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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complement

activity

by lactoferrin