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BIOLOGY
REPRODUCTION
OF
Adverse
Effects
46,
926-931
(1992)
of Cyclophosphamide
on Progeny
Outcome
Mechanisms
in the
Post-Testicular JIANPING
Department
QIU,
of Pharmacolo,gy
and
BARBARA
F. HALES,
and
Can Be Mediated Rat1
BERNARD
Therapeutics, Centre for Montr#{233}al, Qu#{233}bec, Canada
through
ROBAIRE2
the Study of Reproduction, H3G 1Y6
McGill
Universiy
ABSTRACT Previous phamide
studies
may
from
have
post’testicular
effect
phosphamide of two
the
the
dose-related
and
reached
after
increase
single
30,
or 70
the
did
mg/kg
alone.
the
through
point;
treatment
on male
for
7 days
effect
these on the
and
bred
loss
was
whose
progeny
the same
results
demonstrate
of cyclophosphamide
caput
epididymidis
outcome on
and
corpus
the
for
effect
7 days.
ducts
that
short-term
No found
was
ligated
followed
paternal
may
determined.
treatment
be more
to
be
with
affected
any
loss
mg/kg). a single
produced
suggests
that
by cyclophosphamide
of
dose
in pre-implantation However, Third,
ligation
the
a
effect
of the efferent at a
by cyclophosphamide
cyclophosphamide That
was
Second,
of cyclophosphamide
mechanism.
manifested
males
among
post-treatment.
Bilateral
loss as that
period, time
post-treatment.
by administration
or one
with
change
with
rats
(10.0
treated
1 day
4 days
at any
this
saline
Post-implantation
exposure
significant
on
to cyclo-
of male with
treatment
loss
were
after
groups
by a post-testicular
spermatozoa
of each
cyclophos-
elaborate sensitive
by gavage
loss.
rats
testis,
To
of treatment
end
of drug
Male
loss was
ligation
is mediated
post-testicular epididymidis
7 days
after
studied,
of post-implantation
percentage
that
At the
the
most
are
daily
in post-implantation
high-dose
had been
however,
effect
treated
4 days post-treatment. two
cells
on pre-implantation
observed
40%
m transit.
germ
of the
course
1, 4, or
was
in the
spermatozoa
epididymal
epididymis
time
in post-implantation efferent
on
during
rats were
was
nearly
effect
Male
increase
and
observed
outcome;
produced progeny
mg/kg)
I day
no change
animals
effect
10.0
an
testis,
in the
of cyclophosphamide
doses
mg/kg)
not
Together
an adverse adverse
high
time
affect
or
of treatment
in post-implantation
of cyclophosphamide 7 days
4 days
with at either
exerts
First,
to
the
determined.
was
and
was
mg/kg
addition
leave site(s)
undertaken.
progeny
a time-dependent
there
observed
treatment
at which
No
but
of 10.0
were
of their
in
they
outcome.
loss was
dose
to determine
experiments
that, after
(6,8
(10,
for
and
outcome
cyclophosphamide
ducts
cells
suggested
on pregnancy
of treatment
significant
on spermatozoa
of cyclophosphamide
increased
effect
have
effect
to assess the effect
groups,
significantly
laboratory
three
on
doses
mated
of the
on germ
treatment,
cyclophosphamide
were
our
an adverse
(4
4 days
are
or 7 days) required
spermatozoa than
those
effects
of
in in
for
transit
the
cauda
epididymidis.
INTRODUCTION Cyclophosphamide
is a widely
munosuppressive
drug.
A
ferentially used
anticancer
of reports
number
and indicate
of cyclophosphamide Cyclophosphamide, 1 wk, produces an gesting that the drug left the testis while
suggested
Accepted
December
Received
July
Supported pour
22,
Ia Formation
different
germ
cell
stages
are
dif-
epididymal activities
27, 1991. from
the Medical
de Chercheurs Dr. B. Robaire,
University,3655 Drummond 398-7120.
et l’Aide Dept
Research
Council
S Ia Recherche of Pharmacology
of Canada
treatment, is parallel to its onset when given at a low dose for increase in post-implantation loss, may they
affect spermatozoa are maturing
in
[12]. only sug-
after they have the epididymis
of
toxicity carnitine
in terms of functional acetyltransferase
and
parameters, e.g., acid phospha-
tase, and an increase in the number and size of clear cells [13, 14]. However, the consequences of these changes in epididymal biochemistry and physiology are not known. Therefore, the experiments in this study were designed to determine whether, indeed, paternal treatment with low
and the Fonda
(Qu#{233}bec). and Therapeutics,
cyclophosphamide:
However, an effect of the drug after 1 wk of treatment does not exclude the possibility of a testicular mechanism, possibly mediated at the level of the rete testis, due to the variations in epididymal spermatozoal transit time. To clearly demonstrate a post-testicular effect, it was necessary to establish that this effect occurs after shorter treatment periods and/or after ligation of the efferent ducts. Previous research from our laboratory has shown that treatment with low doses of cyclophosphamide for 9 wk produces limited
1991.
by grants
2Correspondence:
that
the
[10].
rats mated with drug-treated males; these effects are also dose-related [8-111. On the basis of the timing of the onset of the effects of paternal cyclophosphamide treatment, it been
to
maximum post-implantation loss has been obtained when testicular germ cells are first exposed as spermatids. The timing of the decrease in the incidence of post-implantation loss after cessation of treatment, i.e., reversal of the effects
imthat
cyclophosphamide alters human fertility [1-31. Adult patients treated daily with cyclophosphamide (1-2 mg/kg) for more than 4 mo have oligozoospermia or azoospermia [1,2,4]. Animal studies have shown that single high-dose treatment of mice with cyclophosphamide (50-100 mg/kg) leads to decreased testicular weight [5], transitory oligozoospermia [6], and decreased DNA synthesis in spermatogonia and RNA and protein synthesis in spermatids [7]. Chronic low-dose exposure of male rats to cyclophosphamide for 1-11 wk produces a dramatic and time-dependent increase in pre-implantation or post-implantation loss, or congenital malformations in the litters of female
has
sensitive
McGill
Street,Montr#{233}al, Qu#{233}bec, Canada, 113G 1Y6. FAX: (514)
926
CYCLOPHOSPHAMIDE
doses
of cyclophosphamide
outcome
via
produces
an effect
so, to elucidate
the
on
time
an adverse
post-testicular course
germ
of such
POST-TESTICULAR
AFFECTS
progeny cells,
an effect.
this information, three experiments were done: study of the effect of paternal cyclophosphamide
and,
if
duct ligation on progeny
combined outcome.
with
cyclophosphamide
MATERIALS
AND
dry,
treatment
Time treatment
of Rats
Adult Sprague-Dawley male (350-400 g) and female (225250 g) rats were obtained from Charles River Canada Ltd. (St. Constant, QC, Canada). 14L: 1OD cycle with food The
animals
were
kept
Animals and water in this
1 day, males that randomly divided
were capable into groups
clophosphamide according the end of treatment, each 2 females killed with
were maintained provided ad
experimental
for 1 wk; at the end of this week to assess their copulatory activity
on libitum.
a
the male rats were prior to treatment.
10.0
mg/kg)
females by saline
were or cy-
the male epididymides,
rats
prostate, seminal vesicles, and spleen were removed, blotted, and weighed. The spermatozoa! contents were determined for one testis and one epididymis (divided into Caput-corpus and were homogenized 1-mm interval, merthiolate, sistant
cauda in
and
condensed
by means
segments). The testis for two 30-sec periods, 10 ml of 0.9% sodium
0.5%
Triton
spermatids
X-100. and
of a hemocytometer
percentage counting matozoa
and epididymis separated by a chloride, 0.1%
Homogenization-re-
spermatozoa [15].
were
To assess
motility, a drop of fresh caudal epididymal pended in PBS at 35#{176}C, placed on a slide and examined under a phase-contrast light
for
spermatozoa!
fluid was suswith a coverslip, microscope. The
motile spermatozoa was determined the total number of motile cells per 100 observed in each visual field.
ent but
study minor rats
of Progeny
In the
morning
of gestation,
female
spermatozoa. sarian moved,
female,
mating,
considered
checked female
for rats
numbers of implantations, determined. Pre-implantation
calculating lutea and and
the difference the number
post-implantation
the
as Day presence
underwent
was
of
the
monitor
each
determined
effect male in orgeneral
studies in the
[8pres-
progeny outcome system. At the
male
was
bred
to
of cyclophosphamide system and on
a single Male
different single doses of saline 30, or 70 mg/kg). Each treated 4 days
2
preg-
above.
rats with outcome.
Pregnancy rats on
drug distal daily the
as described
and
rats
gavaged daily (6.8 mg/kg or
dose rats
of
were
or cyclophosmale rat was
post-treatment
with
2
outcome was assessed in Day 20 of gestation as de-
above.
Effect treatment imals
of efferent duct on the progeny
each
were
used.
received
saline
received
cyclophosphamide
was
ligation outcome.
One
by gavage
ducts
plus
group
daily
was
cyclophosphamide Four groups of 6 anused as control and
for 7 days. daily
The
second
by gavage
(10.0
group mg/kg/
Groups 3 and Group 4 underwent efferent ducts and were then treated to Groups 1 and 2. Ligation of the
done
under
ether
anesthesia.
The
testes
of each animal were exposed by means of median suprapubic incisions. Ligatures were tied around the efferent ductules at points immediately adjacent to the testes. Parcare
was
bred
nancy
was
taken
to avoid
impeding
the
testicular
treatment began immediately after At the end of the drug treatment,
overnight
outcome
with
was
two
determined
females
efferent each
in proestrus.
as described
blood duct male Preg-
above.
cae-
Statistical
resorptions, and loss was as-
Statistics
assessed
to
The effect reproductive
females in proestrus. sperm-positive female
ovaries were reThe uteri were
between the of implantation loss
0
and
period,
at 1 day
transection.
on Day 20 of gestation. The corpora lutea were counted.
opened, and the live fetuses were
each
were
Sperm-positive
section and
sessed by of corpora
rats
each
dose
have major effects on on the male reproductive
was
overnight
scribed
of male
were
On the basis of previous that the two doses chosen
treatment
with (10,
q’clophosphamide groups
to determine
Effect of treatment of male cyclophosphamide on progeny
ticular
Outcome after
drug
in proestrus. on the male
outcome
gavaged phamide
malformations.
as they pass through the The animals were weighed
would effects
vessels. Drug Analysis
1, 4, or 7 days
the
of each
efferent
by sper-
for external
of 6 animals) or cyclophosphamide
day) for 7 days. Both bilateral ligation of the in a manner identical
counted
of
adjust
it
bred
were ventral
examined
consisting (control)
of the animals. was expected
nancy
to the experimental design. At male was mated overnight with mating, testes,
to
health 10],
female exposure
bred After
the number of implantafetuses. Fetuses were blot-
course of the effect of paternal on progeny outcome. Nine
group saline
end
environment
of inseminating and gavaged
in proestrus. After an ether overdose;
and
on the spermatozoa reproductive tract.
METHODS
927
Design
(each with
der
Treatment
weighed,
Experimental
on progeny outcome, a study of the effect of treatment of male rats with a single dose of cyclophosphamide on progeny outcome, and a determination of the effect of efferent
CELLS
culating the difference between tion sites and the number of live ted
To obtain
a time-course treatment
GERM
number sites for by cal-
Evaluation
Statistical OK). of
Data variance
analyses
System were
(CSS)
were
done
computer
analyzed
(MANOVA,
relationship) and one-way lowed by the Neuman-Keuls
with program
by two-way dose-dependent
the
aid of a Complete (Statsoft
Inc., Tulsa,
multivariate analysis and time-specific
analysis of variance (ANOVA) foltest [16]. Comparable data were
QIU
928 derived
from
experiments
done
animals considered
from
the same significant.
shipment.
at the
same
A value
time of p
