[CANCER RESEARCH 54,1027-1034, February 15,19941
Hormonal Protection from Procarbazine-induced Testicular Damage Is Selective for
Survival and Recovery of Stem Spermatogonial
Marvin L. Meistrich: Gene Wilson, Wei-San Ye: Belma ~ u r d o ~ l uNandakishore
?
Parchuri, and
Nicholas HeA. Terry
Department of Experzrnental Radiotherapy, The University of Texas 1W. D. Aildevson Cancer Cente~,Hou~ton,Texas 770.70
ABSTRACT
Procarbazine produces long-term sterility in the male by killing stem
spermatogonia. The degree and selectivity of protection of stem spermatogonia in rats from procarbazine by pretreatment with steroid hormones
were investigated. Male LBNFl rats were treated for 6 weeks with Silastic
implants containing testosterone plus 17P-estr.diol. The hormone-treated
were given a single injection of graded
rats and sham-treated
doses of procarbazine and the hormone implants were removed the next
day. Spermatogonial stem cell survival and function, assessed by the repopulation indices and sperm head counts 10 weeks later, showed that
stem spermatogonia were protected by testosterone plus 17P-estradiol
treatment from the toxic effects of procarbazine with a dose-modifying
protection factor of about 2.5. In contrast, there was no hormonal protection from the procarbazine-induced killing of differentiating spermatogonia, preleptotene spermatocytes, and spermatocytes in meiotic prophase
or from the delay in maturation of round spermatids, assessed 9 days after
procarbazine injection by histological or flow cytometric methods. In
addition, there was no hormonal protection from the procarbazine-induced decline in body weights and lymphocyte counts, indicating that the
gastrointestinal, neurological, and hematological systems were not protected. The specificity of protection indicates that the hormonal protection
of the stem spermatogonia is not the result of a systemic or overall testicular decrease in drug delivery, decrease in bioactivation, nor increase in
drug detoxification, except possibly within the stem cells themselves. We
conclude that the degree of hormonal protection and its specificity would
be appropriate for clinical application provided that the mechanism of
protection is elucidated and appears applicable to humans.
INTRODUCTION
Treatment of rats with analogues of GnRH5 or with gonadal steroids results in a cascade of events including decreased lutehizing
hormone secretion, Leydig cell atrophy, decreased intratesticular testosterone levels, loss of Sertoli cell androgen-dependent functions,
and suppression of spermatogenesis completion (1-3). All of these
effects are reversible uPon withdrawal of the hormone treatment.
When hormone-treated
are given procarbazine injections, the
recovery of spermatogenesis at 8 to 14 weeks after the last procarbarine injection is dramatically increased compared with that in nonof 'permatogenesis
hormone-treated rats (4-8)* Since the
must occur
surviving stem
these results demonstrate that
hormonal pretreatment protects stem spermatogonia from the dam%ing effects of procarbazine.
Chemotherapy induces sterility in male rodents and humans largely
by killing stem spermatogonia (9, 10). Thus, hormonal treatment that
rats
Received 9/13/93; accepted 12/14/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must
be hereby marked udvertisernent in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Grant CA-17364, Core Grant CA-17762, and Training Grant T32HD07324, all from the NIH, and the Katherine Unsworth Lead Annuity Trust.
M.
Andenon
To whom requests Ior
be addressed, at
Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.
Present address: Department of Histology and Embryology, Peking Union Medical
College, 5 Dong Dan San Tiao, Reijing 100005, People's Republic of China.
Presenl address: Institute of Oncology, University ofIstanbu1, Capa, Istanbul, Turkey.
The abbreviations used are: GnRH, gonadotropin-relea5ing hormone (same as luteinizing hormone-releasing hormone); T S E , testosterone plus 17P-estradiol; RI, repopulation index; PF, protection factor; LI, diploid cell labeling index; BrdUrd, bromodeoxyuridine.
'
protects these cells may minimize long-term sterility in patients undergoing cytotoxic treatments for cancer. However, extending results
obtained in rats to the treatment of men is problematic because the
mechanism of this protective effect is unknown. The originally proposed mechanism (ll), that the hormonal treatment places the stem
~ermatogoniain a resting state, cannot be valid because such treatment does not alter the kinetics of spermatocyte proliferation or differentiation (12) and, to maintain the spermatocytes, the stem cells
must also be proliferating.
Various alternative mechanisms may be proposed to explain the
protective effect. Some of these and their consequences are listed in
Table 1. Several imply that protection of multiple organs, tissues, or
cell types within the testis should occur. To test whether any of these
proposed mechanisms are involvcd, we examined whether there was
protection of other tissues or other germ cells in the testis under
conditions that resulted in protection of stem spermatogonia. Animal
mortality, weight loss, and reduced white blood cell count were used
as measures of toxicity to other tissues. Survival of differentiating
spermatogonia and preleptotene spermatocytes was of particular interest as measures of toxicity to other testicular cells, because they are
located adjacent to the stem cells in the basal compartment of the
seminiferous tubules; if protection is mediated by the levels of active
this compartment, they should be protected as well,
drug
MATERIALS AND METHODS
Animals. Adult LBNFl male rats were obtained from Harlan SpragueDawley (Indianapolis, IN). They were maintained on a 12-11112-h lightldark
cycle.
Hormone Treatment. At 15 wccks of age, the hormone-treated rats were
given s.c. implants of 2-cm-long Silastic capsules containing testosterone and
0.5-cm-long capsules containing estrogen (170-estradiol). Capsules were prepared from silicone tubing (602-305; Dow-Corning, Midland, MI) as described previously (8, 19). The hormonal treatment was continued for 6 weeks
until procarbazinc administration. Previous studies using Sprague-Dawley rats
(1) have shown that nearly identical treatment with implants results in no
change in average serum testosterone levels and a 3-fold increase in serum
estradiol levels (from 45 to 125 pg/ml).
rats, 12 weeks of age, were implanted with 3-cm-long Silastic
capsules containing cholesterol for 2 weeks prior to injection of procarbazine.
The different ages and times of implantation were used for the 2 groups so that
they would be approximately
weight-matched at the time of procarbazine
..
injection. In both groups, the capsules were removed 1 day after procarbazine
injection.
I'rocarbazine Administration. Fbcarbazine {N-(l-mcth~leth~l)-4-[(2methylhydrazine)methyl]benzainide monohydrochloride} was a gift from
Hoffmann-La Roche (Nutley, NJ). It was dissolved in normal saline and
injected i.p. into rats in volumes of between 0.25 and 5 ml. To minimize
possible variations due to injection technique, equal paris of the desired volume were injected at 3 sites in the peritoneal cavity. All treatment groups
consisted of 8 to 10 rats.
Assessment of Reduction in Lymphocyte Counts. One day after the
injection of procarbazine (at the tirne of removal of hormonal implants), about
500 of blood were withdrawn by retr0-orbital sinus puncture and placed into
heparinized tubes. Twenty-five p l of blood were diluted into 475 p1 of 2.86%
acetic acid for total white blood cell counts. Smears were prepared and
Wright's-stained for differential leukocyte counts.
of
white
Preliminary experiments showed a
centration between days 1 and 14 after procarbazine injection and also a drop
L,
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Research.
PROCARBAZINE-INDUCEDTESTICULAR DAMAGE
Table 1 Some of the possible mechanisms to explain the protection of stem spermatogonial survival/function from procarbazine-induced damage with hormonal treatment
Mechanism
Reason for considering
Consequence
Alteration of systemic metabolism of procarbazine
Liver function is affected by androgens (13)
Protection of multiple organs and tissues
Decreased drug delivery caused by a decrease in
testicular blood flow
LH regulates testicular blood flow (14)
Protection of all cells within testis
Decreased bioactivation of procarbazine within
testis
Procarbazine requires bioactivation involving P-450
enzymes and molecular oxygen (15) Levels of
P-450 enzymes in Leydig cells lowered by
hormone treatment (16)
Protection of multiple cell types within testis
Reduced levels of thiols within testis
Sulfhydryl reducing agents detoxify active metabolites
of procarbazine (17)
Protection of multiple cell types within testis
Increase in number of stem spermatogonia at
time of procarbazine treatment
Stem cells may increase in number in response to
depletion of differentiated cells (18)
Protection would be specific for stem cells; protection
factor should decrease at higher doses
in lymphocyte percentage between days 1 and 4. The concentrations of lymphocytes were most markedly reduced at days 1, 2, and 4 and were the most
sensitive measure of procarbazine hematological toxicity. Day 1 was chosen
for assay because the rats were anesthetized for implant removal at that time.
Assessment of Weight Loss. Rats were weighed 1 and 2 days before and
at the time of procarbazine injection, and the average of the 3 weights was
taken as the weight prior to procarbazine injection. Preliminary experiments
showed that the maximum weight loss occurred at days 3 and 4 after procarbazine injection. Hence, the average weight on these 2 days minus the weight
prior to injection was used as a measure of weight loss.
Flow-Cytometric Assessment of Survival of Differentiating Spermatogonia and Spermatocytes. Nine days after procarbazine treatment, rats were
Histological Assessment of Spermatid Development and Survival of
Differentiating Spermatogonia. Rats were killed 9 days after the injection of
procarbazine, and the left testes were fixed in Bouin's solution. Tissues were
mounted in plastic (JB4 embedding kit; Polysciences, Inc., Warrington, PA),
and 4-/xm sections were cut and stained by the periodic acid-Schiff-hematoxylin method. From each rat, one transverse cross-section through the testis was
prepared for histological analysis.
It had been shown previously that procarbazine doses of 100 mg/kg delayed
the development of round spermatids (20). To assess the development of these
cells, we used the frequencies of occurrence of each of the stages of the
seminiferous epithelium cycle, since the stages are defined by the step of
development of the round or elongating spermatids (21). For example, tubules
containing step 5 spermatids are considered to be in stage V, and those with
step 8 spermatids in stage VIII. One section from each testis was scanned along
parallel lines I mm apart under a X 100 objective; the stage of the seminiferous
epithelium cycle was determined for each of the first 200 tubules that came
into view. In T+E-treated rats, the exact stage could not be determined reliably in stages IX to XIV tubules because development of spermatids past
step 8 was markedly reduced by hormone treatment. Hence, tubules in stages
IX to XIV, identified in the T+E-treated rats by 2 generations of spermatocytes, no round spermatids, and occasional elongating spermatids, were
grouped into one category.
To measure the survival of spermatogonia and early spermatocytes, we
counted preleptotene spermatocytes in stage VIII of the seminiferous epithelium cycle and pachytene spermatocytes in stage V. In each testicular section,
all tubules in these stages, up to a maximum of 10, were analyzed. The nuclei
of Sertoli cells showing the nucleolus were also counted, and the numbers of
spermatocytes present at the indicated stages divided by the numbers of Sertoli cells were taken as measures of germ cell survival. No corrections were
made to account for section thickness and differences in nuclear diameters
(22), since relative, not absolute, numbers of spermatocytes per Sertoli cell
were most important. Working backwards using the kinetics of spermatogenesis (23), we determined that the preleptotene spermatocytes in stage VIII
and the pachytene spermatocytes in stage V were A3 spermatogonia and
preleptotene spermatocytes, respectively, at the time of procarbazine treatment. Therefore, the above assay should be a measure of the survival of these
latter cell types (24).
For the identification of spermatocytes at a particular stage to be valid, it
must be ascertained that the stages of the seminiferous epithelium cycle are
unaltered by the treatments (hormonal, procarbazine). To do so, we compared
the frequencies of the stages of the cycle of the seminiferous epithelium in
different treatment groups of rats (analysis of variance, t test).
given injections of 30 mg/kg BrdUrd and killed 1 h later. Pieces of the right
testis weighing approximately 100 mg were fixed in cold ethanol as described
previously (25). The left testis was fixed in Bouin's fluid for histological
analysis.
Testis cell nuclei were prepared for flow-cytometric analysis by treatment
with pepsin, acid denaturation, incubation with the monoclonal antibody IU-4
(which recognizes BrdUrd in DNA), incubation with a fluorescein isothiocyanate-conjugated antibody, and staining for DNA with propidium iodide (26). A
total of 50,000 cells were analyzed in each sample for both DNA content and
BrdUrd incorporation (25). Histograms were analyzed using the "Multi" series
software (Phoenix Flow Systems, San Diego, CA). The average coefficient of
variation of the diploid cell peak was 2.4%.
Since the diploid peak contains primarily nonproliferating, nongerminal
cells, which are not lost as a result of hormone treatment and are not killed by
procarbazine, it was used for normalization. The small number of G1 spermatogonia and preleptotene spermatocytes and secondary spermatocytes contained within the diploid peak should have only a small quantitative effect on
the results.
One-dimensional DNA histograms were fitted using the MultiCycle program. The numbers of cells in the 1C peak, 2C peak, S-phase region between
2C and 4C, and 4C peak were calculated. The 4C peak consists mainly of
primary spermatocytes in meiotic prophase, plus a small number of G2 spermatogonia. The ratio of 4C to 2C cells was used as a measure of the numbers
of primary spermatocytes in meiotic prophase (i.e., those between the preleptotene stage and meiotic divisions).
BrdUrd-labeled cells counted by flow cytometry can be used as a measure
of spermatogonial survival (27). The labeled cells should be mostly preleptotene spermatocytes (50%), B spermatogonia (25%), and intermediate spermatogonia (12.5%), with various types of A spermatogonia comprising the
remaining 12.5% of the labeled cells.
Bivariate histograms were analyzed using the Multi2D program to calculate
the numbers of cells in a given area of the histogram. A diploid cell LI is a
percentage calculated by dividing the number of cells that were labeled (all
diploid cells) by the total number of diploid cells (labeled and unlabeled).
Since there were some aggregates (clumps) of diploid cells with haploid cells
that were not excluded by the analysis gate, 3C signals (a diploid cell plus a
haploid cell) were included with the unlabeled diploid cells and signals between 4C and 5C (S-phase diploid cells plus a haploid cell) were included with
the labeled cells.
Assessment of Stem Spermatogonial Survival and Recovery. Rats were
killed 10 wk after procarbazine injection. The left testis was fixed in Bouin's
fluid and processed for histology as described previously (28). The histological
sections were stained with iron-hematoxylin, and the RI, which is the percentage of tubules showing repopulation, was determined. Tubules were considered
to be repopulating if they contained at least 3 cells at the B spermatogonia stage
or later. The tunica was removed from the right testis, the testis was weighed,
and the tissue was processed for counts of sperm heads (nuclei of sonicationresistant elongated spermatids) (28). Sperm heads were counted using a hemacytometer.
Protection Factors. The degree of protection was quantified by PF (8). The
PF is the ratio of the procarbazine dose required to produce a given effect
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PROCARBAZINE-INDUCED "I'ES'I'I(?tJLAR DAMAGE
when the protective treatment is given to the dose necessary to produce
the same effect in control (cholesterol-treated) rats. The PF was computed at
data points obtained in the T+E-treated rats. The dose to produce the same
effect in control rats was determined graphically by the intersection of a horizontal line through the data point in the hormone-treated rats with the straight
line drawn connecting points on the dose-response curves for the control rats.
|
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The weights of the cholesterol- and T+E-treated rats at the time of
O..
tu
procarbazine injection were very nearly matched. The cholesterolp"O- -e
0.4
treated rats weighed 333 _+ 20 (SD) g, while the T+E-treated rats
weighed 346 _+ 18 g.
I
No cholesterol-pretreated rats died as a result of procarbazine inCb)
jection at doses up to the maximum of 300 mg/kg. No T+E-pretreated
100
D------:~--------~PF>2.7
rats died at procarbazine doses up to 340 mg/kg, but 4 of 6 rats
v
receiving 380 mg/kg died. All deaths occurred within 48 h after
80
r
injection. Since all control animals survived 300 mg/kg and more than
%
half of the T+E-treated animals died at 380 mg/kg, there cannot be
%
60
0
much protection against procarbazine-induced lethality with T + E
._
%
0
%
treatment (PF < 1.3).
40
~L
Survival and Recovery of Stem Spermatogonia. The survival
0
and recovery of stem spermatogonia were assessed by testis weight
r
20
IZ
measurements, counts of repopulating tubule cross-sections, and
sperm head counts at 10 weeks after administration of graded doses of
I
'
t
'
,
'
I
procarbazine. The results are shown in Fig. 1.
(c)
~
Protection was observed with all 3 endpoints. At 160, 220, and 300
i
mg/kg, all values obtained in T + E rats were higher than those obw
08
""'0"~- PF-2.8
tained for the cholesterol-treated rats.
11
PFs were between 2.4 and 2.7 for testis weight, at least 2.7 for
%
"~
10 7
repopulation index, and 2.3 to 2.6 for sperm head counts. The value of
c
%
the PF for repopulation index is likely to be appreciably higher than
0
r~
2.7 (300/110) since at the highest dose for which we have adequate
1o
10 s
o
data in the T + E rats (300 mg/kg), the RI was 98 _ 2% (SEM), while
ID
-Iat the lowest dose used for the control rats (110 mg/kg) the RI was 82
E
105
l
Cholesterol
\
-+ 6%. Furthermore, the 6 T+E-pretreated rats that survived following
6
qD
procarbazine doses of 340 or 380 mg/kg had RIs of >--99%.
[3 T + E
\
Q.
U1
Body Weight Changes. To examine whether T + E treatment inlO4
l
*
I
'
I
'
~
duced systemic protection, body weight losses at 3 to 4 days following
0
1 O0
200
3,00
procarbazine injection were measured. Control rats showed a doseProcorbozine
Dose (mg/kg)
responsive change in the percentage of initial body weight lost, reachFig.
1.
Dose-response
curves
for
stem
spermatogonial
survival and recoveryassayed
ing a decline of about 14% at doses of 300 mg/kg. The decline in body
by (a) testis weights,(b) seminiferoustubule repopulationindices,and (c) sperm head
weight in T+E-pretreated rats was almost identical. PFs ranged from counts after procarbazine administrationto cholesterol-and T+E-pretreated rats. Bars,
1.1 to 1.4 in the dose range of 100 to 300 mg/kg (Fig. 2a). Since the SE. PF were calculatedat several isoeffectlevels.
T+E-pretreated rats had a slightly greater average initial body weight
at the time of procarbazine injection (346 g v e r s u s 333 g for the
control rats), the weight loss data were also expressed as the absolute
normal at 300 mg/kg (Fig. 2b). An essentially identical decline to 23%
loss of body weight in grams (data not shown). In this case, the PFs
were between 1.0 and 1.1 over most of the dose-response curve, was observed with this dose of procarbazine in T+E-pretreated rats.
except for the highest dose point. Thus, we conclude that T + E pre- PFs along the dose-response curve from 100 to 300 mg/kg ranged
treatment offers no significant protection against procarbazine-in- from 0.7 to 1.2, indicating that hormonal pretreatment did not protect
duced loss in body weight.
the rats against the hematological toxicity of procarbazine.
Development of Spermatids. Since no systemic protection from
Hematological Changes. The systemic effects of procarbazine
and the effect of hormonal pretreatment were also assessed by per- procarbazine toxicity was found, we next investigated whether other
forming measurements of total white blood cell counts and the per- testicular cells besides the stem cells were protected. To assess this by
histological means, it was first necessary to ascertain that the frequencentage of lymphocytes in control and T+E-treated rats. T + E treatcies of stages of the cycle of the seminiferous epithelium were not
ment reduced the total white blood cell count from 12,200 _ 700
(SEM)/mm 3 to 9,700 - 500/ram 3 and the percentage of lymphocytes altered by the hormone treatment. The relative numbers of tubules
from 92.3 ___ 0.8% to 88.1 +_ 1.0% (and hence a decline in the found in each of stages I-VIII and in stages IX-X1V combined in
lymphocyte count from 11,200 _+ 700/ram 3 to 8,500 _+ 400/ram3).
cholesterol-treated and in T+ E-treated rats not receiving procarbazine
The scales on which the dose-response curves (Fig. 2b) are plotted are were not significantly different (t test).
The frequencies of all these stages were not affected by procarbatherefore adjusted so that the values at zero dose overlap.
In control animals, procarbazine resulted in a dose-responsive de- zine at doses up to 65 mg/kg in the cholesterol-treated rats and up to
cline in lymphocyte counts at 1 day after injection to about 18% of 40 mg/kg in T+E-treated rats at 9 days after drug treatment. In the
1029
.m,
ira*
+,
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Research.
PROCARBAZINE-INDUCED
t
..1..,,
JE
.m
'
I
'
I
'
I
g
l-
E
-4
o.
--8
r-
0
E
~"
9 PF= 14
-12
9 Choleaterol
X
[] T+E
~. ,,
I
12000
~E
E
'
'
I
Q
I
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o
(b)
8000
g 10000
E
[]
8000
6000
I
6000
O
w
+
?
\~---PF=O 7
4000 Q~
O
~
4000
9
2000
Table 2 Effect of procarbazine treatment on spermatid development assessed
by the frequencies of tubules at stages V to VIII of the cycle of
the seminiferous epithelium
3
E
~
I
0
'
I
100
Procorbozine
<
i
-
I
200
Dose
DAMAGE
of spermatocytes than those receiving cholesterol. Since the preleptotene and pachytene spermatocytes scored had been A 3 spermatogonia and preleptotenes at the time of treatment, we conclude that
these latter cells are sensitive to procarbazine and exhibit no evidence of hormonal protection. In fact, hormone treatment may even
sensitize the differentiating spermatogonia and preleptotene spermatocytes to procarbazine.
Survival of Differentiating Spermatogonia and Primary Spermatocytes (Flow Cytometry). Because of uncertainties in the histological assay of spermatogonia or spermatocytes at procarbazine doses
of 65 mg/kg and above due to its effects on staging, flow-cytometric
assays were also performed. The flow-cytometric DNA histogram
from cholesterol-treated (control) rats (Fig. 4a) shows 3 major peaks,
at 1C, 2C, and 4C DNA contents, corresponding mainly to early
(noncondensed) spermatids, somatic cells, and spermatocytes in meiotic prophase, respectively. The region between the 2C and 4C peaks
contains cells with S-phase DNA content (preleptotene spermatocytes
'
(o)
0
TESTICULAR
-
PF= 1
"0
I
'
'
2000 =
Frequency of tubules in stages V to VII (mean -+ S E M )
at indicated dose of procarbazine
0
300
(mg/kg)
Fig. 2. Dose-response curves for (a) the percentage of body weight lost at 3 and 4 days
and (b) lymphocyte counts at 1 day after procarbazine administration to cholesterol- and
T+E-pretreated rats. The lymphocyte counts for the cholesterol- and T+E-pretreated rats
are plotted on different ordinates so that the values at zero dose overlap. Bars, SE.
Hormone treatment
0 mg/kg
25 mg/kg
40 mg/kg
65 mg/kg
Cholesterol
Testosterone + estradiol
38 -+ 1
41 ___ 2
41 + 1
--
39 +_ 1
42 _ 1
38 --- 1
56 - 2 a
a Significantly different from frequency in rats similarly treated with hormones but
receiving no procarbazine at P < 0.001 (t test). N o other differences were significant at P
< 0.005.
I
T+E-treated rats, the frequencies of stages I to IV were not affected
by procarbazine at 65 mg/kg; however, stages V through VIII were all
increased by procarbazine treatment (significant for stage VI, P =
0.003, t test). When the frequencies for stages V through VIII were
added together (Table 2), it was even more highly significantly increased. There was a concomitant decrease in tubules in stages IX to
XIV in the T + E group. These results indicate that this dose of procarbazine delays the development of spermatids that would normally
have progressed beyond step 8 during the 9-day interval, resulting in
an accumulation of tubules with spermatids at steps 5 to 8. Since this
effect of procarbazine was noted at 65 mg/kg in T+E-treated rats but
not in cholesterol-treated rats, we conclude that hormonal treatment
actually increases the sensitivity of spermatids to induction of developmental delays.
Survival of Differentiating Spermatogonia (Histology). Counts
of preleptotene and pachytene spermatocytes were performed 9 days
after procarbazine injection to determine whether there was protection
of differentiating spermatogonia by the hormone treatment. However,
it was first necessary to determine whether the hormone treatment had
any effect on the numbers of these cells in the absence of procarbazine
treatment. The number of preleptotene spermatocytes per Sertoli cell
nucleolus was 4.31 _ 0.09 in control rats and similarly was 4.12 ___
0.23 in the T+E-treated group. The numbers of pachytenes per Sertoli
cell was decreased from 5.22 ___ 0.08 in the control group to 3.38 +
0.21 in the T + E group. Because of the hormone effect on the numbers
of spermatocytes before procarbazine treatment, the ordinates of the
dose-response curves were adjusted (Fig. 3) so that the values at zero
dose overlapped.
Dose-response curves for the effect of procarbazine on spermatocyte counts (Fig. 3) showed that 40 mg/kg reduces the numbers of
preleptotene and pachytene spermatocytes similarly in both
cholesterol- and T+E-pretreated rats. At procarbazine doses of 65
mg/kg, the rats pretreated with T + E had significantly lower counts
(a)
o
t
'
I
Preleptotene
t
-
I
'
Stage
I
5
VIII
4
3
3
2
._
Q)
-6
E
~
o_
I1~
>,
~
@
if)
I.~
D
1
+
r'rl
i
I
(b)
:
',
:
Pachytene
',
-
:
',
Stage
:
4 ? ,Q
V
q
o
3
t'll
--i
'-'v
E
Q_
~
2
1
t
0
:
I
20
Procorbazine
:
I
40
Dose
:
I
60
'
I
80
0
(mg/kg)
Fig. 3. Dose-response curves for the numbers of (a) preleptotene spermatocytes in
stage VIII and (b) pachytene spermatocytes in stage V at 9 days after procarbazine
administration to cholesterol- and T+E-pretreated rats. The counts for the cholesterol- and
T+E-pretreated rats are plotted on different ordinates so that the values at zero dose
overlap. Bars, SE.
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Research.
PROCARBAZINE-INDUCED
TESTICULAR
DAMAGE
with S-phase (particularly late S) DNA content. The bivariate histogram (Fig. 4d) shows that the increase in cells with late S-phase DNA
content was due primarily to unlabeled cells with late S-phase D N A
content. Note also that most of the remaining labeled cells also had
late-S DNA content. We interpret the increase in late S as a procarbazine-induced delay in the completion of S-phase of the preleptotene
and spermatogonia). The bivariate histogram shows peaks of unlabeled cells at 1C, 2C, and 4C and BrdUrd-labeled cells uniformly
distributed between 2C and 4C (Fig. 4b).
The most apparent effect of procarbazine on the DNA histogram of
testis cells from control rats was to decrease the number of 4C cells
(Fig. 4c). However, there was a nearly concomitant increase in cells
5600
,i
G4
(a)
. . . .
ib)
SL
48.
3200
9~
40
2C
"~
32.
~400
4C
24
1600
16,
800
6
::-----N
L~
,~.
'
s'~176
t
'(c)
56O01
4,8
.
-~oo t
4~176
!-
32~176
t
Fig. 4. Flow-cytometrichistogramsof testis cell
nuclei prepared from rats 9 days after receiving
injections of procarbazine, a, c, e, and g, DNA
histograms, with cells with haploid (1C), diploid
(2C), S-phase (S), and tetraploid(4C) DNA content
indicated, b, d, f, and h, bivariate BrdUrd-DNA
histograms represented as contour plots with
BrdUrd-labeled S-phase cells (sL), unlabeled late
S-phase (late Su), and minor peaks of clumped
cells (*). a and b, control rats (cholesterol-pretreated, 0 procarbazine); c and d, 250 mg
procarbazine/kg body weight (cholesterol-pretreated); e and f,, T+E-pretreated rats (0 procarbazine); g and h, T+E-pretreated rats given 250 mg
procarbazine/kg body weight. Bars, SE.
t~
6
a4001
11~,
o
'22t
'~
.Q
56001
i
'(e)
Z
4"600 t
4000 t
32
3a~176
1
0
24
24001
16
16001
800 i
0
64
i
(g)
56
I0000
48
4G
32
24
oo:i i
4O
8
e'o
x~o
i'~'~
2oo
q
Channel Number (DNA content)
1031
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Research.
,
PROCARBAZINE-INDU('ED TESTICULAR I)AMAGE
spermatocytes; some of these cells were still synthesizing DNA while
most had ceased DNA synthesis.
Despite this increase in cells with S-phase DNA content, the bivariate histograms from procarbazine-treated rats (Fig. 4d) show a
decrease in the LI. In particular, there was a loss of cells with early and
mid-S-phase DNA contents. We interpret this result as indicating a
loss of cells from all parts of S-phase but with the addition of another
population whose exit from S-phase was delayed by the procarbazine
treatment.
The DNA histograms from T+E-treated rats (Fig. 4e) indicate that,
compared with control rats, there was a slight reduction in cells in the
4C peak, and a significant reduction in the 1C peak (from 118 _ 9%
of the 2C peak in control to 26 + 4% in T + E treated). This is
consistent with the known slight reduction in numbers of pachytene
spermatocytes and marked reduction in spermatids as a result of
hormone treatment (29). Analysis of the bivariate histogram (Fig. 4f)
shows that there was no reduction in the LI as a result of hormone
treatment (Fig. 5b). This confirms that the hormone treatment did not
put spermatogonia into a resting state.
The most dramatic effect of procarbazine on the T+E-treated rats
shown in the DNA histogram (Fig. 4g) was the reduction in the 4C
peak (plotted in Fig. 5a), indicating loss of most primary spermatocytes. The most dramatic effect of procarbazine seen from the bivariate histogram (Fig. 4h) was a reduction in the LI (plotted in Fig. 5b).
The procarbazine-induced losses of cells with 4C DNA content and
those that were synthesizing DNA were greater in the T+E-treated
rats than in controls.
DISCUSSION
The purpose of this study was 3-fold. (a) We wanted to quantify the
hormonal protection of spermatogonial stem cell survival/recovery
from the cytotoxic effects of procarbazine. (b) We wished to determine whether the protection was specific for the testis in general and
the stem spermatogonia in particular. (c) We wanted to utilize these
data to test some of the possible mechanisms of stem spermatogonial
protection.
Using 2 endpoints of stem spermatogonial survival, RI and sperm
head counts, protection factors in the range of 2.5 to 3 were obtained.
These values are consistent with the rough estimates made earlier (8)
based on partial dose-response curves. PFs of 2.5 to 3 are encouraging. If similar protection were obtained in a clinical situation, hormonally pretreated patients could be treated with procarbazine at 6000
mg/m 2 and still display no more long-term testicular toxicity than
those treated with only 2000 to 2400 mg/m 2 without hormones. Since
about 90% of patients treated with 6 cycles of mechlorethaminevincristine (Oncovin)-procarbazine-prednisone chemotherapy (procarbazine dose, 6000 mg/m 2) were azoospermic at a median of 3 years
after treatment but none treated with about 2000 mg/m 2 was azoospermic at 2.5 years (30), this degree of protection would be beneficial to
patients. Whether or not this degree of protection is achieved should
be testable in a clinical trial.
Because no other authors have generated dose-response curves to
allow calculation of PFs, we cannot compare these results. However,
we can compare the amounts of protection in the studies of Ward et al.
(5) and Glode et al. (7) at doses that yielded similar RIs to points along
our dose-response curve. Averaging the results of Ward et al. (5)
obtained at 50 and 90 days (we used 70 days), we calculate a RI of
about 5% in the procarbazine-treated piebald variegated rats and 94%
- 0.8
in the same rats that had been pretreated with GnRH prior to procarbazine treatment. At a procarbazine dose of 300 mg/kg, we obtained
a RI of 4% in our strain of rats; pretreatment with T + E raised the RI
0.6
to 98%. Thus, the amount of protection obtained with GnRH agonist
pretreatment was comparable to that obtained here using T + E pre0.8
'',
treatment. In contrast, the degree of protection reported by Glode et al.
0.4
(7), who also used T+E, was less than that found here. They found a
-o
0.6
RI of 2% in the control Sprague-Dawley rats receiving 10 injections
of procarbazine but only 25% in the T+E-treated rats. The lower
~0.4
0.2 ~ "
protection may be a result of the use of outbred v e r s u s inbred rats (5)
or the variability observed in the response of Sprague-Dawley rats to
~ g 0.2
procarbazine (8), which would affect the near-zero values in the
control more than those in the protected rats.
The second question, whether protection from the systemic toxiciz
~
~
(b) B r d U r d - l o b e l e d
cells
0.15~
ties
of procarbazine would be observed with the hormonal treatment,
(~ 0.15
3
O -is important because if systemic protection were observed, changes in
-,-, O
t"
hepatic metabolism of procarbazine might be involved and protection
of tumors would be likely. Procarbazine is known to be toxic to the
0.10
o
9
O.lO
neurological, gastroenterological, and hematopoietic systems (31).
O
The death of some rats within 48 h of injection of high doses of
O
procarbazine appears to be a result of the neurotoxicity. The weight
.r
O
loss is likely a result of both the gastrointestinal toxicity and neuro0.05
n0.05
toxicity of procarbazine. The rats were noticeably lethargic for about
1 day after procarbazine injection, and this likely affected their food
intake; subsequently many developed diarrhea. Although hematological toxicity has been generally ascribed to killing of hematopoietic
0.00
I
I
!
I
I
I
0.00
cells in the bone marrow (31), we found that the most sensitive
0
50
100 150 200 250
endpoint for hematological toxicity was the decline in lymphocyte
Procarbazine
Dose (rng/kg)
count at 1 or 2 days after treatment. This is most likely due to
Fig. 5. Dose-responsecurves for the numbers of (a) 4C cells and (b) BrdUrd-labeled chemotherapy-induced apoptotic death of mature lymphocytes (32)
cells, normalizedto numbers of diploid cells, at 9 days after procarbazine administration and not to death of lymphocyte precursors. There was no protection by
to cholesterol- and T+E-pretreated rats. The ratios for the cholesterol- and T+E-prehormonal pretreatment against either the procarbazine-induced lethaltreated rats are plottedon differentordinates so that the valuesat zero dose overlap.Bars,
SE.
ity, body weight loss, or the drop in lymphocyte count.
1032
1o
?
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PROCARI3AZINE-INDUCED TESTICULAR DAMAGE
Several endpoints of testicular damage by procarbazine were examined. Histological assays showed that hormonal pretreatment had
no significant effects on the sensitivities of A3 spermatogonia or
preleptotene spermatocytes at doses of 40 mg/kg, which is the highest
dose at which precise staging was possible in T+E-treated rats. At 65
mg/kg, the cells were actually more sensitive in the T+E-pretreated
animals; however, there could have been some error in staging because of the delay in spermatid maturation in the T+E-pretreated rats
at this procarbazine dose. Any such delay in spermatid maturation
would result in the spermatogonia and spermatocytes actually being at
a later stage than they appeared based on their association with spermatids. But this shift in staging cannot account for the increased
procarbazine sensitivity of these cells in the T+E-treated rats, because
if the cells were at stages just later than A3 or preleptotene they should
have become more resistant/'
Flow cytometry allowed independent and quantitative analysis of
cells based on DNA content or DNA synthetic abilities without relying
on staging tubules. We anticipated that the numbers of 4C cells would
provide a measure of meiotic primary spermatocytes and that the LI
would provide a measure of preleptotene spermatocytes and spermatogonia. The apparent delay in completion of S-phase of the preleptotene spermatocytes made this distinction less clear.
The loss of 4C cells in the DNA histograms was a result not only
of the killing of 4C primary spermatocytes and of type A4 spermatogonia through preleptotene spermatocytes that would have progressed
to meiotic prophase within 9 days, but also of the blockage of surviving preleptotene spermatocytes in premeiotic DNA synthesis. In
either case, the loss of 4C cells measures the toxicity of procarbazine
on differentiating spermatogonia and early primary spermatocytes.
The LI obtained from the bivariate histograms measures not only
the surviving intermediate and B spermatogonia and preleptotene
spermatocytes, but also the spermatocytes in meiotic prophase that
were still in DNA synthesis. The cells being measured in the LI assay
were therefore A1 through intermediate spermatogonia at the time of
procarbazine treatment. However, the reduction in LI is an underestimate of the killing of differentiating spermatogonia because of the
contribution of meiotic spermatocytes still completing S-phase.
Analysis of both the 4C cells from the DNA histograms and LI from
the bivariate histograms shows that the differentiating spermatogonia
and early primary spermatocytes are more sensitive in the hormonepretreated rats than in nontreated rats (Fig. 5). The uncertainties as to
the stage of cells with S-phase DNA content after procarbazine treatment do not affect this conclusion, as we performed the measurements
objectively in both cases. It should be emphasized that stem spermatogonia constitute only 1.3% of the spermatogonia plus preleptotene spermatocytes, and their labeling index is only 10%, which is
lower than that of the later stages of spermatogonia or preleptotene
spermatocytes. Thus, the stem spermatogonia contributed negligibly
to the labeled cells assayed by flow cytometry; a similar argument can
be used to show that stem cells contributed negligibly to the 4C peak.
The greater sensitivity of spermatids to procarbazine-induced toxicity in the hormone-treated animals may be a result of the androgen
deprivation, which alone results in many of them degenerating at step
7 anyway (33). The greater sensitivity of the spermatogonia and
preleptotene spermatocytes was unexpected, since these cells are
hardly affected by the hormonal treatment. In either case, there was no
evidence of hormonal protection of any of these cells from the cytotoxic or delaying effects of procarbazine.
In contrast, differentiating spermatogonia and/or spermatocytes
have been shown to be protected from the mutagenic effects of procarbazine; there were fewer postimplantation deaths of fetuses in
female rats mated to males treated with medroxyprogesterone and
6 N. Parchuri, and M. L. Meistrich,unpublishedobservations.
testosterone prior to procarbazine (6). The use of a different strain of
rat or the use of medroxyprogesterone instead of estradiol along with
testosterone in the suppression protocol could be responsible for this
difference, but these explanations seem unlikely. The following conclusions are based on our observations of selective protection of stem
spermatogonia; the different results of Velez de la Calle and Jegou (6)
remain as a caveat in the arguments.
The results obtained here address the possible mechanisms of hormonal protection of stem spermatogonia from procarbazine-induced
damage. One mechanism, an alteration in the systemic metabolism of
procarbazine, is unlikely since there was no protection against lethality or loss of weight or lymphocyte count. Although it is not certain
that the same metabolite of procarbazine is responsible for both the
stem spermatogonial toxicity and the other toxicities, metabolism of
procarbazine to the azoxyprocarbazine isomers is required for all
proposed pathways of toxicity (17, 34). Therefore, systemic metabolism to this level cannot be affected by the hormonal treatment. A
decrease in testicular blood flow, resulting in delivery of less procarbazine to the testis, is also unlikely to be the mechanism by which
stem spermatogonia are protected, because other germinal cells, the
differentiating spermatogonia, primary spermatocytes, and round
spermatids, are not protected.
Other possible mechanisms of protection include decreased bioactivation or increased detoxification of procarbazine within cells of the
testis. If these occurred in nongerminal cells such as the Leydig,
peritubular, or Sertoli cells through which the drug might pass and be
metabolized before reaching the germinal cells, then all of the germinal cells, and particularly the stem and differentiating spermatogonia
and preleptotene spermatocytes, which are adjacent to each other in
the basal compartment of the seminiferous tubules, should be protected equally. That they were not indicates that the mechanism of
protection cannot involve changes in procarbazine metabolism elsewhere in the testis; however, these experiments do not rule out the
possibility that the hormonal treatment selectively alters the ability of
the stem cell to bioactivate procarbazine or to detoxify it, perhaps
through an intracellular thiol, such as glutathione. In any case, mechanisms involving drug delivery or bioactivation of procarbazine are
unlikely to be the dominant cause of protection since hormonal treatment protects stem spermatogonia from radiation damage as well. v
Another possible mechanism is that under conditions of hormonal
suppression of the completion of spermatogenesis, the number of stem
cells increases, perhaps as a homeostatic response to the loss of
differentiated cells. If the number of cells increases without any
change in their individual procarbazine sensitivities, the dose-response curve for stem cell survival would be shifted upwards and to
the right by the hormonal treatment without a change in slope. A
consequence of such a shift would be a decrease in the PF with
increasing dose. Although the present data are not sufficient to unequivocally rule out a decrease in PF with increasing dose, the PFs
appear to be relatively constant with dose (Fig. 1), indicating that there
is a change in the sensitivity of the stem cells, and not merely an
increase in their number. The observation that stem spermatogonia are
protected from the mutagenic effects of procarbazine by hormonal
treatment (6) also argues against the protection of stem cells being due
solely to an increase in their numbers; rather it indicates that the
amount of DNA damage fixed in these cells is reduced.
Therefore, the suggested mechanisms of hormonal protection from
procarbazine-induced damage listed in Table 1 are considered unlikely. Other possible mechanisms of protection, besides specific bioactivation or detoxification of procarbazine in stem cells, include
increased DNA repair within stem cells, decreased stem cell sensitiv-
7 B. Kurdoglu,G. Wilson,W-S. Ye, N. Parchuri and M. L. Meistrich.Protectionfrom
radiation-induced damage to spermatogenesisby hormone treatment, submittedfor publication.
1033
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Research.
PROCARBAZINE-INDUCED-I'ESTI('UI.AR DAMA(IE
ity to procarbazine induced by altered secretion of cytokines by Sertoli
cells (35), subtle cell cycle alterations of stem cells, and increased rate
of recovery of spermatogenesis from an unaltered number of surviving
stem spermatogonia. Further experiments are required to test these
possibilities.
ACKNOWLEDGMENTS
The assistance of Kuriakose A b r a h a m with histological preparations, the
technical help of Yun Zhang, and editorial advice of Walter Pagel are appreciated.
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1034
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Research.
Hormonal Protection from Procarbazine-induced Testicular
Damage Is Selective for Survival and Recovery of Stem
Spermatogonia
Marvin L. Meistrich, Gene Wilson, Wei-San Ye, et al.
Cancer Res 1994;54:1027-1034.
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