BIOLOGY

OF

47,

REPRODUCTION

Murine

Pregnancies

72-75

(1992)

Predisposed

to Spontaneous

Concentrations RL.

Departments

of Leukotriene

GENDRON,2’3’4

R

of Physiology,3

Microbiology

Reproduction,

McGill

Resorption

Alterations

B4 and Prostaglandin and

FAROOKHI,3

& Immunology4

Universuy,

Show

M.G.

and

Montreal,

in the

E21

BAINES4

the McGill

Quebec,

Centre

Canada

for

H3A

the

Study

of

2B4

ABSTRACT CBA/J

Female

Second

nancy.

BALB/c

males

We

(1)

The

measured

primigravid

CBA/J We 42)

in vitro,

that with

the

LTB4

without

effect.

in

deficiency

mean

the

killer

and induced the infiltration

GM1-positive is ample evidence

results

the suggest

(RES);

metabolites

in the

local

fetal resorption and activation

regulation

(570.2

by treating the cells inhibitor indomethacin

neously deficient anoid

resorbing CBA/J in an NK-inhibitory fraction

nancies be

extracts

with The

(2)

45.5

decreased

effect

[5]. These

important

a critical

role

for

nonspecific stages

killer

NK activity

16-8].

natural

the

other in vitro.

5-lipoxygenase

in the

cells [1-4]. of arachidonic

that

cytotoxic the

has activity role

are

of the known

reported

at concentrations

of LTB4

matings

in RES fetoplacental

capacity,

pregwith

NK-sup-

NK

activity

at Day

8 of gestation

(SEC);

and

in averting

(3)

in from

primigravid

NK-mediated

to augment

that

biphasic.

alone

either

early

spontaneous

some

pg/mI;

n

=

RES extracts, Addition

stimulated

resorption.

fetal

studies

of LTB4 NK activity

shown

no

aug-

[9]. Both LTB4 and POE2 have tissue during early pen-implantation

been

destages

than

Day

lipid

extracts

X DBA/2

PGE2 The

undertook the local differed

results

have

studies,

however,

6 of gestation. from

were

We

Day

primigravid

not

ex-

demonstrated

8 fetoplacental

matings

were

units

deficient

[5]. The absence of this similar to the resorption

trations

may

in normal

of LTB4

early

ing exogenous biphasically

of

presence may be

of the

during

NK

versus

here

in

activity ocfrequency

LTB4

of RES necessary

matings. concen-

primigravid

to RES extracts.

extracts. for the

determine of LTB4 and

decreased

Since LTB4 we examined the

to

resorption-prone

demonstrate

units. matings,

modulated

early

investigation concentrations

in resorption-prone

DBA/2 fetoplacental decreased in RES

of

the present fetoplacental

reported

concentrations the effects LTB4

X

were of add-

supplementation

NK-regulatory

These normal

CBA/J

activity

in

the

results suggest that LTB4 regulation of NK activity

gestation.

of i0’#{176}M

in NK activation

11.8

39, respectively).

=

supplemented

110]. These

further

We whether

to modulate a product

understood;

n

[5].

preg-

E series

was

LTB4

±

pg/mI;

we

an NK-inhibitory activity curred with a frequency

be

(176.4

units

± 59.5

to RES extracts

whereas

of CBA/J

units are in eicos-

during

420.2

in RES matings,

of LTB4

previously

synsponta-

eicosanoids unit

B4 (LTB4), been

first mated

in a lipophilic to modulate

units

x DBA/2

n = 21 and

tended

maternal

of a population

effects

elcosanoids

CBA/J

in rat pregnancy

There acid

of normal

fetoplacental

Leukotriene

pathway,

However,

stages

activity

inhibitory

known

females

of 5-10%.

a deficiency

are

menting effects tected in uterine

pregnancy. to

prostaglandins,

and

cells

of normal

In addition

early the

pg/mi;

LTB4

in mice is asof a population

fetoplacental recoverable

suggest

in regulating

second

addition

NK suppressive

of nonspecific

X DBA/2 activity

observations

and

fetoplacental

NK suppression

of the

extract’s

with the prostaglandin [4]. Furthermore,

during

of individual

of LTB4 ±

antifetal immune reactions. Decidual NK and lymphokineactivated killer cell activity in pregnant mice is diminished by decidua-derived prostaglandin E2 (PGE2). Activity can restored thesis

cells

in their

in CBA/J

a frequency

with

E2 (POE,)

entirely

natural killer (NK)-like for the involvement

of

(NK).Iike

in extracts

INTRODUCTION Spontaneous sociated with

occurs

prostaglandin

concentration

units

pg/mI).

increased

These

POE2

matings

is associated

(0-500

pg/mI

locus)

(30-35%)

rate

resorption

(CON).

decrease

LTB4

of 30-125

and

and

resorption

in contrast,

histocompatibility (LTB4)

resorption-prone

spontaneous

(15-20%).

of natural

B4

SEC fetoplacental

and

exogenous

range

or was

CON

rate

infiltration

of LTB4

matings

a significant

with

major

leukotriene

X DBA/2

control

H-2

fetoplacental

concentrations

CBA/J

To confirm in the

the

X BALB/c detected

compared

by

an increased

resorption

at the

eicosanoids

exhibit

males

a decreased

to DBA/2

is preceded

activity.

vitro.

show

(identical

Resorption pressive

mice mated with DBA/2

pregnancies

MATERIALS

is not

AND

METHODS

Animals Accepted June 5. 1991. Received March 9, 1991. ‘This work was supported by a grant from the Medical Research Council of Canada (248-43). R.L.G. is supported by a Doctoral Bourse d’Etude from the Fonda de Ia Recherche en Sante du Quebec. ‘Correspondence: RL. Gendron, Rm 404 Lyman Duff Building, 3775 University St. Montreal, Quebec, Canada H3A 2B4. FAX: (514)-398-7052.

Virgin from

female

Jackson

mice Laboratories

of the

the DBA/2 and BALB/c strains River (St. Constant, PQ). Mice Three 72

different

groups

CBA/J

(Bar

strain

Harbor, were were

of matings

were ME).

purchased Male

mice

of

purchased from Charles used at 8-9 wk of age. were

conducted.

In the

first,

virgin

CBA/J

(control

females

matings,

CON).

ALTERATION

OF LTB4 AND PGE2

were

with

In

mated

the

second,

BALB/c

virgin

CBA/J

CBA/J DBA/2

nancies checked inal

X DBA/2 litters males to yield

(SEC). Females the following

plug.

The

of gestation. cation on diately

of plug

snap-frozen

separated

on

from

overnight for the

detection

Pregnant mice Day 8 of gestation.

and

were

day

paired morning

the

was

C02,

frozen

mated preg-

and

uteri.

designated

Day

by cervical removed

disloimme-

fetoplacental

units

Individual

0

-

Chemicals,

Materials,

and

first and contents.

immediately

before

fetal

ratories

bovine

use.

(McLean,

were

Eicosanoid

C18

purchased

were

medium

purchased

from

ether

and

procedure were octadecylsylil

from

Extraction

Eicosanoids

culture

were

VA). Petroleum

used in the extraction Sigma. The SEP-PAK tridges

Tissue

serum

Waters,

and

30 sec

1640)

Flow

Labo-

methyl

formate

purchased silica (ODS)

Bedford,

from car-

MA.

extracted

from

by hand

in PBS,

pH

15%

effector

less than checked

curves

by

of

cell:target

cell

CBA/J

virgin

mice,

taining various concentrations ples were added to the wells 51-labeled

then

added

cell

ratio

YAC-1

target

to all wells of 50:1

in a final

lysis

measured

by use

of RIA kits

purchased

from

Advanced

were

Laborato5% heat-

(%CML)

was

cells! from

wells

(1

con-

X 104/well)

a constant

well

volume

cell:target

of 200

pA. Plates

were percentage

calculated

were

effector

Supernatants and the

well. counter,

at 37#{176}C was har-

counted in of cell-me-

according

to the

a

follow-

formula:

% CML

Maximum

=

(cpm (cpm

release of

test max (cpm

cpm cpm

-

max)

resuspended

spontaneous) spontaneous) was

target

(cpm

containing lymphocytes and were counted to measure effector:target

ratio

of

50:1

were

carried

out

Differences

in fetal

were

Wallis large

nonparametnic sample size.

Mag-

imal

significance

Data was

by counting

alone was

while

targets with extract cpm test. Control was

sponta-

determined

by

medium from wells of experimental and/or %CML

determined

the values of %CML from wells containing alone or extract buffer (PBS containing assays

100%

determined cells

spontaneous)

by the Chi-square test. individual fetoplacental

extracts

to

vested from each Beckman gamma

measuring the radioactivity tuted sample. Recoveries

in the

Assays

x g for 5 mm, incubated 150 pA of supernatant

Statistics

POE2

using

in which target

(Flow with

were centrifuged at 200 for 4 h, and recentrifuged;

of the fetoplacental unit (0.01 mI/mg tissue) and stored at -80#{176}Cuntil assay (not more than 1-2 days later). Recoveries for the extraction procedure were monitored by adding tnitiated LTB4 or PGE2 to the tissue homogenates and

and

[11].

added

cells

to yield

samples of supernatant target cells only. Samples

of LTh4

assay. with

dilution

ratios

were

counting taining

tween 83 and 90%. The concentrations

1%

of extract and/or LTB4. Samin a volume of 50 il. Chro-

justed to a pH of 3.5-4.0 with 1 N HC1. The supernatants were applied to SEP-PAK C18 columns that had been prewetted with absolute ethanol and distilled water. After washes with 15% ethanol and absolute petroleum ether, the col-

reconstiwere be-

than

bovine serum. Effector cells (5 X i0 as whole spleen cell suspensions

release

recovered in the final for both LTB4 and POE2

the

10% for either for parallelism

by a 4-h micro-assay mixed with radiolabeled

neous

umns were eluted with absolute methyl formate. The methyl formate eluants were dried under a stream of nitrogen. The extracts were reconstituted in PBS, pH 7.4, containing 1% ethanol at volumes adjusted according to the initial weight

with

analysis.

centrifuged at 400 X g for were collected and ad-

The homogenates were at 40#{176}C. The supernatants

less

sample

regression

aliquots

ethanol. 10 mm

to show

96-well, v-bottomed plates 1640 medium supplemented

inactivated fetal well), prepared

fetoplacental

containing

were always

was measured cells were

at fixed

mium

supplied

Measurements

conducted in ries) in RPMI

as described prewas homogenized 7.4,

supplier

linear

NK activity mouse spleen cells

the

antisera

other related and nonrelated arachiAssay sensitivities were 4 and 8 pg/ml respectively. The interand intraassay

standard

NK Activiy

ing

individual

by

computer-assisted

diated

Measurement

units with SEP-PAK C18 ODS cartridges viously [5]. Briefly, weighed frozen tissue at 40#{176}C for

(RPMI

reported

Specific

of variation assays were

spleens

Reagents

Eicosanoids (LTB4 and POE2) were purchased from Sigma Chemical Company (St. Louis, MO) and diluted into culture medium from stock solutions (1 mg/ml absolute ethanol) and

were

MA).

corresponding

fetopla-

in separate groups of CBA/J X DBA/2 matings by gross inspection of the uterine

(Cambridge,

coefficients Individual

cental units were placed in individual freezer vials, weighed, and stored at 80#{176}C until extraction (not more than 1 day later). Resorption frequency was assessed at Day 11-12 of gestation second

kits

73

FETAL RESORPTION

cross-reactivity with donate metabolites, for LTB4 and POE2,

with males were presence of a vag-

were killed Uteri were solid

fedeliv-

1-2 wk previously were second CBA/J X DBA/2

SPONTANEOUS netics

males

males were mated with DBA/2 males (resorption-prone matings, RES). In the third, CBA/J females that had ered with

IN

by

conwells LTB4 at an

averaging

either medium 0.5% ethanol). All

in triplicate.

resorption LTB4 and units were

frequency

test for multiple are expressed determined

were

analyzed

POE2 concentrations in analyzed by the Kruskal-

at p

comparisons with a as mean ± SE. A min