BIOLOGY

OF

REPRODUCTION

46,

(1992)

721-732

Phagocytosis of Immunoglobulin G and C3-Bound Human Sperm by Human Polymorphonuclear Leukocytes Is Not Associated with the Release of Oxidative Radicals1

J.

OSMOND

Departments

of Obstetrics

and

D’CRUZ,3

BAO-LE

Gynecology3

WANG,4

and

and

City,

G.

University

Pathology,4

Oklahoma

GILBERT

Oklahoma

HAAS,

JR.2’3

of Oklahoma

Health

Sciences

Center

73190

ABSTRACT antibody

Antisperm by phagocytic with

swim-up

itored

sperm

by the

release

were

used

zymosan

when

compared release

PMN

maximally

were release

with

in the

was

as positive

and C-positive burst

when

of ASA-positive

with

sera,

ASA-positive

and to either

limited

release

by PMN

not

requires

fails

the

binding

to release

reactive

to the

ingestion

be coupled

polymorphonuclear of defense against

malignant burst

nucleated

following

activated

cells,

phagocyte

reactions

including

Superoxide released

oxygen

oxygen

initial anion

species;

formation

of sperm

respisub-

toxic

hypochlorous

and

singlet are that

oxygen

the are

in the

cells

occurs

follow[4]. The

the

fertilizing

malian

sperm

sperm

are

December

4, 1991.

Received

September

25,

‘Presented

in part

OK

at the

of Reproduction,

2Correspondence: 73190.

Gilbert FAX: (405)

oxidative

highly

Accepted

Immunology City,

from

damage

susceptible

by PMN.

to oxidative

complement assay,

sperm does

by not

Annual

Meeting

G. Ham,

Jr.,

of the

Virginia, MD,

P.O.

American

June Box

23-27,

26901,

for

Patient

with

PMN

high

level

of unsaturation

also because, to institute motile sperm

results,

for PMN/sperm [8]. whether

the

tract, alternative sperm/phagocyte on PMN/sperm

possibly

because

chemotaxis respiratory

(C)

and

burst

found requires

involve

the

an

that

ability

to trigger

in vitro

PMN/sperm

ASA-

and

intimate release

plays

killing

a role

of

motile

effect of sperm on PMN oxof antisperm antibody (ASA)

their

Using

none

fraction of seminal

[7] or oxidative

immune-mediated

we studied the in the presence

of

unlike somembrane from acti-

C-dependent

killing contact

oxygen

oxi-

interac-

PMN/sperm

of toxic

AND

PMN

of but

metabolites.

METHODS

Sera was

collected

from

22 infertile

patients

(12

men

and 10 women) positive and 3 control patients (2 men and 1 woman) negative for serum IgG ASA as determined by an indirect immunobead test (IBT) [10]. The sera were stored at - 70#{176}C. The serum IgG ASA titers were tested using five-

the

1991. 4SP-720

associated

performed with a purified whole semen or unselected

MATERIALS

damage

Society

conflicting

PMN

Mam-

Serum

Charlottesville,

we

that

of ASA-

phagocytosis

processes

of the

were rather,

metabolites.

incubated

indicated

results

fatty acids and lack a mechanism to protect the

and

by PMN, functions

dative

1991. 11th

used

nonimmune-

sperm idative

tion

tic cell reaction addition, the

[51. In

were

burst response To ascertain

two powerful known to kill

stituents of sperm or seminal plasma have been shown either to be chemotactic [6,7] or to trigger an oxidative burst of the PMN [8]. The potentially toxic armamentarium of PMN could be destructive for both microbes and spermatozoa within semen unless mechanisms have evolved to protect

leukoc

had

acid

cervix

of PMA-induced of PMN

2)

resulted

sperm.

because

membrane cells, they [91. Hence,

mol-

and

types involved in this PMN and macrophages

3) metabolic C-bound

interaction

in

human

surface;

and

of these studies motile sperm;

agents

are con-

main cell phagocytic

C to the sperm

production In contrast,

kinetics

These

mon-

opsonized

also

phagocytosed.

The

contact.

0

sperm

preparations

and

and

vated phagocytes in the female genital mechanisms must be operative at the membrane level. Early studies performed

process,

of the whereas

of other

(H202),

(0H),

their matic repair

to a variety

product (Ok),

anions and H202 by the phagocytes

deposition

and

of ASA-positive

not

Cytocentrifuge

at the site of PMN/sperm ASA

metabolic

In this

a variety of cells in vitro [31. and massive influx of leukocytes

efficiently A rapid the

to the

radicals

target.

molecular

peroxide

hydroxy

ecules. oxidants

ing

lead

an oxidative their

[1, 21. The superoxide

hydrogen

(Hod),

with

reduces

of toxic metabolites ratory burst is the sequent

undergo

interaction

were

C.bound

(PMN)

activity,

(PMA)

did not enhance

or PMA.

zymosan

sperm.

of both

(PMN), the body’s malignant or non-

that

burst

acetate and

in part

leukocytes

respiratory

by PMN

sperm

by phagocytes

leukocytes microbes and

PMN

myristate

of ASA-positive

opsonized

C-bound

for

sperm

to be caused

is thought

polymorphonuclear

Phorbol

C-bound

C-negative

and

INTRODUCTION Blood first line

and

of ASA-positive 0

(H202).

Phagocytosis

#{248}in response

revealed

sperm

human

sees and assayed

peroxide

ASA-negative

to human

purified

ASA-negative

hydrogen

presence

by PMN

may

co-cultured

to release sperm

sperm

with

compared

sperm

and

and

Phagocytosis

of sperm

by the

activity

(Ofl

injury

immune

we

process,

of ASA-positive

controls.

and C-bound

(C)-mediated

this

anion

stimulated

of motile

respiratory

presence

unaffected

1) phagocytosis

complement

investigate

of superoxide

of H202

ASA-positive

positive

and

To

to incubation

in minimal O

(ASA)-

neutrophils.

Oklahoma

271-8547.

721

722

D’CRUZ

fold

serial

serum

dilutions

ranging

from

1:25

to

1: 15 625.

ET

AL.

viability

PMN;

Routine clinical tests for serum IgG, IgA, and 1gM ASA by the indirect IBT in previous batches of sera (6-9 mo) revealed a significant increase in IgG ASA, and only 3 of 22

test

ASA-positive

Opsonization

of 1:15625. when 20% or more binding (6-9

of antimo). In

directed exclusively to head, and in 15 of the

directed to multiple regions the sperm head. Six of the

exhibited an indirect IBT titer rected to the acrosomal region

of the 22 sera

for IgG ASA of >1:3 of the sperm head.

125

Semen 12)

was

obtained

who

were

by masturbation consistently

ciated IgG and IgA by a direct ples that met the requirements

from

negative

fertile

for

men

sperm-asso-

IBT [11]. Only of the American

semen samFertility So-

ciety 1990 guidelines for normal donor semen were used for this study. The semen was allowed to liquefy for 30-60 mm at 37#{176}C, an aliquot was removed for a conventional semen analysis, and the with Tyrode’s salt solution

were

smears.

serum

recentri-

60-90

mm

at 37#{176}C in 5%

C02/95%

air.

After

Chemical

Co.)

was

of 12 mg/mi, g for

X

8 mm,

suspended boiled

washed

in 0.9%

for

three

1 h, centimes,

this

As:say

(100

with PMN was assessed microscopy of fixed and l)

of the

by direct stained cell

suspension

PMN

(106)

from

each

of 22 IgG ASA-positive

and

(Fisher

Diagnostics, PMN

was

Orangeburg,

NY). The

determined

by

Adherence per PMN;

were

was defined 100 PMN cells

expressed

Flow Cytometric and C5b-9

as the

of three

determinations.

of Sperm-Bound

C3d

of 22 IgG ASA-positive sera and 3 ASA-negative for 60 mm at 37#{176}C. The deposition of activated

tration stored

ucts

of the

Isolation

1% BSA, and within 1-2 h.

of PMN

Peripheral volunteers

venous blood was obtained from healthy (n = 5) of both sexes 2 1-40 yr of age.

was

into

10-mi

heparmn-coated View, CA).

drawn

The

glass PMN

centrifugation ical Corp.,

on Polyprep Westburv, NJ)

instructions. hypo-osmotic The

croscopic Wright’s

tubes

(Becton separated (Accurate according

containing Dickinson, by density

60 USP

(HBSS,

then

washed

Gibco

examination stain revealed

twice

Scientific and Chemto the manufacturer’s

Laboratories,

with

Hanks’ New

balanced Island,

NJ).

To assess

salt Mi-

of the cell suspension stained by that more than 95% of the cells were

the

were

C-fixing

incubated

initial

ability with

ml

of a 1:10

Scan

detail for

(Becton

[12].

surface-bound

Dickinson).

deviations sera was

Transm&ion

elsewhere

(dsb-9)

dilution

of

control prodon

in

terminal

donor

antiusbeen

concomitantly

and

0.5

sera,

C pathway

described

(C3d)

of ASA-positive

donor sperm were detected with polyclonal rabbit bodies to human C3d and C5b-9 complex, respectively, ing immunofluorescence flow cytometry. The assay has

standard negative

Mountain gradient

Erythrocyte contamination was minimized by lysis in buffered 0.15 M ammonium chloride.

PMN were

solution

vacutainer beads were

adult Blood

3 ASA-

of at least The re-

each sera

10 sperm/mi in Tyrode’s temperature (RT) and used

X

of sperm-

sperm

X

were

microscopy

as the binding were evaluated.

mean

Determination

number

light-field

interval of “swim-up,” the supernatant containing more than 90% motile sperm was aspirated and centrifuged (500 x g for 5 mm). The resulting sperm pellet was washed once with 5 ml of Tyrode’s 1% BSA, resuspended to a concenof 100 at room

and

in 0.9% NaC1. 5 ml of a 1:5 di-

particles/mI.

108

samples

sults

(500 x g for 4 mm) to form a pellet. The tubes were capped and incubated at a 45#{176} angle for approxi-

mately

prepara-

negative sera were added to a final dilution of 10%, and the tubes were incubated at 37#{176}C in a water bath. After 2030 mm, 20 pA of the reaction mixture was plated on a glass slide, air-dried, fixed, and stained with a Leukostat stain kit

aliquots,

and

used.

A (Sigma

Aliquots

resuspended,

0.5-mI

exclusion

placed into 12 x 75-mm tubes to which 100 pA of 10 106 motile sperm in Tyrode’s 1% BSA was added. Diluted

(1 000x). 3 sperm

into

until

of sperm using light

was was

divided

ice

Association visualization

The sperm pellet 1% BSA. The pellet

fuged loosely

on

Interaction

centrifuged at 600 X g for 6 mm. washed twice with 5 ml of Tyrode’s

1%

PMN

PMN/Sperm

bound

7.4

The

of Zymosan

to

pended

and

pH

by the

lution of ASA-negative serum and allowed to incubate for 30 mm at 37#{176}C, washed four times in HBSS, and resus-

BSA)

BSA,

to 95%.

at 1 300

(Tyrode’s

1%

dye

equal

resuspended to 20 X 108 particles/mI zymosan pellet was opsonized with

threefold St. Louis,

containing

blue

or

at a concentration

trifuged then The

Trypan

than

stored

Zymosan NaC1

was diluted Chemical do.,

MO)

remainder (Sigma

tions

as assessed

greater

di-

of Sperm

Preparation

=

1gM response

positive occasions

IgG ASA were of the sperm

22 sera, the ASA were sperm surface, including

(n

antisperm

was

Sperm

C3d A degree

and of

were C5b-9

assessed by a FAd-

fluorescence

two

above the mean fluorescence of 3 ASAused to identify sera with C-fixing ASA.

Electron

Microscopy

(TEM)

of PMN/Sperm

Interaction To prepare

sperm

bound

to or ingested

by PMN

for TEM

examination, the cell suspension was washed in PBS and resuspended in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.5, for 60 mm at RT [13]. The fixed sperm pellet was washed in PBS, dehydrated in graded alcohols (35-

PMN/SPERM

INTERACTION

723

75%), and embedded in L. R White (London Resin do., London, U.K.) at 55#{176}C for 48 h. Ultrasections of 100 nm were cut with a diamond knife and collected on nickel grids. The sections were post-stained with uranyl acetate and lead

with

U/mg; Sigma) or 0.2% proteinase Sigma) or BWW without protein protease-treated and untreated

K (type XXVII; 2.4 U/mg; for 30 mm at 37#{176}C. Both sperm were washed twice

citrate

with

for

before

being

viewed

on

an

electron

microscope.

O

was

quantitated

reduction

incubated

with

sence fixing the

by the

motile

of sera (5%) ASA-positive, presence

superoxide

of ferricytochrome sperm

(10)

p.M

as a positive control. O by the addition

the

The assay of superoxide

concentration for 30 mm were

absorbance at 550

in control

tubes

and

heart,

specific for (3 000 U/mg, of 60 p.M. Afin a 37#{176}C water

collected

by centrif-

of the supernatants was nm, and the background

containing

ab-

3 non-Cpatients in

volume of 1.5 ml. 13-acetate (PMA, (DMSO) was used

was rendered dismutase

supernatants

ugation at 4#{176}C. The in a spectrophotometer sorbance

presence

in the

were

(106)

C (bovine

in a total 12-myristate sulfoxide

Co.) to a final of the suspension

cell-free

in the

ferricytochrome

type V, Sigma Chemical Co.) PMN incubated with phorbol 75 ng/tube) in 5 pA of dimethyl

bath,

PMN

from each of 3 ASA-negative, and 3 C-fixing ASA-positive

of 60

Sigma Chemical ter incubation

dismutase-inhib-

C [14].

only

buffered

Tyrode’s

1% BSA,

read abcyto-

presence

dismutase . 10’ PMN formation by [15]. There-

bursts, absence

sperm were

for then

cubation

30 mm tested with

sayed for O In studies

PMN-Mediated

transferred field

microscopy

of O generation induced for 60 mm at 37#{176}C with

mean

of two

sperm 5 ml of

a 1:10 dilution of C-fixing, ASA-positive serum in Tyrode’s 1% BSA. The sperm suspension was washed three times with 5 ml each of Tyrode’s 1% BSA and resuspended to sperm/0.1

ml.

control. In studies motile up

teinase Whitten,

to evaluate

sperm

donor

Sperm

on sperm

PMN were

K. Twenty-five and

Whittingham

incubated the

without effect

respiratory

of limited oxidative

exposed

to

million

motile

medium

serum

either (BW)

served proteolysis

function, trypsin sperm were

in

of swim-

or

re-

pro-

Biggers, incubated

that of PMA (75 above. effect

treated O

and release

ng)

of sperm

on

in the C-coated

untreated following

zymosan

or

PMA

trapping

PMN in-

and

as-

of O

by

were preincubated for 5 mm at the absence of 5 p.g/ml cytochalCo.)

in DMSO

and

then

incubated

i0 sperm in the presence of or ASA-positive serum. Sperm as a control. above.

The

1.5 in-

O

re-

Assay

cvtotoxic

sperm

effect

(6.5

X

containing

to

nonmotile

of PMN were

106)

either

10%

a slide,

and

sperm/100

the

sperm

oxidative added

metab-

to 2 x

C-fixing

(200

x).

assessments

percentages was

106

ASA-positive

The

results

of 100

of motile

enumerated were

sperm

The counts obtained were compared ilarly processed control suspensions out

ASA-positive

C-inactivated (56#{176}C/30 mm) ASA-positmve serum, or alone. After 60 mm at 37#{176}C, 20-pA aliquots were with

and bright-

expressed from

as the

each

sample.

with counts from simof motile sperm with-

phagocytes.

Quantitative NBT methods

as a

or

the

ative and C-fixing ASA-positive sera or medium alone, and the release of O was determined as described above. In studies to evaluate the effect of sperm on the kinetics

with opsonized sperm, swim-up incubated for 30 mm at 37#{176}C with

and

were preincubated 10’ ASA-positive and

X

Spermicidal

To evaluate

serum, medium

and

inhibitory

without serum served was assayed as described

in HBSS

PMN PMA.

(106)

30 mm at 37#{176}C with of 5% ASA-negative

PMN

experiments x 106) were

motility,

serum

opsonized

Chemical

motile

For (50

1X; 10000

(106)

at 37#{176}C. Both for cumulative

either

D (Sigma

for ml

the

PMN of 5

PMN, the phagocytes 37#{176}C in the presence

olites,

sperm (10); sperm without

sperm

release as described above. to evaluate the intracellular

fore, in studies to evaluate the effect of changes in PMN to sperm, 106 PMN were incubated in duplicate with motile sperm at ratios of 1:5, 1:10, 1:20, and 1:40. Each of these suspensions was incubated with a 5% dilution of ASA-neg-

by PMA, the PMN were incubated PMA (75 ng) in the presence or

to test

oxidative presence and

cubated leased

nonopsonized opsonized

type

Aliquot 3 incubated without serum The relative capacity of protease-treated to trigger PMN respiratory bursts in the

In experiments PMN

tion in the absence and presence of superoxide and expressed as nmol O production mm using an extinction coefficient of E = 21.1.

and with

assessed

pancreas

presence of serum was compared with under identical conditions as described

chrome C was subtracted. All experiments were performed in duplicate. The quantity of O produced by the PMN was calculated from the difference in ferricytochrome C reduc-

absence of opsonized were also incubated

(porcine

of 5% ASA-negative

serum, respectively. served as a control. and control sperm

asin

The rate of oxygen consumption and O PMN is dependent on the ratio of PMN : particle

ttypsmn

suspended to 10’ sperm/0.1 ml in Tyrode’s 1% BSA. Both protease-treated and untreated sperm were divided into 3 aliquots. Aliquots 1 and 2 were incubated with PMN

02 Assay itable

1 ml of 0.25%

added

Reduction reduction of Nathan

to the

tubes

was

Tetrazolium

in duplicate:

0.4

ml

sera,

of 3 non-C-fixing

of PMN

by the

addition

of

(106);

the were 0.5

0.1% NBT (iO); 50 p.l

ASA-positive

ASA-positive sera; or 75 ng was incubated at 37#{176}C for

stopped

(NBT)

by use reagents

of phenol red dye) containing Co.); 50 pA of motile sperm

of 3 ASA-negative or 3 of C-fixing reaction mixture

Blue

of PMN was measured et al. [16]. The following

ml of HBSS (free (Sigma Chemical

reaction

of Nitro

sera,

of PMA. 60 mm.

of 1 ml of 0.5

The The

N HC1.

D’CRUZ

724 The tubes were the supernatant

centrifuged was aspirated,

at 400 X g at 4#{176}C for and the reduced dye

15 mm, was ex-

tracted with the addition of 1 ml of DMSO. The absorbance of the solubilized formazan from the reduced NBT was determined in a spectrophotometer at 572 nm against a DMSO blank.

ET AL. B

60

o 60 0. #{149}0

20

z 0.,

20 Sperm

0

Localization

of Reduced

Direct

NBT

microscopic

visualization

of NBT

reduction

was

performed after the cell suspension had been washed in HBSS and 20-p.l aliquots of the reaction mixture were plated on glass slides. The cell smear was air-dried, fixed in methanol,

stained

croscopy

with

for

0.2%

the

eosin,

presence

and

of dark

observed blue

by

grains

light

40

60

Pos,tioe

80

0

20

(5)

for C3d

40

Sperm

60

Pos,t,oe

for

80

C5b-9

00

(5)

FIG. 1. Correlation between the percentage of PMN with adherent sperm and the percentage of sperm positive for sperm-bound C3d (A) or C5b-9 (B) following incubation with 22 lgG ASA-positive sera. The binding of C3d and C5b-9 to sperm was assessed by direct and indirect antibody fluoresceinated probes using flow cytometry to determine sperm fluorescence.

mithe

of formazan.

significance

son’s

of

correlation

relationship

was

determined

by

Pear-

coefficient.

F1202 Aav Hydrogen

peroxide

mediated

and

release

horse

was

radish

quantified

peroxidase

by the

oxidation of phenol H2O2 concentration

red dye [17] using a standard (1-60 p.M). PMN (1 06) were

with motile sperm samples from each

(10) in the presence of 50-p.l serum of 3 ASA-negative sera, 3 non-C-fixing

curve incubated

of

Chemical Co.) in a total was incubated for 30 mm

at 37#{176}C, the was added

tubes were centrifuged, and to each tube. The samples

spectrophotometry

at 610

PRS to which were expressed

Binding Presence

plasma

is known

binding or PMN/microbe To evaluate the effect of sperm

incubated in the treated

with

5

to

to contain

X

both

factors

oxidative of seminal PMN,

15

10’ PMN

and

incubated was

1 ml

of

burst plasma X

106

in the

heat-stable for

and

PMN /sperm

response [18, 19]. on immune admotile

C-fixing,

presence or absence of 10% fresh (56#{176}C,30 mm) seminal plasma.

ture was suspension

of

was added. The results 10 PMN 30 mm.

immunosuppressive

herence

a blank

C-Bound Sperm to PMN Seminal Plasma

of Autologous

Seminal

10 p.1 of 1 N NaOH were analyzed by

against

10 p.1 of 1 N NaOH as nmol H2O2

of ASA- and

heat-labile

nm

sera or PMA containing

sperm

MA-positive

were serum

autologous or heatThe reaction mix-

at 37#{176}C. At 10-rn in intervals, 20 p.1 of the plated onto a glass slide, air-dried, fixed,

and stained by a Leukostat stain kit (Fisher Diagnostics) for the localization of PMN/sperm rosettes. Duplicate observations of 100 PMN were made, and the results were expressed as the mean ± 1 SD.

Statistical The termined ables

Analysis significance of differences by Student’s t-test. The

was

analyzed

by least

square

between relationship regression

means was debetween varianalysis,

and

C-Bound

the

on

sperm

adherence

of

PMN

bation with 22 and the capacity sperm.

and

examined

percentage

50 p.g volume

VI-A, Sigma suspension

of ASA-Positive first

C pathway

MA-positive solution

(PRS)

Binding We

MA-positive sera, and 3 C-fixing, (75 ng) in buffered phenol red of HRP (type of 1 ml. The

RESULTS

H2O2-

(HRP)-dependent

The

with

to PMN

of MA to PMN

adherent

IgG MA-positive of these sera

binding

Sperm

importance

and

the

sperm

that became 0.001) and

1 shows

incu-

and 3 MA-negative to fix C3 or C5b-9 on

of homologous

Figure

between the flow cytometric

the

following

d3d

and

a significant

to moand

positive

nonflow

correlation

percentage of PMN forming rosettes quantification of the percentage positive for sperm-bound C5b-9 (r = 0.595, p

sera donor

C5b-9

tile sperm after a 60-mm incubation in immune immune sera was quantified by immunofluorescence cytometry.

intact

by comparing

and the of sperm

C3d (r = 0.706,p 0.001) after incubation


0.05).

incubated unaffected

sperm

oxidative

trapping of O. For this purpose cytochalasin D, which has been

O

PMN

PMA.

with

control PMN. not suppress

Next, we tive products

C-bound

PMN with opsonized incubating the PMN

or

of PMN

and to release

not inhibit the subsequent interval by either opsonized

Q)

>

of PMN

D Q)

and

with untreated sperm Sperm motility was

proteolysis.

ability

press

C

compared conditions.

mechanism

PMN

to activate

possibilities: on the

sperm

1) a specific surface;

burst in PMN; an extracellular to sperm

was

a respiin-

2) sperm

3) PMN/sperm O; or 4) the

a) C-)

C 0

-a 0 U)

-o

membrane-bound.

The possibility of an inhibitor was tested after limited proteolysis of donor sperm and incubation with PMN in the presence of MA-negative or C-fixing MA-positive sera. Figure 6 reveals that pretreatment of sperm with limited amounts of protemnase K or trypsin did not increase O

Buffer

ASA

C

Sero

ASA+

C

Sera

ASA+

C

Sero

PMA

FIG. 5. NBT dye-reducing capacity of PMN and motile sperm incubated in the presence of ASA-negative and ASA-positive sera. PMN were incubated with sperm in the presence of HBSS, sera, or PMA. Each test was performed in duplicate, and the vertical bars represents the mean ± SD.

PMN/SPERM

C-fixing, mologous

Buffer

C

E

1.8

ASA

Serum

z

1.6

ASA+

Serum

a-

1,4

(0

1.0

cytosis immune

CN 0.8

0 U, 5)

ni nHn

0

E C

FIG.

6.

Effect

that

8, A and

Control

Proteinase

of sperm

proteolysis

at the failed

did

not

bind

B). Because

PMN/sperm to confirm

K

PMA

Trypsin

on the

to PMN

a localized

ability

were

release

of PMN

to produce

the and

NBT-negative of O

point of contact but the presence of an

present studies indicate that non induced by MA-positive

(Fig.

was

ASA- and

C-Bound

Seminal

plasma

idants

that

has the

been PMN

shown

on

phenomeis locally

the Binding

to possess

oxidative

of

both

response

to

antioxinteracof PMN-

bound sperm to trigger O was due to seminal plasma antioxidants associated with the sperm surface. When the kinetics of PMN/sperm association was compared in the

(Fig.

9).

autologous plasma,

After

seminal a marked

20 mm,

90%

of

plasma inhibition PMN

and heat-inactiwas observed

formed

rosettes

MA-positive and C-bound sperm. By contrast, only 35% of PMN were associated with sperm in the of fresh However, 30 mm

and heat-inactivated despite the continued after

the

addition

centage of PMN ward the control inal

involves acrosome,

ing

the

second

to

plasma

on

plasma,

that formed rosettes was values. Thus, the inhibitory PMN/sperm

interaction

the

was

micrographs

firmed the phagosome. activated defensins

sperm.

of sperm

intracellular In addition

The

oc-

an inleadstep

is

by sperm-bound C5bC3 alone can opsonize ingestion by PMN. The

activity

motile sperm the intracellular lytic action In previous

lationship

was

positive for came positive relationship the percentage

Since

in

PMN

toxic peptides of metabolically

that analogous recently have

[23].

by

the

con-

sperm within the enzyme arsenal, called active

amphophilic been shown present

pepto have

study

only

were ingested by PMN, it is conceivable that killing of the live sperm may be aided by

membrane-active

the

ingested

PMN are known to secrete in response to ingestion

spermicidal

C-mediated immothus play a role in in a woman who

destruction of to the lysosomal

[22]. It is of interest termed magamnmns

in a manner

analogous

to

of serum C5b-9. studies using flow

defensins

cytometry,

a biphasic

re-

observed

the

IgG for was of

between

percentage

of sperm

versus the percentage of sperm that besperm-bound C3d or C5b-9 [12]. A similar also observed in the present study when PMN forming rosettes was compared with

with

19% and presence

mean

shifted effect

of

balance between these two factors, i.e. bilization and immune adherence, may determining the fate of fertilizing sperm has antisperm antibodies.

Buffer

seminal plasma, respectively. presence of seminal plasma,

of seminal

immobilization

sperm can be directly immobilized 9, conversely, binding of MA and the motile sperm to enhance their

the

microbes

[19, 20] as well as inhibitors of sperm/phagocyte tion [18]. We determined whether the limited ability

presence of vated seminal

[21]. The present studies suggest that cell-mediated injury to MA-positive and C-bound sperm may

Electron

the previous studies oxidative burst, the

PMN oxidative C-bound sperm

Seminal Plasma Sperm to PMN

limit

to fix hoto promote

The first step PMN and sperm

cells tides of Autologous

which were shown surface, were able

cur in a two-step sequence. timate contact between the

detected

restricted. Effect

sera, sperm

associated with nonoxidative intracellular digestion of the sperm. In the presence of adequate levels of C and MA,

02 in the presence of ASA-negative and ASA-positive sera. Ten million untreated, trypsin-treated, or proteinase K-treated motile sperm were incubated with PMN in the presence of ASA-negative and ASA-positive sera. PMN and sperm incubated without serum served as controls.

Sperm

MA-positive C onto the

efficiently the binding of sperm to PMN. Inasmuch as C-fixing, MA-positive serum is a prerequisite for sperm binding to PMN, this process appears to be similar to microbial phago-

1.2

0

729

INTERACTION

Preincuboted ASA+ mid C+

with

sperm

C

E

per-

back toof sem-

Control

z a(0 0

short-lived.

c’-J

0

DISCUSSION Previous C are results

required of the

studies

have

for immune present study

demonstrated

(1) 5)

that

both

destruction of sperm suggest a mechanism

C plays this role. In examining with PMN in vitro, we found that to PMN in the absence of C-fixing,

the interaction motile sperm MA-positive

MA

and

[12]. The by which

of sperm did not bind serum. Only

0

E C

FIG. 7. Effect of preincubation of PMN with opsonized sperm on subsequent 0 release by opsonized zymosan or PMA. PMN were preincubated with ASA-positive and C-coated sperm followed by addition of opsonized zymosan or PMA.

730

D’CRUZ

FIG. 8. cultured with stained with rows) sperm the PMN. X

is that

of sperm-bound IgG, C3d, and C5b-9 revealed of the binding of IgG and C deposits observed C-fixing MA-positive sera [12]. One possibility

serum

C can

be

activated

the fluid phase by directly tivation of sperm surface ing

the

to such

C5b-9

complex

a possibility

cleaving protease

on the because

AL.

Light micrographs of NBT reduction at sites of PMN/sperm contact. A and B: PMN and sperm were coC-fixing, ASA-positive serum in the presence of NBT. The cell smears were then air-dried, fixed, and eosin. PMN-bound formazan positive (long arrows) and non-PMN-bound formazan negative (short arare evident (A and B). Inset (A) shows formazan deposition localized on the sperm head in contact with 1 000.

the percentage of sperm-bound C3d or C5b-9. Sperm-bound C5b-9 was more often detectable by flow cytometry than was sperm-bound C3d. In addition, immunofluorescent localization dissimilarity with some

ET

sperm sperm

by MA-bound

sperm

acrosomal

Our

data

enzymes

to activate serum nents in the fluid cleaves C3

in

serum C3 following acand selectively depositsurface.

the potential to cleave native surface protease of a protozoan

point have

flow bring

C3 to C3b [24]. Moreover, the parasite was recently shown

C by activating phase where the

the terminal C composurface protease directly

C3 and not by the surface deposition of activated Despite the lack of binding of C3 as detected by cytometiy, the binding of C5b-9 alone is sufficient to about sperm dysfunction in the absence of a mem-

[25].

brane recovery The binding receptors

on

mechanism of IgG and phagocytes

from C attack. C3 to corresponding is known

to generate

Fc and

C3b

a common

PMN/SPERM

INTERACTION

hanced

Inn

#{149}-#{149}10% #{149}-#{149}with

E

ASA+ C+ serum 10% Seminal plosma

80

(I-)

r

seminal

(‘l

plasma

#{149}

with

10%

a) U,

0

40

a:

have

been

adversely

E

0 U-

1‘0

0

30

20

Incubation

Time

was

(mm)

amounts

FIG. 9. Effect of human seminal plasma on the kinetics of ASA-and C-dependent immune adherence of sperm to PMN. Results are expressed as the mean ± SD of duplicate counts.

message

sequent positive stimulate

that

stimulates

phagocvtosis

oxidative bursts [26]. However, and C-bound sperm by PMN the release of extracellular

ferrictochrome tified by NBT ization

and

C-bound

zymosan)

stimulus.

of PMA C-bound

with PMN sperm did

Furthermore,

in the presence of not affect the kinetics

inability

of MA-positive

and

to stimulate

the

release

of O

be

inactivation

fore,

of the

of O

that

the

and

C-bound

the

respiratory

cannot

O-generating

by non-ingested

recognition sperm

and are

burst

C-bound attributed

in these

from

cells.

or

It is likely,

It is of interest

lular

pathogens

survive

within

phagocytes

dition, certain C3-binding target internalized without an oxidative

the

of

that

fail-

In

ad-

ods: superoxide reduction and

ferricytochrome addition of superoxide

metabolites

[29].

In

contrast,

the

unlikely from

failure

and

by sperm

because seminal

soluble

to trigger

of the plasma,

lummnol-en-

PMN

[19]. O

re-

presence of sperm-bound since sperm did not affect

to other stimuli. The transient seminal plasma on PMN/sperm

observed in this study may in the female genital tract.

somatic

cells

C

is known

to be

of reactive

factor

to induce

olites.

Thus,

the the

oxygen

limited

PMN

membrane-bound,

tween locally

be beneficial to sperm After the migration of

C5b-9

complex

stimulus

metabolites

for

[30]. The

ability

O, particularly [9, 31]. In the pres-

for the acrosomal

enhanced and

finding

specific region.

zona

sperm pellucidae

bound to sperm also to zona-free hamster

of MA-

and

C-dependent

oxygen

metab-

to sperm

physical

may

contact

be-

sperm. Alternatively, the weight O may be readily head

membrane channels,

deposition

to sperm sperm head

of these

heterologous

C-fixing MA ing of sperm

requiring

the sperm transmembrane

damage of the ability

of toxic mechanism

IgG and C-bound low-molecular

permeable through the C5b-9-induced account sperm’s

release

cytotoxic thus

PMN and generated

Oxidative peroxidation

meth-

for extracelluas well as other

ox-

stimuli

ent study, CSb-9-bound sperm did not by themselves reduce ferricvtochrome C, and they remained NBT-negative. However, upon contact with PMN, a localized release of O was evident, indicating the requirement of a PMN co-

be

sugoxi-

by two

oxygen

study,

lease was antioxidant

particulate

of sperm cells to spontaneously generate by abnormal sperm, is well documented

dative damage to fertilizing sperm. In this study, O was measured

reactive

In our

to both

to

an adequate PMN:sperm ratio protective mechanism prevents

specific by O

response

the production

environment gested that

makes the measurement O whereas NBT is reduced

idative

activation [18,20].

the presence of a low-molecular was shown to inhibit the PMN

either

host from oxidative damage. Our obsperm were incapable of triggering oxygen metabolites directly into the

dismutase lar free

studies suggested antioxidant that

a membrane

antigens are preferentially burst as one of the mech-

dismutase-inhibitable NBT reduction. The

Recent weight

or H2O2

interaction.

of sublytic

affecting intracel-

different

PMN/sperm

on

anisms to protect the servations that motile the release of reactive despite a similar

during

sperm

there-

[27, 28].

formed

in the female genital tract. It is of interest that deposition

triggering

ure to trigger oxidative metabolites without phagocytosis is one mechanism by which certain

of nonsperm Our experiments little or no oxi-

sperm from the seminal plasma, the ensuing toxicity of PMN/ microbe interaction would be evaded by the fertilizing sperm

of MA-positive the

produced could

plasma is known to suppress immune adherence of target cells to phagocytes

adhesion survival

MA-positive and of PMA-induced

phagocytosis

dissociated

not

incubation

system sperm.

did

other paththat pre-

(0.30 vs. 0.61 nmol mm 10 peroxidative degradation of low

the PMN oxidative response and reversible nature of

when chal(opsonized

simultaneous

The

scavenging

sub-

as quaninternal-

by PMN

O release a particulate

release.

to the

O and

sperm

the ability of PMN to trigger with a soluble (PMA) or

and

presence

ejaculate. generate

to

[8] lacks may reflect

presence of immune complexes The amount of measurable

that of O a possible

of H2O2

Seminal or immune

phagocvtosis of MAin suspension did not O as quantified by

C reduction or intracellular reduction. In addition, binding

of MA-positive

affect lenged

lower than suggesting

PMN),

by the

in the sperm

dative response in the activated C components.

z

intracellular

investigators bursts and

PMN oxidative bursts by chemiluminescence

affected

cells or senescent sperm demonstrated that motile

C

a.

by earlier

PMN oxidative oxygen species

vious studies investigating by seminal cells as quantified

II)

O

used

sperm-induced for the individual

generation of metabolic products derived from ways of oxygen utilization. It is likely, therefore,

60

C U,

chemiluminescence

quantify specificity

5)

a.

731

of is known membrane to bind [31].

aided

by could

on

the

which

formazan to induce resulting both

homologous

It is of interest

promote abnormal eggs [32]. Therefore, production

lipid in an

of reactive

that bindour ox-

D’CRUZ

732 ygen

metabolites

tween

the

only

at the

phagocyte

one mechanism function due

and

the

of MAto immune

C-independent result in changes

oxidative in the

and cell signalling. antioxidants to

eggs,

to be

contact

membrane

sperm/egg C-dependent

in the

sperm may of the sperm

the potential the formation

enregulaof exogeof reactive

of the membrane of antibody-bound

presence

12.

of follicular

IYCruz

OJ, Haas GG Jr. Wang

by lgG

anusperm

13. Haas

injury (XI

drying

BM, Kipnes

15. Green

MJ, Hill

anion

formation

16. Nathan

06,

acknowledge

gen

HAO,

produced

Britigan

BE,

DJ, Rosen

Hassett

cvtotoxicitv. 4. Pandva

Blood

1980;

J-P, D’azambuja

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lJ, Cohen

matozoa. 5. Barratt

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in the

J. The Steril

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male

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leukocvtic

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female

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Cooke

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