Jan 2, 1974 - testicular extract. (Sadri et al., 1969). Further progress in this area of research requires a single, highly purified antigen and the development.
BIOLOGY
OF
11,
REPRODUCTION
108-115
(1974)
Immunological
Impairment by Lactate
JERROLD Department
E.
of Biological
of Pregnancy
in Mice
Dehydrogenase-X
LERUM
Sciences, Accepted
GOLDBERG
ERWIN
AND
Northwestern
University,
January
2,
Evanston,
Illinois
60201
1974
LDH-X, the sperm specific isozyme of lactate dehydrogenase was used as an antigen to induce infertility in female mice. Injection of rabbit anti-mouse LDH-X serum on Days 1-4 after coitus significantly reduced the number of pregnancies in these animals below the level observed in mice receiving serum from nonimmunized rabbits. Pregnancy frequency in mice injected with LDH-X in Freund’s Adjuvant was significantly decreased within 6-8 wk after the primary immunization. As a model to explain the mechanism of antibody action, we propose that supernumerary sperm within the perivitelline space of fertilized mouse ova provide specific recognition sites which are available to antibody to LDH-X. The antibody-antigen complex confers upon the embryo a heteroantigenicity which leads to its immunological rejection.
Numerous
immunological
means
of fertility
has
been
tracts
of
Kiddy 1968,
et
al.,
Rollins,
1969;
Bell
1973).
et
McLaren, of
accomplished
to a mouse
serum
et al.,
and
1964;
Bell,
in mice
injections
of
testicular
Further
progress a
the
muile
that
development
response. an
berg,
in
1963;
ham
et
gested
and to
1964; this
Goldberg,
attractive
reproductive
studies.
first
detectable
the
was
#{174} 1974 of
reproduction
by
The in
Society any
for form
the
immuno-
in
testes
of
is
Goldberg, demonstrated
and
Gold-
distinct
(Goldberg, to mouse
from
1971). LDH-X
can
(Goldberg and immunization of female mice
LDH-X
fertility and
lactate
1972). Furthermore, convincingly that
in
1972), and with mouse
reduced
other and
immunologically
pregnancy
crystalet al.,
chemical
(Hawtrey
and LDH-5 antiserum
passively
as
for
Study reserved.
properties 1970; been
the in
1968; Mouse
of
actively
reduces
fertil-
present examination
report
female
mice
immunized
of both with
LDH-X.
sug-
serve
Hawtrey,
from
isozymes
and
meiosis
1968).
(Goldberg, 1973). The describes a more detailed
a
MATERIALS
AND
METHODS
Animals The mice a random-bred
of 108
Copyright All righta
and
Goldberg,
It is different
rabbits
(Zink1965),
antigen
(Goldberg
ity
1963)
might
primary
in
and
Lerum,
im-
(Gold-
spermatozoa
when
suppress
observations
testes
testes
LDH-1 Rabbit
antigen
Zinkham,
protein
particularly
is
and
the
LDH-X
dehydrogenase,
sperm
specific
al.,
LDH-X
lactate
of
Blanco
that
the
found
only
berg, it has
of research
of a monospecific
human
and
unique
area
is
enzymic
(Sadri
purified
Therefore,
isozyme
LDH-X,
this
in
highly
single,
spermatocytes 1967)
1973).
anti-
extract
Hawtrey,
dehydrogenase
Erickson,
pregnancy
and
LDH-X has been isolated in pure line form (Goldberg, 1972; Adams
1969).
requires and
by
1959;
1972),
1970;
the time (Goldberg
Hawtrey
(Menge,
al.,
at
is occurring
ex-
guinea
Katsh,
Edwards,
and
in
rabbits
1964;
Termination
was
1959;
Menge
(McLaren,
with
testes
1973),
1970a,b;
mice
or
a
infertility
by immunization
appear
mice
to as
Female
spermatozoa
and
sought
phenomena
control.
induced
(Isojima
pigs
have
investigations
exploit
Reproduction.
used
in these experiments Swiss Webster strain
were from maintained
PREGNANCY
in
this
obtained
caged
under
at
Rabbits
laboratory.
were a
a
12-h
temperature
a diet
of
The
for of
(1
saline,
immunizations.
was
given
to
Mice
one-half
were
cg)
in
each
week
Rabbits the
artery. at
blunt
time
centrifugation
to
IgC the
tein
was
fraction procedure
concentration
metrically
10
serum Campbell
for the
and
1
isolated
clot mm.
according (1970).
was
and
Mating
Injection
units
of
sexually
(7-10-wk-old)
female
mice
with
(12-20-wk-old)
male
were
established.
a
copulatory
as
Day
1
at
this
time.
neous rabbit serum nancy
uterus
of
plug pregnancy
was
observed and
the
one
female
mature
fertile
a proven was
The
day
designated was
to
were
lutea
corpora
young
were
day The
immunized
one.
Pregnancy
at
or,
In
from
were
as
determined
the former the breeding
analyzed by by Student’s
noted,
mouse
alone
was
autopsy.
removed
of birth. results
with
or saline-adjuvant
or at
parturition
were by case, all cage on
chi t
square test.
RESULTS Specificity
isolated
Each female received a single subcutaor intraperitoneal injection of 0.1-0.3 ml anti-mouse LDH-X serum (AS) or control (CS) daily on Days 1-4 after coitus. Pregwas determined either by examining the at autopsy or allowing parturition to occur.
of Immune
Ouchterlony
a single,
of
identity
between
extract
(MTE)
nonimmunized
cipitin The with
lines antiserum mouse
attached several sue
Response
gel
sis revealed testes
five
for
by
Protocol
consisting
which
methods,
the enzymic equivalence point technique as described by Markert and Holmes (1969) and applied by Goldberg (1971). An equivalence point value of 1.0 would represent the inhibition of 1.0 IU of LDH-X by 1.0 ml antiserum. If th serum was not used at once, it was stored frozen.
Mating
examined
in adjuvant
mated
the
measured
were
of sacrifice.
Females
Pro-
spectrophoto-
titer
ovaries
time
LDH-X
then
30
The the
overnight
mm)
ci al.
determined
antibody
clot
56#{176}Cfor
was
a
venous
from
at
of
was
and
for
heating
of
inserting
stored
separated
(12,000g by
by to
then
from from
retroorbital
allowed
and
or
obtnined
or
the
were
serum
decomplemented The
sacrifice
temperature
(50 adminis-
puncture were
3
FIG. 1. Precipitin lines of identity formed by LDH-X and crude testis homogenate. The center well contained rabbit anti-mouse LDH-X serum. Wells 2 and 4-purified mouse LDH-X; wells 1 and 3-an equivalent amount (in terms of LDH-X activity) of crude testes homogenate.
6 wk.
into
samples
room
2-4#{176}C. The
by
of
__
or
LDH-X
adjuvant
cardiac
samples
tube
The
h at at
the
least
by
Blood
capillary
plexus.
at
prihad
intradermal ml
and
the titer level.
one 0.05
saline
bled
after
maximum
of
of
for
were
ear
mice
or
injection
antiserum
with
series
physiological
weeks
its
injection
adjuvant
tered
of
A
FCA
adminis-
booster in
the
immunized
intraperitoneal
was
LDH-X
when
ml. and
A single
several
immunization,
physio-
saline
A
mg
4
pure
10.0
injections
(1
mg
complete
of
LDH-X/animal). alone
10
sterile
volume
control
mg
declined
proce-
Freund’s and
final
intradennal
iv)
mary
a
physiological
0.05-mI
antigen
purified
published of
of
of sterile
u#{176}edfor
of
ml
(Calbiochem) to
1: 1 mixture
tered
on
water.
was
by
consisted
5
(FCA)
20
to
experiments
antigen
saline
was
maintained
access
testes
LDH-X,
adjuvant
regimen
1971).
provoking
crystalline
of
these
mouse
(Goldberg,
logical
and
fre.
were
light
Procedures
LDH-X
The
with
109
White)
animals
artificial
22-25#{176}C
chow
extracts
dures
Zealand All
daily
of
Purina
Immunization from
(New
commercially.
LDH-X
BY
TERMINATION
immunodiffusion intense LDH-X
(Fig.
rabbits
with
analy-
precipitin
either
did ovary,
extraembryonic concentrations
not
and
1).
Serum
did not LDH-X show
uterus,
line mouse
cross or
from
form preor MTE. reaction fetus
membranes of antiserum and
with
at tis-
homogenate.
In order spermatozoa,
to observe mouse
the effects epididymal
of AS on sperm
110
LERUM
were
treated
with
from
rabbit
antiserum
cedure
the
resulted
tion
of
the
cessation
only
slight
fraction
in
motility,
as
motility
serum
adjusted
to
was specific all
mice
isozyme. able
the
same
received
injections
were
wk
antigen.
after
serum
intense
between
diaphragm) fusion
treated
(mouse
AS)
the
or rat
testes
from
MTE.
was
extract
prior
injection immuno-
differences
of
cies a
identity
Furthermore, with fetus,
of
to those
of both antisera
other and
immunodif-
in
in both
When anti-mouse cant
mixed
with
to disc
gel
MTE electro-
injected
mode utilized,
reduction
in
pregnant
CS injection
for
and rabbit
1971).
1972)
mice
reduced these
reported
that
animals
appeared to be jected on Days in terminating 4-7
These study
passage protocols
and
of rabbit
into
pregnant
number below
in mice receiving nized rabbits. The
Days
injection
serum the
(Goldberg
of the
serum effect
antifemale
pregnancies
level
observed
from of the
nonimmuantiserum
most pronounced when in1-4 and nearly as effective pregnancy when used on
focusing
ble
for
are
the
values
2)
prompted on the
and implantation. were compared
a time
comprehenof oviducal
Various and, in
injection addition,
pregnan-
with rabbit was a significompared
significantly In view the
receiving values
different. of these
parameters
the proanimals
are
not
percent
differ-
pregnanIgG
groups.
se is not
suppression
in mice 2). The
responsi-
can
be
saline obtained
results,
we
of antibody
injections are not may
titer
ignore
and
mode
in calculating pregnancy 81.1% for experimental and
trol
respectively.
groups,
difference
the conclusion bodies against pregnancy
appears
to be
t test)
The
