with the results of our previous autoradiographic studies of 10-60-day-old rats that had received
3
H-thymidine in vivo; those results demonstrated that cells
labeled with 3 H-thymidine are seen throughout the ventral
prostate at all ages [11].
We also show in the present paper that the number of
cells in the distal segment is significantly greater than that
in the proximal segment of prostates of Day 10-60 rats.
This accounts for the observation that expression of 3H-thymidine incorporation as cpm per prostatic segment revealed significantly greater levels of incorporation into the
distal than the proximal segment at each age. Undoubtedly,
DNA SYNTHESIS IN RAT VENTRAL PROSTATE
the fact that there are greater numbers of labeled cells in
the distal than in the proximal segment explains the conclusion, derived from whole-mount autoradiographic analyses [9, 10], that DNA synthesis occurs predominantly, if not
exclusively, at the distal tips of the prostate. Clearly, those
studies were not designed to illuminate the extent to which
cell proliferation occurs throughout the gland.
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