Peptide fragments of lactate dehydrogenase-C4. (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study.
BIOLOGY
OF
REPRODUCTION
32,
1201-1210(1985)
Binding of Antibodies against Antigenic Domains Lactate Dehydrogenase-C4 to Human and Mouse Spermatozoa’
of Murine STAN
A.
THOMAS
BEYLER,3
Department
E. WHEAT,
and
of Biochemistry, Molecular Biology Northwestern University Evanston,
Illinois
GOLDBERG2
ERWIN
and
Cell
Biology
60201
ABSTRACT Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing two of these sequences. Rabbits were immunized with peptides designated MC515 and MC211320, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are first absorbed with the native LDH-C4 molecule, significant inhibition of binding to sperm results. Antisera to both DT-MC5,5 and DT-MC211220 hind to human sperm with similar but weaker patterns than seen with mouse sperm. These data indicate that the immune response to synthetic peptides containing antigenic surface LDH-C4,
of
sequences sperm. including
of LDH-C4 In addition, the MC,15
includes antibodies that there are shared antigenic and MC211.220 peptides.
INTRODUCTION Reduction
or
females
can
be
antibodies
to
experimentally and/or
testes
1983).
and
women
view
see
via
immunization
has
been
highly
purified,
well
presence
of
is unique
achieved with
see
sperm
provided an
immunologic
re-
of
the
impetus
on
based
eliciting
vaccine sperm-specific
that
of
and
is
the
comprised antigen.
to
antibody.
LDH-C4
compartments
of
approximately ciated
10% the
1973),
1974), Accepted January 7, 1985. Received November 1, 1984. ‘This work was supported by NIH grant HD-05863 and by the Program for Applied Research on Fertility Regulation, Northwestern University under a cooperative agreement with the Agency for International Development (AID/DPE-0546-A-0O-1003). The views of the authors do not necessarily reflect AID policy.
and
with cant
tial
reduction
practical that
this
Present Royal Oak,
address: Michigan
William 48072.
Beaumont
we
have
(Wheat
1201
and
et resulted
rabbits Goldberg, a!.,
this
fertility. the
identified
requires a
large
scale.
fragments
antigenic 1981;
potenvaccine,
approach on
1981)
in signifi-
subsequent
available
contain Goldberg,
that
is asso-
a contraceptive of
be
that
and
demonstrate as
antigen
LDH-C4
Hospital,
female
their
results
antigen
Therefore,
of
(Goldberg
application the
found
activity
(Lerum
LDH-C4 in
these
of
mice
mouse
Although
subcellular
They
membrane.
baboons
purified
and
three
LDH-C4
plasma
acces-
quantitated of
immunization
(Goldberg,
is Alvarez
sperm. of
1973;
therefore
each
rabbit
by
membrane
(Goldberg,
and
in
in
with
localized
biochemically
activity
Active
1971).
has been the plasma
Furthermore, have
lactate
somatic
sperm
1975)
(1984)
ap-
One
al.,
is immuno-
(Goldberg,
on
of
enzyme (Zinkham
and
the
mouse
et
Storey
this
spermatozoa
from
methods
rabbit
This
1965),
dehydrogenase-C4
sible
antibodies
Development a
for
contracep-
to
Goldberg,
isozymes
men
isozyme
(LDH-C4).
distinct
Lactate
on the human
sperm-specific
specific
1964;
asso-
(for
immunologic
the
dehydrogenase
both
documented
and
al.,
logically
Goldberg,
infertility in
et
is
this enzyme mouse and
dehydrogenase
Erickson
have
methodology
requires
the
antibodies
of
proach
lactate
1980).
findings
spermatozoa.
in
review
clinical
development
against
fertility
been
bind to between
antigen
of
has
(for
antisperm
Menge,
These
tive
This
extracts
with
the
with
sperm.
Additionally,
dated
such
elimination
associated
specifically sequences
Goldberg
of
sequences et
a!.,
BEYLER
1202
1983;
Wheat
and
amenable
are
to
Goldberg, synthesis.
with carrier
these antigenic protein produced
with
the
native
Prevatt
et
Wheat
et al.,
This mouse
Rabbits
1982;
against
Solid-phase fluorescent
radioimmunoassay techniques were
antibody
these
tive
a
et
(Gonzalesa!., 1983;
binding of
to both antibodies
binding
synthetic
to
and to
to the selection of use in an immunologic
to
bindwill
antigenic contracep-
se-
AND
killed, and cauda epiwere removed and placed in 20 ml of PBS at 4#{176}C.The tissues were then minced with scissors, and sperm were gently expressed in a Teflon/glass tissue grinder. The suspensions were filtered through one layer of paper tissue, and the sperm suspension was centrifuged at 1000 X g for 10 mm at 4#{176}C. The sperm pellets were washed twice more with 10 ml concentration
and Preparation
Lactate
mature
of Antigens
dehydrogenase-C4
mouse
testes by affinity Wheat and Goldberg
5
was purified chromatography
from
as
tion
an NH2-terminal to carrier protein. Each peptide was
to facilitate
cysteine conjugated
conjuga-
toxoid reagent according to the Three groups of four rabto
diphtheria
via a heterobifunctional method of Lee et al. (1980). bits were immunized with either mouse LDH-C4, DTMC,,,, or DT-MC2,,220. Each rabbit received 2 mg of the peptide conjugate dissolved in 1 ml phosphatebuffered saline (pH 7.5) and emulsified with an equal volume of complete Freund’s adjuvant. Injections were given intradermally at multiple dorsal sites. Booster injections of I mg peptide conjugate in in(DT)
complete
Freund’s
adjuvant
were
administered
at
4
and 8 wk after the primary immunizations. The group of animals that received LDH-C4 were immunized in a similar manner, except that 1 mg of antigen was used for the primary injection and 0.5 mg for booster injections.
Preparation
of
Antisera
Blood was collected from each rabbit prior to immunization, and weekly thereafter. Sera were separated, decomplemented at 56#{176}C for 30 mm, and stored
frozen.
Antiserum
samples
from
individual
mals were thawed and equal volumes from pooled into respective groups. Antipeptide obtained
8-10
Cilbertsville, mixture was first
phate-buffered
were
sera were wk after primary immunization and for anti-LDH-C4 sera. The pooled sera with a mixture of activated charcoal
after 25 wk were absorbed (Nonidet T) and land,
ani-
each
mouse
liver
acetone powder (Rockpowder and charcoal
PA). The liver washed twice
saline
(PBS).
with
An equivalent
pH
7.5
to
phos100
PBS, and fmally suspended in PBS at a of 5 X 106/ml. Human sperm samples from a single donor of proven fertility. were diluted with 3 volumes of PBS at
and
sperm,
one
washed such that
layer in
of paper tissue, and manner as for final concentration was
the
the
same
x 106/ml.
Solid-Pbase
Radioimmunoassays
Solid-phase radioimmunoassays (RIAs) were performed in a manner similar to that described by Wolf et al. (1982). Fifty microliters of antigen (2.5 X iO mouse or human sperm, or 5 pmol of mouse LDH-C4) were added to each well of polyvinyl chloride micro-
described by (1977). Two antigenie peptides representing residues 5 to 15 and 211 to 220 (Wheat and Goldberg, 1985) were customsynthesized (Peninsula Laboratories, San Carlos, CA) with
of
centrifuged
METHODS
mouse Identification
Swiss mice were and vasa deferentia
were obtained Semen samples 4#{176}C, filtered through
agent. MATERIALS
male
didymides
and
for relative These results
50
Preparation
Ten
immunodemon-
spermatozoa,
mg charcoal was used took place at 4#{176}C overSera were then clarified by centrifugation at 20,000 X g for 30 mm, and stored at -60#{176}C until further use. A pool of nonimmune serum that was used as a control in the radioimmunoassay and the iinmunofluorescence studies was subjected to the same procedures. Sperm
antigens.
used
compare the various antisera ing ability and localization. for
to reacted
in preparation).
paper describes the and human spermatozoa
contribute quences
weight liver powder and per ml of serum. Absorption night with constant shaking. dry
that
immunized
molecule Goldberg
developed
strate
and
peptides conjugated antibodies that
LDH-C4
a!.,
1985)
ET AL.
mg
titer plates (U-shaped wells; Dynatech Labs, Alexandria, VA). Sperm were bound to the bottom of the microtiter wells by air drying overnight at room temperature and were stored at -20#{176}Cfor up to 2 wk until use. Lactate dehydrogenase-C4 was bound to microtiter wells by overnight incubation at 4#{176}C without drying. These plates were always prepared immediately
prior
to assay. assay, microtiter plates were washed twice with 0.2 ml of 0.1% bovine serum albumin (BSA, Fraction V; Sigma Chemical Co., St. Louis, MO) in PBS (BSA/PBS), and 0.2 ml of 2% decomplemented goat serum (CS; Gibco, Grand Island, NV) in PBS
For
(GS/PBS)
each
was
added
to block
nonspecific
antibody
binding. After 1.5 h at 4#{176}C,plates were washed twice with 0.2 ml of BSA/PBS, Sera to be tested were diluted 1:4 serially with BSA/PBS and 0.05 ml was added to each of triplicate wells. Controls, omitting antigen or serum, were included on each plate. After a 1.5-h incubation, excess serum was decanted and wells were washed twice with 0.2 ml BSA/PBS. Binding of
antibody to antigen on the microtiter wells mined by adding a ‘25I-labeled goat antirabbit noglobulin G (IgG). The radioactive label was each well (5 X io cpm/weIl). After 1.5 wells were washed three times with 0.2 ml Bound antibody was quantitated with counter. Indirect Clean approximately suspensions
Immunofluorescence glass
microscope
was deterimmu-
added
to
h at 4#{176}C, BSA/PBS. a gamma
Microscopy
slides were coated with 0.1 ml of either human or mouse sperm (prepared as described above). Slides were
LDH-C4
ANTIBODY
BINDING
then air dried at room temperature. All incubations were done at 4#{176}C in a dark, moist chamber. Slides were first incubated with 0.2 ml of CS/PBS for 1.5 h to block nonspecific antibody binding. Slides were then washed thoroughly with BSA/PBS and incubated for 1.5 h with 0.2 ml of the antiserum to be tested at 1:20 dilution in BSA/PBS or with nonimmune serum of the same dilution. Each slide was incubated overnight with 0.2 ml of fluorescein isothiocyanate-labeled goat antirabbit lgG (Miles, Elkhart, IN) diluted 1:16 in GS/PBS. Slides were washed in several changes of BSA/PBS, covered with Pro-Texx mounting medium (Scientific Products, McGaw Park, IL), and examined immediately. Fluorescence was observed with a Leitz Ortholux microscope equipped with a darkfield condenser. An HBO 200 mercury vapor high-pressure lamp was used for excitation with filters BG-12 and BG-38. A K530 barrier filter was also employed. All sperm were photographed with a 1-mm exposure on ASA 400 Ektachrome film. RESULTS
Binding
of Antibodies
Synthetic
Peptide
Binding LDH-C4
of
to LDH-C4
antipeptide
antibodies by
the
data
immune
Fig.
and
that
This
binding of
titer
that
than
Binding to Mouse
could
be
the
native
readily
and had
to
both
contain
antiprotein.
detected
it seemed a somewhat
at
that the higher
of DT-MC211_220.
of A ntibodies and Human
Sperm
Antiserum binding to intact mouse sperm was similar to that observed with purified mouse LDH-C4 as the target (Fig. 2). Binding of immunoglobu!ins from antisera to DTMC5_15 detectable
and
DT-MC211_220 at dilutions
with
maximal
1:16
to 1:64. Immunoglobulins
binding
binding
occurring
also
(Fig. was
to mouse sperm was greater than 1:1000,
to
were
spermatozoa
mouse 1. Con-
with
1:1000, antiserum
and pre-
Antisera
DT-MC5_15
cross-react
dilutions DT-MC515
to LDH-C4
respectively.
DT-MC211_220 bodies
1203
of antiserum
serum,
conjugates to
in
SPERMATOZOA
trols consisted
against Conjugates
is illustrated
TO
the
shown
3).
in the synthetic
peptide
to bind
to human
Although
60-70%
of
that
range
the seen
amount with
of
of
mouse
7.
8-
-j -j
5.
IL’
0
4-
z 0 a. U
3.
2
1-
I
I
I
I
1
2
3
4
5
6
LOGIO DILUTION
A...A,
FIG. 1. Antibody anti DT-MC515
binding serum;
X -X, of mouse LDH-C4. s--., control serum.
Anti-LDH-C4
serum;
u---u,
anti
DT-MC211220
serum;
1204
BEYLER
8.
ET AL.
a
7-
6-
-j
5-
-j
w 0
z
4-
0 3.
a.
U
2
.
1#{149}
..-.
I
I
I
I
I
I
I
1
2
3
4
5
6
7
LOG1O DILUTION FIG.
2. Antibody
sperm,
it
1:1000.
was readily The binding
tive control what higher, (anti-LDH-C4) mouse of the
sperm. antisera
mouse
LDH-C4
to mouse
binding
sperm.
detected associated
Symbols
at dilutions of with the nega-
(preimmune) serum was someand that from the positive control serum was lower, than seen with Also, were
the less
or mouse
differences pronounced
in binding than with
sperm.
are
the
same
sera binding absorption
and
The
(i.e., binding demonstrated which prior
to
antisera RIA.
to in
sperm-associated competition
were When
binding
to sperm
LDH-C4) experiments
preincubated 1:128 dilutions
with
was in
LDH-C4 of antisera
human
microscopy
sperm the
to
that synthetic
a pattern
similar
to
spermatozoa,
strong
with
serum.
slight mouse
decrease sperm
seen the control sera and the slight increase
and
2).
binding to in control
obtained
fluorescence
and was
1
anti-LDI-I-C4 control. The
that
with
conjugates with
antisera to LDH-C4 (Figs. 4 and 5). On mouse sperm, the antibody binding was directed toward the tail segment with little or no binding to the head or midpiece. With human
strongest fluorescence, DT-MC211_220 serum
(Tables
obtained for as a positive
mouse
incubated
peptide
was
reduced
of
were
seen on the tails, but some associated with the midpiece anti-LDH-C4 serum treatment
significantly
LDH-C4
Sperm
to DT-MC5_15 or DT-MC211_220 were preincubated with 0.5 mg of mouse LDH-C4/ml, antibody binding to both human and mouse sperm Similar results were sera, which served
following
Localization to Human
Binding
Immunofluorescent
showed
of antibody
to human sperm not significant.
Mouse
and
Experiments
specificity
1.
was
Immunofluorescent of Antibody
antisera Competition
as in Fig.
anti
DT-MC515
did not bind tions of this
was
again
binding was also and head. Again, resulted in the binding by the less than that
to mouse sperm assay. There was
Nonimmune under a very
anti seen sera
the condifaint non-
LDH-C4
ANTIBODY
BINDING
TO
SPERMATOZOA
1205
5
0
4-
-j -j
Iii 30
z 0
2-
#{163}
.
a.
-.-.-#{176},.
#{149}
C.)
..,.
1-
1
specific human
binding
3. Antibody
FIG.
halo
to human
of fluorescence
sperm
I
I
I
I
2
3
4
5
LOG1O
DILUTION
sperm.
associated
Symbols
with
are
the same
terns DISCUSSION
present developed
antigens
derived
study
demonstrates against synthetic
from
both to the purified enzyme on the surface
TABLE
1. Inhibition
mouse
antipeptide bind
LDH-C4
native enzyme of sperm.
of antibody
that
and
to
sperm
Mean
Seruma
Untreated
Control Anti-DT-MC515
1131 3941
±
Anti-DT-MC,11220 Antimouse LDH-C4
3209 9906
aAil sera bAbsorbed cSiiflcandy
diluted
0.5
different
mg
Student’s
cpm
indicate that the amino acid and MC211_220 are antigenic
the
molecule recognition
two
by preabsorption
bo und/well
antigenic
of antisera
±
solidpat-
LDH-C4,
results MC5_15
(Figs.
mouse
1-3).
These sequences sequences
that are exposed for on sperm. Furthersequences
with
are
shared
LDH-C4.
SD
LDH-C4
%
absorbedb
Inhibition
±
±
162
2489
±
283
5022
t-test).
mouse sperm
1025 2464
LDH-C4/ml. (P
