BIOLOGY
OF REPRODUCTION
43, 712-717
Lactoferrin C.
Center
for
(1990)
Binding
J. THALER,2
0.
Reproduction
and
Molecules
in Human
A. VANDERPUYE,
J.A.
Transplantation
Immunology,
Indianapolis,
Seminal
McIN1YRE,
Plasma1
W.
and
PAGE
MethodIst
Indiana
FAULK
Hospital
of Indiana,
Inc.
46202
ABSTRACT During
ejaculation,
tigens.
Ptiyslcochemlcal
These
differences binding
lactoferrin.
SP
been
SP
were
In
molecules
of approxImately that
associate
munoprecipitated
molecules
In
binding
60,
SP are free appears
and
lactoferrin peaks, and
in SP was
one
that
of which
others
to change
are
No binding
was
associated
This
with
ligand
binding
thus
for
results
These molecules.
could
by coincubatlon
The
transferrin.
its
binding
bound
that
in SP Ian.
some
of
lactoferrin
biologic
activity
to SP of mol.
presence
Lactoferrin suggest
Binding
influence
biotinylated
Lactoferrin
lactoferrin
Biotinylated
studied
have of
inhibited
an-
lactoferrin.
We binding
Immunoelectrophoresis.
crossed
and
purified
of
or transferrln.
IgG
biotinylated
lactoferrin.
lactoferrin
tested
competitively
blotting.
with
by using
properties
was
of sperm-coating
those
to lactoferrin. and
human
with
observed
to purified
Its physicochemical
plates
binding
and
component from
macromolecules
done
SDS-PAGE
studied
differ
microtiter
incubatlons
by using
further
a major
seminal
lactoferrin.
corresponded
forms
on sperm
and and
onto
coated
by control
30 kDa.
(SP)
of unknown
biotinylated
affected
to sperm
plasma
were
characterized
120,
with
binding
samples
bind
not
biochemically
as two
molecules
was
binds
In seminal
to the
SP. The SP
In but
molecules
ecules
of lactoferrin
to specifically
found
lactoferrin
lactoferrin
protein
attributed
molecules
was
unlabeled
binding
Iron
properties have
lactoferrin with
the
lactoferrin
to lactoferrin and its affinity
to sperm.
INTRODUCTION Lactoferrin teins that bind
and transferrin ferric ions. Both
the
are structurally related proare present in seminal plasma
cells monas fluence
[7], and
a role
in proliferation
the induction
vaginalis [8]. Much of lactoferrin on
of cultured
of metabolic attention humoral
tively
on the inimmuno-
hibit ment
of monocytes [9]. Lactoferrin also was the formation of the classic C3 convertase [10] and to down-regulate the secretion
cyte-macrophage of lactoferrin
colony-stimulating in SP is not
known.
shown
forms
the basis
to in-
of granulo-
[11, 12]. The
During
ejaculation,
Biotinylation
role
The
lac-
molecules
of
molecules that have These molecules are putabinding factors. The characterand their binding to lactoferrin
AND
METHODS
of Proteins of Bayer
lactoferrin
et al. [19] was and
transferrin
modified
IL) and
Rockford,
cervical mucus [17]. It is not known how lactoferrin binds to sperm during ejaculation or how it is released within
tein dobiotin
solution
was
adjusted added
(NHS-Biotin;
4#{176}C against 0.01 (PBS). Biotinylated
Foundation. 1701
and TransBoule-
712
dialyzed
to 1 mg/ml. To each 120 pA of N-hydroxy-succinimi-
Sigma
Chemical
M phosphate-buffered proteins were
x g for 1 h to remove were determined, and
N. Senate
that
Chemical were (Pierce, ml of pro-
Co.)
up to I mg/ml in dimethylsuiphoxide. After bound NHS-biotin was removed by dialyzing
for Reproduction
so
(Sigma
Company, St. Louis, MO) were reconstituted and against 0.1 M NaHCO3, pH 8.4, Protein concentrations measured by using the Coomassie protein assay
the
Inc.,
in
results
of this report.
method
lyophilized
toferrin binds to sperm and appears to represent a major component of the sperm-coating antigens [13-16]. Lactoferrin binding to sperm is reversible, because lactoferrin is gradually released from spermatozoa after they penetrate
Health
if factors The
of comple-
factor
Accepted June 12, 1990. Received February 26, 1990. ‘This work was supported in part by the Methodist 2Correspondence: Christian J. Thaler, M.D., Center plantation Immunology, Methodist Hospital of Indiana, vard, Indianapolis, IN 46202.
of these
asked
binding.
SP contains
lactoferrin.
lactoferrin
ization
have
uefaction at room temperature (RT), samples were centrifuged at 10 000 X g for 5 mm to remove cells and spermatozoa SP was stored at - 20#{176}C and studied immediately after thawing.
logic functions. Lactoferrin was reported to inhibit antibodydependent cellular cytotoxicity and to augment natural killer activity
designated
that
We
Seminal plasma samples from 6 healthy men were studied. Samples were monitored according to Thaler et al. [18] to exclude genito-urethral infections. After spontaneous liq-
in tricho-
has focused and cellular
in lactoferrin
MATERIALS
leukemia
enzymes
tract.
involved
studies indicate specific affinity for
[1]. About 80% of SP iransferrin is secreted by Sertoli cells [2, 3]. Transferrin binds to early spermatogenic cells but not to mature sperm [3]. Lactoferrin is secreted by prostate and seminal vesicles and is absent from testes and epididymides [4]. A variety of highly diverse functions have been described for lactoferrin. These include antimicrobial
[5,6],
reproductive be
our
(SP)
effects
female
SP could
overnight
at
0.15 M NaC1, pH 7.4 ultracentrifuged at 100 000
aggregates. aliquots
freshly made 4 h at RT, un-
were
Protein stored
concentrations at -20#{176}C.
LACTOFERRIN
Binding
BINDING
Assays
Wells
of Immunlon II microtiter plates (Dynatech Labs., Alexandria, VA) were coated overnight at 4#{176}C with SP diluted I :20 in PBS. Control plates were directly coated with biotinylated lactoferrin and transferrin at different concentrations. Wells were washed twice with PBS containing 0.05% Tween 20 (PBS-Iween), blocked with 2% BSA in PBS for 1 h at RI, and washed once with PBS-Tween. Biotinylated lactoferrin and transferrin diluted in PBS-Tween were added for 30 mm at RI. For competition studies, 0.1 pM biotinylated lactoferrin was incubated with different amounts of unlabeled lactoferrin, transferrin, or human IgG (Sigma Chemical Co.). The wells were were washed 3 times with PBS-Tween, oxidase
incubated
for
(HRP)-conjugated
30
mm
with
Streptavidin
horseradish
(Sigma
per-
Chemical
Co.;
diluted 1:1 000 in PBS-Tween), and washed 5 times. Color reactions were developed by adding 100 pA of substrate buffer containing 0.42 mM tetramethylbenzidine (Miles Co., Naperville, IL) and 0.0045% H202 in 0.1 M sodium
acetate
buffer,
pH
6.0. The
color
reaction
was
IN
MOLECULES
cubated for 2 h at 4#{176}C by using an Immunetics Miniblotter 25 (Cambridge, MA). The membranes were washed for 1 h in PBS-Tw#{232}en with several changes, then incubated with HRP-conjugated swine anti-rabbit lg (DAKO Corp.; 1:250 in PBS-Tween)
described
wells. All assays were accomcontrol that was incubated only with
HRP-conjugated streptavidin, and net OD5 were calculated by subtracting the background OD values. With biotinylated lactoferrin and transferrin coated directly to the plate, only
the incubation strate reaction
with
assay
variations,
were
compared.
streptavidin and subwas the influence of interonly data obtained within the same assay The intraassay thplicate variability was 8.6%.
Gel Electrophoresis, Polyacrylamide continuous
4-16%
HRP-conjugated done. To avoid
Immunoblotting
and
gel electrophoresis gradient
Ligand
separating
gels
Blotting
was done
(PAGE)
and
4%
in
stack-
ing gels with 0.1% SDS (Research Organics, Cleveland, OH) according to Laemmli [20]. SP samples were solubilized by boiling for 3 mm in 2% SDS containing 125 mM TRIS-HCI, pH 6.8, and 20% (v/v) glycerol. For immunoblotting, an SP sample of 0.25 mg protein was loaded per well. Markers of low molecular mass (Pharmacia Inc., Piscataway, NJ) were used to calibrate relative molecular mass (Mr). For ligand blotting, 0.1 mg of protein was loaded per well, and the rvlW-SDS-Blue Kit (Sigma Chemical Co.) of prestamned molecular mass markers was used according to the technical bulletin
Before
provided. blotting,
incubated for 2 h in 4 M urea buffer, pH 8.0, containing 10 mM TRIS and 50 mM NaC1 to allow renaturation [21]. Transfer to 0.2-p.m pore size
nitrocellulose
Centre, NY) ethanolamine,
a transfer Francisco,
SDS-PAGE
gels
membranes
was performed 25 mM glycine,
were
(Bio-Rad
Labs.,
electrophoretically and
20%
were
washed
ex-
Co.) and
diluted 1:400 developed as
above.
stopped
coefficient
triplicate
at RT. Membranes
conjugated Streptavidin (Sigma Chemical in PBS-Tween. Membranes were washed
calculated
from
for 30 mm
tensively in PBS-Tween, and reactions were visualized in color development buffer containing 60 mg of 4-chloro-1naphthol (Bio-Rad Labs.) in 20 ml methanol and 60 p.1 of 30% H202 in 100 ml of PBS, pH 7.4. For ligand blotting, membranes were cut, and individual lanes were incubated for 4 h at RT in 0.2 p.M biotinylated lactoferrin, transferrin (in PBS-Tween containing 0.5% BSA), or buffer only. After washing, membranes were incubated for 1 h at RI in HRP-
Statistical
by a PBS-Tween
713
PLASMA
blocked for 1 h with 2% BSA-PBS. For immunoblotting, rabbit anti-lactoferrin (DAKO Corporation, Santa Barbara, CA) and normal rabbit serum (1:500 in PBS-Tween) were in-
30 1.11of 2 M H2S04, Optical density (OD) of each well was measured at 450 rim in a Multiscan Microplate Reader (Flow Labs., McLean, VA). Mean OD readings were
by adding
panied
SEMINAL
The
Analys&c assessed for lacof the correlation of variance was used to and trarisferrin binding
comparability of biotinylation and transferrmn by calculation
toferrin
(r). A two-way analysis compare OD values for lactoferrin to SP from six individuals [23]. Crossed
Immunoelectrophoresis
Crossed
from
immunoelectrophoresis
containing
was
et al. [24]. SP samples
Axelsen
analyzed,
was
from
with 50 p.1 SP electrophoresed Tricine buffer IV (Bio-rad
done as modified 3 individuals were in 1% agarose
Labs.,
Richmond, for 3 h at constant
gels VA), cur-
pH 8.6. First dimensions were done rent of 13 mA per plate. Second dimensions were done overnight with 8 mA. Rabbit anti-lactoferrmn (DAKO Corp.) was used at dilutions ranging from 1:50 to 1:200, and rab-
bit anti-transferrin (DAKO Corp.) was used at 1:200. The system was standardized by electrophoresing 20 p.1 purified lactoferrin (1mg/mi in PBS) and 50 p.1 transferrin (20 p.g/ ml in PBS). Lactoferrin preparations with different iron saturations were compared. Lactoferrin from Sigma Chemical Company was completely iron saturated, and lactoferrin from ICN
Biochemicals
(Cleveland,
OH)
was
22%
saturated.
After electrophoresis, plates were rinsed extensively, dried, and stained for 30 s by using 0.5% (w/v) Coomassie Blue R Ultrograde (LKB Instruments, Inc., Gaithersburg, MD) in 45% ethanol with 10% acetic acid and H2O. Plates were destained with a mixture of 25% propanol, 10% acetic acid, and
H20.
Rockville
RESULTS
in 25 mM
methanol
apparatus from Hoefer Scientific Instruments CA) overnight at 0.15 amps [22].Membranes
by using
Binding
(San
lactoferrin
were
assessed
studies and
SP were transferrin. The
for lactoferrin
with
and
done extent
transfernn
by using biotinylated of biotinylation was preparations that were
714
THALER
ET AL. Net
OD45Onm
0.4
0.7
0.6 0.5
0.3
0.4
0.3 0.2 0.2
0.1 0.1 0 16
3.2
400
80
2000
protein IpMI
biotinylated #{176}-Lactoferrin
---
Transferrin
0 Lactoferrin
FIG. 1. Extent of biotinylation compared for lactoferrin and transfernin. Different concentrations of biotinylated lactofemn and transfemn were coated to microtiter plates. 00 values were significantly correlated (r = 0.99, p < 0.001), indicating that the lactoferrin and transfernin used for binding studies were
0.6
comparably
labeled.
Transfernin
FIG. 4. Binding of 0.1 M biotinylated lactoferrin and transfernin to SP samples from 6 males. Data points that correspond to individual samples are connected. When compared as groups, 00 values for lactoferrin binding were significantly (p < 0.0001) higher than for transferrin binding.
#{176}tj45OnmI
0.5
0.4
0.3
0
0.2
0.1
0 100
50
25
12.5
6.25
[nMl -
-
Lactoferrin
Transfernin
-4-
Buffer
Only
FIG. 2. Binding of biotinylated proteins to seminal plasma coated to microtiter plates. Lactoferrin bound dose dependently to SP. Transferrin showed only background binding (buffer control). Lactofernin did not bind to wells not coated with SP (OD ± STD for 100 nM biotinylated lactoferrin: 0.003 ± 0.003). Net
OD
0.7
1450nm1
0.6 -e 0.5 0 -
0
10
20
40
80
160
4-
Unlabelled
320
protein
unlabelled Lf
4-
640
1280
-
2560
5120
InMI
Unlabelled
Trf
FIG. 3. Competition studies: binding of 0.1 M biotinylated lactoferrin to SP-coated microtiter plates was studied with different concentrations of unlabeled lactoferrin and transferrin present. Unlabeled lactoferrin but not transferrin dose-dependently inhibited the binding of biotinylated lactoferrin to SP.
FIG. 5. Seminal plasma was separated by SOS-PAGE (4-16% linear gradient). Proteins were electrophoretically transferred to nitrocellulose membranes. Membranes were incubated with 0.2 M biotinylated lactoferrin (LF), transferrin (Tn), or buffer only 0. Lactoferrin bound to bands corresponding to approximately 120, 60, and 30 kda. Left lane shows molecular mass standards.
BINDING
LACTOFERRIN
MOLECULES
coated direcly to microtiter plates. Streptavidin binding to biotinylated lactoferrin and to biotinylated transferrin were
(r
significantly correlated
comparable
biotinylation
Microtiter
plates
Biotinylated microtiter nylated
lactoferrin
plates,
(Fig.
were
of biotinylated
binding
but
transferrin
p
0.99,
=
1).
coated
with
2). Biotinylated
and BSA-blocked of lactoferrin binding biotinylated lactoferrin
Specificity incubating labeled
proteins.
SP
and
tested for
lactoferrin, transferrin, or buffer only. bound dose-dependently to SP-coated no binding was observed with bioti-
(Fig.
Unlabeled
Molecules ther
lactoferrin
did
not
wells. to SP was studied by in the presence of un-
lactoferrin
samples
from to
by
that
by SDS-PAGE nitrocellulose
and
with
were
When lyzed
decrease
lactoferrin
binding
phoresis.
tinylated
lactoferrin
to SP also
bation results
not
with BSA or human IgG (data indicate that lactoferrin binding
restricted
to biotinylated
Studies with SP samples that lactoferrin binding was ual differences were values for lactoferrin
higher
than
noted. binding
for transferrin
3). Binding
not affected
not
of bioby coincu-
shown).
These
to SP is specific and
lactoferrin.
When
were binding
compared significantly
(Fig.
sp
ent
This
showed
Individas groups, OD (p < 0.0001)
For
all
or with
HRP-conjugated from it was
corresponded two peaks same
binding molecules, in SP by using resulted
in two
in a single
6b). if such
ter
of noncovalent
peaks
The
cathodic
differences bonds,
could
be observed
SP was
sp
--
Trf
A
B
5).
of differpeak
lactoferrin. incomplete
4).
Lf
(Fig.
The im-
SP by using the antibody to transferrin resulted peak corresponding to purified
transferrin (Fig. To investigate dissociation
membranes
not shown). in association
precipitation
the position of purified partially fused, indicating identity (Fig. 6A). Analyzing
precipitation
samples,
studied immunoimmunoelectro-
to
rabbit
SP
1 donor were ananoted that 1 20-kda
we crossed
mobilities.
and
SP
trans-
avidin
were
conditions
SP
to bands that corresponded and 30 kd. No reaction was
SP samples experiments,
electrophoretic
munological
from 6 healthy males present in all samples.
electrophoretically
60-kda bands varied in intensities (data To investigate if lactoferrin in SP is present
with lactoferrin reactive lactoferrin
fur-
blotting.
tested. SDS-solubilized
transferrin
with
were
ligand
membranes.
biotinylated
3 consecutive in separate
lactoferrin
and
and
bound 120, 60,
incubated
decreased binding of biotinylated lactoferrin. Incubating biotinylated lactoferrin with unlabeled transferrin did not was
of binding
SDS-PAGE
3 individuals were
biotinylated lactoferrin with approximately observed
715
in SP capable
separated
ferred
dose-dependently
to SP (Fig.
PlASMA
characterized
was
to PBS-coated
bind
0.001), indicating
>
IN SEMINAL
FIG. 6. Crossed immunoelectrophoresis done with SP resulted in two peaks of immunoreactive lactoferrin (A, top). Only the cathodic peak corresponds to purified lactoferrin (A, bottom). The two peaks are partially fused (arrow), indicating incomplete immunological identity. SP transferrin immunoprecipitated as one peak (B, top), which corresponded with purified transferrin (B, bottom).
analyzed
afby
716
THALER
SDS-PAGE
Western a doublet
blot analysis. Rabbit anti-lactoferrin ranging between 60 and 70 kda (Fig.
and
reacted
with
7). This
SDS-PAGE/immunoblot data [25] and with
published analyzing
naturing
purified conditions,
trophoretic
pattern
is in agreement
with
the results we obtained when lactoferrin (data not shown). Under deSP lactoferrin corresponds in its elec-
mobility
with
purified
We
have
studied
the
iron
in SP. Transferrin corresponded
lactoferrin.
binding
to purified transferrin from blood plasma. In contrast, SP lactoferrin analyzed under these conditions resulted in two distinct precipitation peaks. The anodic lactoferrin peak observed in SP could represent a form of lactoferrin that is noncovalently
The
proteins
lactoferrin
analyzed by crossed imin its migration pattern
kDa
bound
formation
known
DISCUSSION
and transferrin munoelectrophoresis
ET AL.
to lactoferrin
of complexes
macromolecules
binding
between
in
factors
(LF-BF).
lactoferrin
SP was
proposed
un-
and by
Hekman
[26], who also observed different electrophorectic mobilities of purified lactoferrin and lactoferrin in SP. The presence of lactoferrin binding macromolecules in SP is supported by our studies done with biotin-labeled lactoferrin and SP samples that were coated onto microtiter plates. With this approach, labeled lactoferrin could compete with SP lactoferrin for binding to LF-BF. Our experiments showed that lactoferrin bound dose-dependently to immobilized
SP. This
by coincubation
with
it is specific and trol incubations
not
binding
was
unlabeled
competitively
lactoferrin,
restricted
to the
inhibited
indicating
labeled
that
protein. Contransferrin,
were done with biotinylated bind to SP samples. Testing biotinylated transferrin lactoferrin that was directly coated to the wells showed comparable extents of biotinylation. Thus, the differences observed for transferrin and lactoferrin binding to SP were not due to differences in biotinylation. The presence of LF-BF in SP was supported by SDS-PAGE and ligand blotting. Molecular masses of LF-BF were estimated as 120, 60, and 30 kda. When serial samples from one donor were examined, the 120- and 60-kda bands were variably expressed. It is possible that the 60- and 30-kda bands represent fragments resulting from proteolytic digestion of the which
94 67
120-kda
did
not and
band
that
The biological
43:-
suggested sociation
that
with
BF are such
occurs
during
liquefaction
role of LF-BF is not known. lactoferrin
other
binding
seminal
proteins,
and
to
proteins.
that
they
sperm
It is possible represent
properties
fer from
of complexes
those
other systems can specifically
2O.1-
between
lactoferrin
[28] in asthat
LF-
of a sperm immunoelec-
part
coating complex. Our studies using crossed trophoresis indicate that physicochemical
3O
[27]. Boettcher occurs
and and
antigenic LF-BF dif-
of purified lactoferrin. It has been shown in that changes caused by binding of cofactors alter affinities of ligands to membrane re-
ceptors [29]. Thus, it is possible that the binding of lactoferrin to LF-BF regulates its affinity to sperm. Speculations on a possible role of lactoferrin-binding to sperm are influenced by the variety of functions that have been described for lactoferrin. Bound lactoferrin, through
14.4-
its anticomplementary antibody-dependent protect sperm in the
b FIG. 7. SP lactoferrin studied under denaturing conditions with the use of SDS-PAGE (4-16% linear gradient) and Western blot analysis. Rabbit antilactofernin (1:500) reacted with a doublet ranging from 60 to 70 kda (Lane a). No reactivity was observed with normal rabbit serum (1:500; Lane b). Left lane shows molecular mass standards.
effect [10,30] cytotoxicity of immunocompetent
and its inhibition of monocytes [9], could environment of the
female reproductive tract. Lactoferrin-binding analogous to the mechanism described in Trichomonas taginalis [8] could induce enzymes of spermatozoal metabolism and aid increased metabolic possibilities might influence
needs indicate
on physicochemical
at the time of ejaculation. Such a biologic role of LF-BF and their properties
of lactoferrin.
BINDING
IACFOFERRIN
MOLECULES
ACKNOWLEDGMENTS assistance. suggestions.
split
PF, Zaneveld ejaculates.
J
UP,
Reprod
Propping Fertil
D, Schumacher
1975;
JD,
Reiter
Biochim
agents. 6. Arnold
RB, Cole
ence
M, Balboni seminiferous
dysfunction
in man.
GC. Immunostaining tubules.
Fertil
Ste-
1977;
A, Vaalasti T, Tuohimaa P. Localization of lactoferrin in the IntJ Androl 1989; 12:179-186. B. Inhibition of bacteria by lactoferrin and other iron-chelating Biophys Acts 1968; 170:351-365. MF, McGhee JR. A bactericidal effect for human lactofernn. Scitract.
197:263-265.
Y, Amagasaki T, Jacobsen cell lines. Blood 1987; 70:264-270,
7. Yamada 8. Peterson
host
and gonadal
L, Vaalasti
reproductive
5. Oram
of human
45:536-541.
4. Wichmann
male
Components
43:249-267.
2. Holmes SD, Lipschultz U, Smith RG. Transferrin Fertil Steril 1982; 38600-604. 3. Vanelli BG, Orlando C, Barni T, Natali A, Serio of transferrin and transferrin receptor in human nil 1986;
GFB.
KM, Alderete
lactoferrin
binding
JF. Iron
DW, uptake
by 7)ithomonas
Green and
B. Lactofernn increased
usgina8s
binding
intracellular receptors.
J
by leukemia activity follow Exp Med 1984;
160:398-410.
9. Nishiya K, Horowitz DA. Contrasting effects of lactoferrin on human lymphocyte and monocyte natural killer activity and antibody-dependent cell-mediated cytotoxicity. J Immunol 1982; 129:2519-2523. 10. Kijlstra A, Jeurissen SHM. Modulation of classic C3 convertase of complement by tear lactoferrin. Immunology 1982; 47:263-270. 11. Broxmeyer HE, Smithyman A, Eger RB, Meyers PA, De Sousa M. Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production. J Exp Med 1978; 148:1052-1067. 12. Broxmeyer HE, Ralph P, Bognacki J, Kincade P, De Sousa M. A subpopulation of human polymorphonuclear neutrophils contains an active form of lactoferrin capable of binding to human monocytes and inhibiting production of granulocyte-macrophage colony stimulatoty activities. J Immunol 1980; 125:903-909. 13. Hekman A, Rumpke P. The antigens of human seminal plasma. With special reference to lactoferrin as a spermatozoa coating antigen. Fertil Steril 1969; 20:312323.
15.
Li T5, Shulman tions
REFERENCES 1. Tauber
SEMINAL
14. Robert TK. Boettcher 1969; 18:347-350.
Bill Bussell for photographic andJudie Aitken for secretarial B. Coulam, M.D., and Alajos Berczi, Ph.D., helped with valuable
We thank Carolyn
IN
BL Identification
of sperm
S. hnmunoelectrophoretic
fractionation
after
717
PLASMA
by various
methods.
coating
antigen.
J Reprod
analysis of human seminal Internat J Fertil 1971;
plasma
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