DURING FASTING 925

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Jul 22, 1991 - RE, Cumming. RB. Induction of translocations in different germ cell ... 1989;. 10:296-303. 27. Wheeler. GP, Alexander. JA. Effects of nitrogen.

SYSTEMIC

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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

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