BIOLOGY OF REPRODUCTION 67, 1796–1803 (2002)
Published online before print 04 October 2002.
DOI 10.1095/biolreprod.102.007013
Spermadhesin PSP-I/PSP-II Heterodimer and Its Isolated Subunits Induced
Neutrophil Migration into the Peritoneal Cavity of Rats1
Ana Maria S. Assreuy,3 Juan J. Calvete,2,4 Nylane M.N. Alencar,5 Benildo S. Cavada,6
Duı́lio R. Rocha-Filho,3 Sabrina C. Melo,3 Fernando Q. Cunha,7 and Ronaldo A. Ribeiro2,3
Departamento de Ciências Fisiológicas-CCS-Universidade Estadual do Ceará,3
Instituto de Biomedicina de Valencia,4 Valencia, Spain
Depto de Fisiologia e Farmacologia,5 Faculdade de Medicina and Depto de Bioquı́mica e Biologia Molecular,6
Universidade Federal do Ceará, Fortaleza-CE, Brazil
Depto de Farmacologia,7 Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto-SP, Brazil
ABSTRACT
Spermadhesins are a group of (glyco)proteins from seminal
fluid involved in various aspects of porcine fertilization. PSP-I/
PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of porcine seminal fluid. Its biological function
remains, however, enigmatic. Using an in vitro chemotaxis assay,
we showed that PSP-I/PSP-II and its isolated subunits induced
migration of purified neutrophils. A possible proinflammatory
activity of PSP-I/PSP-II induced upon injection of the spermadhesin heterodimer and its isolated subunits into the peritoneal
cavity of rats was investigated. Lavage of peritoneal cavities,
thioglycolate treatment, and mast cell depletion were done before spermadhesin administration, and neutrophil migration was
evaluated 4 h after injections. Pharmacological modulation was
also investigated. Resident cell depletion by lavage reduced the
neutrophil migration induced by PSP-I/PSP-II and the PSP-II subunit but had no effect on that induced by isolated PSP-I. Both
an increase of macrophage population by thioglycolate treatment and mast cell depletion potentiated the neutrophil migration induced by PSP-I/PSP-II and by PSP-II. The glucocorticoid
dexamethasone but not indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and BN50739 (platelet activation factor [PAF] antagonist) inhibited neutrophil migration
induced by PSP-I/PSP-II. Coincubation with mannose-6-phosphate (a PSP-II-specific ligand) inhibited neutrophil recruitment
induced by PSP-II but did not alter the PSP-I activity. As a whole,
the data suggested that enhancement of the neutrophil migration-inducing activity of PSP-I/PSP-II and PSP-II involved an indirect mechanism, i.e., via activation of resident cells, probably
macrophages. On the other hand, PSP-I appeared to act directly
on neutrophils. We hypothesize that the neutrophil migrationSupported by grants Programa de Apoio ao Desenvolvimento Cientı́fico
e Tecnológico (PADCT), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal
de Nı́vel Superior (CAPES), and Fundação Cearense de Amparo à Pesquisa
(FUNCAP) from Brazil and grants PB98/0694 and BMC2001-3337 from
Dirección General de Enseñanza Superior e Investigación Cientı́fica
(Spain).
2
Correspondence: Juan J. Calvete, Instituto de Biomedicina, CSIC, Jaime
Roig 11, 46010 Valencia, Spain. FAX: 34963690800;
e-mail: [email protected]; Ronaldo A. Ribeiro, Dept. de Fisiologia e
Farmacologia, Universidade Federal do Ceará, R. Cel. Nunes de Melo,
1127-Rodolfo Teófilo, 60 430.270 Fortaleza, CE, Brazil.
FAX: 55 85 2888333; e-mail: [email protected]
1
Received: 30 April 2002.
First decision: 30 May 2002.
Accepted: 1 July 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
inducing effect displayed by PSP-II might be due to interaction
of its lectin domain with cellular receptors and that neutrophil
recruitment induced by PSP-I may involve protein-protein interactions.
immunology, oviduct, seminal vesicles, sperm, uterus
INTRODUCTION
Spermadhesins, a family of low-molecular-mass (12–16
kDa) (glyco)proteins, are major secretory products of the
seminal vesicle epithelium of certain domestic animals, i.e.,
boar, bull, and stallion [1]. Members of this secretory protein family display heparin- and carbohydrate-binding activities and coat the apical third of the boar sperm acrosome
cap at ejaculation. Most of the spermadhesin sperm-coating
molecules are released during capacitation [2]. This loosely
attached spermadhesin population may serve as decapacitation or acrosome-stabilizing factors protecting the acrosomal membrane from premature acrosome reactions,
whereas sperm-bound spermadhesin molecules are thought
to act as primary zona pellucida glycoprotein-binding lectins during gamete recognition at fertilization [3]. On the
other hand, the two major boar spermadhesins, PSP-I and
PSP-II [4], represent over 50% of the total seminal plasma
proteins. PSP-I and PSP-II form a noncovalent heterodimer
devoid of sperm membrane-binding capability [5] and
hence represent another spermadhesin population.
Spermadhesins are built by a single CUB domain [6].
CUB domains occur in diverse combinations in structurally
and functionally unrelated mosaic proteins [7] and serve as
a structural scaffold onto which different functionalities can
be imprinted [6]. The PSP-I/PSP-II heterodimer expresses
carbohydrate-binding activity associated with its PSP-II
subunit [5], and the PSP-II subunit in addition possesses a
mannose-6-phosphate binding site, cryptic in the heterodimer [8]. The biological functions of the PSP-I/PSP-II spermadhesin complex remain enigmatic, however. Recently,
Yang et al. [9] showed that purified and biotinylated PSPI bound to a subpopulation of porcine peripheral lymphocytes enhanced their in vitro immune activity. The same
group [10] demonstrated that PSP-I and PSP-II triggered
immunostimulatory activity. Noteworthy, oligosaccharides
with the NeuNAca2-6GalNAcb1-4GlcNAc-R motif, which
are present in the PSP proteins [11], block adhesive- and
activation-related events mediated by CD22, suggesting a
possible immunoregulatory activity for PSP-I/PSP-II. These
data suggest a role for PSP-I and/or PSP-II proteins in the
modulation of the uterine immune activity to ensure reproductive success. For instance, entry of semen into the uter-
1796
PROINFLAMMATORY EFFECT INDUCED BY PSP-I/PSP-II
ine cavity of the pig elicits, albeit not fully disclosed how,
a massive invasion of polymorphonuclear leukocytes
(PMNs) into the lumen, where immediate sperm phagocytosis is undertaken [12]. These PMNs are present in the
lamina propia, probably related to the high levels of preovulatory estrogens, immediately subjacent to the lining epithelium at estrus but only infiltrate the epithelium shortly
after semen deposition [13, 14]. There is morphologic evidence that the PMNs cooperate with intraepithelial macrophages during the first 3–6 h postsperm deposition, at
least in the porcine species [12]. Noteworthy, the pig is one
of the species with a large amount of spermadhesins in the
seminal plasma [2–4, 5]. The rationale behind this prompt
invasion of PMNs in a species where spermatozoa reach
the oviductal reservoir within minutes postmating (an area
that is documented free from leukocyte infiltration) is to
provide a foreign cell-free uterine environment for the descending early embryos. Moreover, in many instances, animal mating elicits damage of female genital tract tissues
along with infection of the lower reproductive tract by a
variety of pathogens. Thus, in this context, a rapid recruitment of leukocytes to the site of infection would be advantageous for the animal and for the fertilization process.
Neutrophil migration to the site of acute inflammatory reactions is one of the primary defense mechanisms used by
the organism against foreign agents. Leukocyte activation,
by their interactions with exogenous stimuli or via cell-cell
and cell-extracellular matrix interactions, generates cytokines, leukotriene B4, PAF, C5a, etc. Cytokines and leukotriene B4, mainly from macrophages or mast cells origin,
induce neutrophil migration both in vivo and in vitro and
have been involved in aspects of the immune and inflammatory responses [15–17]. Cell-cell and cell-matrix interactions during the inflammatory reaction involve proteinprotein and protein-carbohydrate recognition mechanisms
mediated by adhesion molecules, i.e., integrins and lectins
of the selectin family. Here we sought to investigate the
possible in vivo proinflammatory effect of PSP-I/PSP-II
and its isolated subunits on neutrophil migration to the peritoneal cavity of a model animal, the rat. In addition, we
investigated the participation of resident peritoneal cells
(macrophages and mast cells) as the source of endogenous
neutrophil chemotactic factors involved in spermadhesininduced leukocyte migration.
MATERIALS AND METHODS
Animals
Female Wistar rats (150–250 g) were housed in a temperature-controlled room with free access to water and food. Investigations were conducted in accordance with current guiding principles for the care and use
of research animals (NIH guidelines).
Isolation of Spermadhesin PSP-I/PSP-II and Its Subunits
The PSP-I/PSP-II heterodimer was isolated by size-exclusion chromatography on Sephadex G-50 of the non-heparin-binding fraction from
the seminal plasma of German Landrace boars. The individual subunits,
PSP-I and PSP-II, were separated by reversed-phase HPLC on a C18 column using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water
(solution A) and 0.1% TFA in acetonitrile (solution B) [1]. The purity of
the proteins was assessed by N-terminal sequencing (using an Applied
Biosystems 473A instrument) and tryptic peptide mapping using a Voyager DE-Pro (Applied Biosystems, Langen, Germany) MALDI-TOF mass
spectrometer. For trypsin digestion, PSP-I/PSP-II (2–5 mg in ammonium
bicarbonate buffer, pH 8.3) was incubated overnight at 378C with 1:100
(w/w) enzyme:substrate ration. Thereafter, trypsin was inactivated by heating at 1008C for 2 min, and the mixture was lyophilized. Completion of
proteolysis was checked by SDS polyacrylamide gel electrophoresis, re-
1797
versed-phase HPLC, and mass spectrometry. Protein concentration was
determined spectrophotometrically using the molar absorption coefficient
(27 332/M/cm) determined by Menéndez et al. [18] or by amino acid analysis (after sample hydrolysis in 6 M HCl for 24 h at 1068C in evacuated
and sealed ampules) using a Beckman Gold Amino Acid Analyzer (Barcelona, Spain).
Drugs and Reagents
Chemicals and biochemicals were of the highest purity grade available
and were purchased from the following companies: trypsin, fMLP (Nformyl-L-methionyl-L-leucyl-L-phenylalanine), compound 48/80, and
mannose-6-phosphate (Sigma, St. Louis, MO); thioglycolate (Lab Difco
Ltda., São Paulo, Brazil); dexamethasone (Prodome Quı́mica e Farmacêutica, Campinas, Brazil); MK-886, L-663, 536 (3-[1-(4-clorobenzyl)-3-tbutyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid) (Merck,
Montreal, Canada); indomethacin (Prodome), and BN 50730 ([3-1,1-dimethyl-ethyl] hexahydro-1,4,4,7b-trihydroxy-8-methyl-9H-1,7 a (epoxymethano) 6aH-cyclopentana-(c) furo [2,3b] furo [3,2,3,4] cyclopenta [1,2d] furan-5,9,12 (4H) trione) (Institute Henri Beaufour, Paris, France). The
fMLP was solubilized in ethanol; thioglycolate in distilled water; compound 48/80, dexamethasone, and mannose-6-phosphate in NaCl 0.1 M;
indomethacin in Tris HCl, pH 8.0; and MK-886 in methyl-cellulose 0.1%
aqueous solution; BN 50730 was solubilized up to 10% of the total volume
in DMSO and diluted to the appropriate concentration in NaCl 0.1 M.
Evaluation of In Vitro Chemotactic Activity of PSP-I/PSP-II
and Its Isolated Subunits
Human neutrophils (PMN) were obtained from heparinized blood of
healthy volunteers by Ficoll Hypaque gradient (d 5 1.114) fractionation.
Isolated neutrophils (90–95% purity) were resuspended to a final count of
106 cells/ml in RPMI 1640 medium containing 0.1% bovine serum albumin. Chemotaxis was measured in a 48-well microchamber (Neuro Probe,
Cabin John, MD) composed of two compartments divided by a 5-mm poresize polyvinylpyrolidone-free polycarbonate membrane [19]. In six different experiments, 28 ml of RPMI (Roswell Park Memorial Institute) medium containing 0.1% BSA (as negative control), fMLP (1026 M; positive
control), PSP-I/PSP-II (1027, 1026, 1025, and 1024 M), PSP-I (1024 M),
and PSP-II (1024 M) in RPMI/BSA medium were placed in the lower part
of microchamber wells. Fifty microliters of the 106 PMN/ml suspension
were added to the upper chamber of each well and incubated at 378C for
60 min under a humidified 5% CO2 atmosphere. Thereafter, the membranes were removed and their upper portions were washed with PBS (20
mM sodium phosphate, 135 mM NaCl, pH 7.3) in order to remove nonmigrating neutrophils. Neutrophils that had migrated to the lower portion
of the membrane were fixed with 70% methanol and stained with DiffQuick Stain Kit (American Scientific Products, McGraw Park, IL). Viable
neutrophils were counted using optical microscopy (1003 objective) in
five random fields of each well (6 wells/group).
Peritonitis Model for the Evaluation of the Spermadhesin
Chemotactic Activity on Leukocyte Migration
to the Peritoneal Cavity
One milliliter of sterile saline solution containing 2–20 3 10210 moles
of PSP-I/PSP-II, 4–40 3 10210 moles of PSP-I, and 4–40 3 10210 moles
of PSP-II were injected i.p. in healthy rats. In control animals, the same
amount of saline solution with no protein was injected. Leukocyte counts,
both total and differential (neutrophils, eosinophils, mast cells, and mononuclears), were performed 4 h after injections by microscopy. Briefly, after
washing the peritoneal cavities with 10 ml of saline or PBS containing 5
IU/ml of heparin, the peritoneal fluid was collected (approximately 6 ml).
For counting total cells in a Neubauer chamber, 20 ml of this fluid were
diluted 1:20 (v/v) with Turk solution. For differential counting (neutrophils, eosinophils, and mononuclears), 30 ml were centrifuged at 400 3 g
for 10 min, applied to a glass slide, and stained with HEMA III. No
discrimination between lymphocytes from monocytes was made. One hundred cells were counted with an optical microscope using an immersion
objective (1003). The time courses of neutrophils and mononuclear cell
migration were determined at 2, 4, 8, 24, 48, 72, and 96 h after injection
of 20 3 10210 moles of PSP-I/PSP-II. To this end, animals were killed
and cells were recovered by lavage of the peritoneal cavity with 10 ml of
sterile saline containing 5 IU heparin/ml. The fluid was recovered for total
and differential cell counts. Results are expressed as mean 6 SEM of the
number of cells per microliter of peritoneal wash of at least five different
animals.
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ASSREUY ET AL.
Depletion of Total Peritoneal Resident Cells
by Peritoneal Lavage
Effect of Mannose-6-Phosphate on the Neutrophil
Migration Induced by PSP-I/PSP-II Isolated Subunits
The method described by Souza et al. [20] was employed. Rats were
anesthetized with ethyl ether and three hypodermic needles were inserted
into the abdominal cavity. Thirty milliliters of sterile saline were injected
through the needle placed near the sternum. The abdominal cavity was
then gently massaged for 1 min and the peritoneal fluid was collected via
the two needles inserted into the inguinal region. The protocol was repeated three times. More than 83% of the peritoneal resident cell population was recovered. Control (sham) rats were manipulated in the same
way except that no fluid was injected or withdrawn. Thirty minutes later,
the resident cells were estimated in this group of control animals by injection of 10 ml of saline-heparin, as described above. PSP-I/PSP-II (20
3 10210 moles), PSP-I (40 3 10210 moles), PSP-II (40 3 10210 moles),
or fMLP (10 3 1029 moles) were injected i.p. into depleted and sham rats
and neutrophil migration was estimated 4 h later.
One milliliter of PSP-I (40 3 10210 moles) or PSP-II (40 3 10210
moles) alone or containing 20 3 10210 moles of mannose-6-phosphate
(PSP-II-specific binding sugar) was injected into rat peritoneal cavities.
The effect was evaluated 4 h after injection and compared with the salinetreated group.
Increasing the Peritoneal Macrophage Population
by Pretreatment with Thioglycolate
The method described by Ribeiro et al. [17] was essentially followed.
Ten milliliters of a 3% (w/v) thioglycolate solution were injected i.p. and
macrophages were collected 4 days later, counted, and compared with the
number of the same cells obtained from a group of control, nonthioglycolate-treated rats. The fMLP (10 3 1029 moles), PSP-I/PSP-II (20 3
10210 moles), PSP-I (40 3 10210 moles), and PSP-II (40 3 10210 moles)
were injected i.p. into control and thioglycolate-treated rats, and neutrophil
migration was evaluated after 4 h. Neutrophils in the peritoneal washes
collected from control rats 4 days after thioglycolate treatment were subtracted from the number of neutrophils counted after administration of
spermadhesin to thioglycolate-treated animals.
Depletion of Peritoneal Mast Cells
Peritoneal mast cell population was depleted either by chronic treatment with compound 48/80 [21] or by i.p. administration of water [22].
Animals were treated i.p. with compound 48/80 during 4 days (0.6 mg/kg
twice a day for 3 days and 1.2 mg/kg twice on the fourth day). On the
fifth day, depletion of the mast cell population was estimated in a group
of treated animals by counting the number of mast cells present in the
peritoneal cavity. The fMLP (10 3 1029 moles), PSP-I/PSP-II (20 3 10210
moles), PSP-I (40 3 10210 moles), and PSP-II (40 3 10210 moles) were
injected into control and compound 48/80-treated rats, and neutrophil migration was evaluated 4 h later. For depletion of mast cells from peritoneal
cavities by water administration, the animals were injected with 10 ml of
warm (378C) bidistilled H2O 48 h before the neutrophil migration assay.
The number of cells obtained from the animals that had their mast cell
population depleted was compared with that of nontreated control rats.
The fMLP (10 3 1029 moles), PSP-I/PSP-II (20 3 10210 moles), PSP-I
(40 3 10210 moles), and PSP-II (40 3 10210 moles) were then injected
into control and H2O-treated rats, and the neutrophil counts were performed after 4 h.
Effect of Pharmacological Modulators on the Neutrophil
Migration Induced by PSP-I/PSP-II
Control animals were injected i.p. with 1 ml of sterile saline. PSP-I/
PSP-II (20 3 10210 moles) was injected in saline-treated animals or in
animals treated with 0.5 ml of either dexamethasone (1 mg/kg, s.c., for 1
h), MK-886 (1 mg/kg, p.o., for 1 h), indomethacin (5 mg/kg, s.c., for 30
min), or BN 50730 (10 mg/kg, s.c., for 30 min). Neutrophil migration was
evaluated 4 h after injections and compared with the respective controls.
Effect of Trypsin Treatment on PSP-I/PSP-II-Induced
Neutrophil Migration
This approach was used in order to demonstrate whether the spermadhesin-induced neutrophil migration was due to the native protein conformation. MALDI-TOF mass spectrometric peptide mapping showed that
both PSP-I and PSP-II were degraded down to the expected tryptic peptides and no undegraded protein or proteolysis-resistant core remained
after trypsin treatment. The heterodimer PSP-I/PSP-II (20 3 10 210 moles),
intact or after trypsin digestion (1:100 enzyme-substrate, 378C, overnight),
was injected i.p. in 1 ml of sterile saline. The neutrophil migration was
evaluated 4 h later using the sterile saline-injected animals as controls.
Statistical Analysis
All results were expressed as mean 6 SEM for n 5 5 experiments.
Statistical evaluation was undertaken by analysis of variance (ANOVA)
followed by the Duncan test. A P-value of less than 0.05 was considered
statistically significant.
RESULTS
PSP-I/PSP-II and Its Isolated Subunits Induced
Neutrophil Chemotaxis
The concentration-dependent chemotactic activity of
PSP-I/PSP-II dimer and isolated PSP-I and PSP-II was
evaluated in vitro using a 48-well microchemotaxis chamber. Maximal activity was found at 1024 M; the three proteins significantly induced migration of PMNs (Fig. 1A).
At this concentration, PSP-I/PSP-II, PSP-I, and PSP-II enhanced neutrophil migration by 900%, 900%, and 800%,
respectively, compared with RPMI medium. This activity
was similar to that induced by 1026 M fMLP, an established
neutrophil chemoattractant used as positive control.
Dose- and Time-Dependent Proinflammatory Action
of PSP-I/PSP-II
PSP-I/PSP-II stimulated in a dose-dependent manner the
migration of neutrophils to the peritoneal cavity of rats 4 h
after i.p. injection. This effect was 117%, 235%, and 356%
compared with the control, saline-treated group upon injection of 2, 7, and 20 3 10210 moles of the heterodimer,
respectively. Administration of 20 3 10210 moles of PSPI/PSP-II showed the maximal response and was chosen for
subsequent experiments. Time-course experiments showed
that neutrophil migration was already significant 2 h after
PSP-I/PSP-II injection, with maximal effect 4 h after spermadhesin administration, decreasing thereafter and reaching control levels 48 h after PSP-I/PSP-II injection (Fig.
1B). PSP-I/PSP-II also induced mononuclear migration
(Fig. 1B). The time course of leukocyte recruitment induced by PSP-I/PSP-II followed the classical curve of leukocyte migration, i.e., the number of mononuclear cells increased along with a neutrophil count decrease. The peak
of mononuclear migration was 96 h after PSP-I/PSP-II injection.
Neutrophil Migration Induced by PSP-I
and PSP-II Subunits
Similar to PSP-I/PSP-II, the isolated subunits, PSP-I and
PSP-II, induced neutrophil migration to the peritoneal cavity of rats in a dose-dependent manner 4 h after administration (Fig. 2). Injections of 4, 14, and 40 3 10210 moles
of PSP-I increased neutrophil migration by 107%, 136%,
and 263%, respectively, compared with control animals
(Fig. 2A). The neutrophil migration induced by PSP-II at
the same dose as PSP-I was 25%, 239%, and 174%, respectively, of control rats (Fig. 2B).
PROINFLAMMATORY EFFECT INDUCED BY PSP-I/PSP-II
FIG. 1. In vitro and in vivo neutrophil chemotactic activity of PSP-I/PSPII and its isolated subunits. A) The chemotactic activity of PSP-I, PSP-II,
and PSP-I/PSP-II (at 1024 M each) was assessed in a 48-well chemotaxis
microchamber using fMLP (1026 M) and RPMI medium as positive and
negative controls, respectively. The results are expressed as the mean 6
SEM of six different experiments. *P , 0.05 compared with random migration in RPMI medium. B) Spermadhesin PSPI/PSP-II was injected intraperitoneally at a dose of 20 3 10210 moles/ml/cavity and the leukocyte
migration was determined at 2, 4, 8, 12, 24, 48, 72, and 96 h after injection. Values are reported as mean 6 SEM of five animals. Values for
neutrophil and mononuclear cell migration induced by saline are represented at time zero by closed and open squares, respectively. *P , 0.05
compared with control.
Neutrophil Migration Activity of PSP-I/PSP-II and PSP-II
Is Enhanced by an Indirect Mechanism
Depletion of 83% (Fig. 3A) of total resident cells by
lavage of the peritoneal cavity reduced the neutrophil chemotactic activity of 20 3 10210 moles PSP-I/PSP-II and 40
3 10210 moles PSP-II to 51% and 74%, respectively. However, the neutrophil chemotactic activity of PSP-I (40 3
10210 moles) or fMLP (10 3 1029 moles) was not altered
(Fig. 3B). These data suggest that PSP-I/PSP-II and the
PSP-II subunit enhanced their neutrophil chemotactic activ-
1799
FIG. 2. Dose-response curve of the neutrophil migration to peritoneal
cavity induced by PSP-I and PSP-II. Induction of neutrophil migration
evaluated 4 h after injection of 4, 14, and 40 3 10210 moles of PSP-I/
cavity (A) and PSP-II (B). Values represent mean 6 SEM of five animals.
*P , 0.05 compared with control (saline-injected) animals.
ity by a mechanism dependent on resident cells. On the
other hand, neutrophil migration induced by the neutrophil
chemotactic reagent fMLP and by PSP-I was independent
of resident peritoneal cells, indicating that these agonists
may act directly on neutrophils.
Increase of Macrophage Population Potentiates
the PSP-I/PSP-II and PSP-II Neutrophil Infiltration
of the Peritoneal Cavity
Intraperitoneal injection of thioglycolate (3% w/v) 96 h
before the neutrophil migration assay increased by 130%
1800
ASSREUY ET AL.
FIG. 3. Reduction of resident cell population by peritoneal wash impaired neutrophil migration induced by PSP-I/PSP-II and
PSP-II but not by PSP-I and fMLP. A) Number of total resident cells in sham (S) and
washed (W) peritoneal cavities. B) Neutrophil migration evaluated 4 h after intraperitoneal injections of 1 ml of saline (control, Sal) or 1 ml of PSP-I/PSP-II (20 3
10210 moles/cavity), PSP-I (40 3 10210
moles/cavity), PSP-II (40 3 10210 moles/
cavity), or fMLP (10 3 1029 moles/cavity)
into sham (S, open bars) and washed (W,
hatched bars) cavities. Results are reported
as mean 6 SEM of five animals. A) **Represents P , 0.05 compared with sham
rats. B) *Represents P , 0.05 compared
with control animals injected with saline
and # denotes P , 0.05 compared with
neutrophil migration to sham animals.
the number of mononuclear cells in the peritoneal cavity
(Fig. 4A). The neutrophil-inducing activity of 20 3 10210
moles of PSP-I/PSP-II and 40 3 10210 moles PSP-II was
potentiated (267% and 177% of the value obtained with
control animals, respectively) by prior treatment with thioglycolate (Fig. 4B). On the other hand, neither fMLP (10
3 1029 moles) nor the PSP-I subunit (40 3 10210 moles)
had an effect on the neutrophil counts (Fig. 4B). These data
suggest that the peritoneal cells involved in the neutrophil
migration recruitment activity of PSP-I/PSP-II and PSP-II
might be macrophages. The mechanism used by the PSP-I
subunit seems to be independent of these cells.
FIG. 4. Thioglycolate potentiates the neutrophil migration induced by PSP-I/PSP-II
and PSP-II but not by PSP-I. A) Number of
macrophages counted in control (C) and
thioglycolate-treated animals (Tg). B) Neutrophil migration evaluated 4 h after intraperitoneal injections of 1 ml of either saline (Sal) or PSP-I/PSP-II (20 3 10210
moles/cavity), PSP-I (40 3 10210 moles/
cavity), PSP-II (40 3 10210 moles/cavity),
and fMLP (10 3 1029 moles/cavity) in
nontreated (-) and thioglycolate-treated
rats. Results are the mean 6 SEM of 5 animals. **P , 0.05 compared with controls;
*P , 0.05 compared with Sal; and #P ,
0.05 compared with thioglycolate-untreated animals.
Mast Cell Depletion Potentiates the Neutrophil Influx
to the Peritoneal Cavity Induced by PSP-I/PSP-II
and the PSP-II Subunit
Figure 5A shows that chronic treatment of animals with
compound 48/80 completely depleted the mast cell population in comparison with the control (C) group. Compared
with nontreated rats, the neutrophil migration induced by
PSP-I/PSP-II (20 3 10210 moles) and PSP-II (40 3 10210
moles) was potentiated in the mast cell-depleted animals in
366% and 410%, respectively (Fig. 5B). However, injection
of 40 3 10210 moles of PSP-I into the peritoneal cavity of
PROINFLAMMATORY EFFECT INDUCED BY PSP-I/PSP-II
1801
FIG. 5. Mast cell depletion potentiates
the neutrophil migration induced by PSP-I/
PSP-II and PSP-II but not by PSP-I. A, C)
Peritoneal mast cell counts in control animals (C) and in rats treated with compound 48/80 or with 10 ml of H2O, respectively. B, D) Neutrophil migration
evaluated 4 h after intraperitoneal injections of either 1 ml of saline (Sal) or 1 ml
of PSP-I/PSP-II (20 3 10210 moles/cavity),
PSP-I (40 3 10210 moles/cavity), PSP-II (40
3 10210 moles/cavity), or fMLP (10 3 1029
moles/cavity) in control (-) and 48/80- and
H2O-treated rats. Results are reported as
mean 6 SEM of five animals. A, C) **P ,
0.05 compared with C. B, D) *P , 0.05
compared with Sal; #P , 0.05 compared
with 48/80- or H2O-untreated animals.
compound 48/80-treated rats did not alter the onset of neutrophil migration. Moreover, the neutrophil migration induced by fMLP (10 3 1029 moles) was not affected by
depletion of mast cells that followed after treatment with
compound 48/80. In addition, another strategy was used to
deplete the mast cell population from peritoneal cavities of
rats. Intraperitoneal injection of 10 ml of water also reduced
the mast cell population to 14% of control animals 48 h
after the treatment (Fig. 5C). Similar to compound 48/80
treatment, injection of water also potentiated the neutrophil
migration induced by PSP-I/PSP-II (20 3 10210 moles) and
PSP-II (40 3 10210 moles) by 275% and 201%, respec-
tively, whereas the neutrophil migration induced by PSP-I
(40 3 10210 moles) and fMLP (10 3 1029 moles) was not
altered (Fig. 5D).
Dexamethasone but Not Indomethacin, MK886,
or BN50739 Inhibited the Neutrophil Migration
to Rat Peritoneal Cavities Induced by PSP-I/PSP-II
Figure 6 shows the effect of the cyclooxygenase inhibitor indomethacin, a leukotriene inhibitor MK886, a PAF
antagonist BN 50730, and the glucocorticoid dexamethasone on neutrophil migration induced by i.p. injection of
1802
ASSREUY ET AL.
moles) was not altered by coincubation with mannose-6phosphate. Mannose-6-phosphate alone did not induce neutrophil migration (data not shown). The results suggested
that the lectin domain present at the PSP-II subunit could
be involved in the proinflammatory activity of the spermadhesin.
FIG. 6. Dexamethasone but not indomethacin, MK 886, or BN 50730
inhibits the neutrophil migration to the peritoneal cavity of rats induced
by PSP-I/PSP-II. Animals were treated with either saline or 0.5 ml of indomethacin (5 mg/kg), MK 886 (1 mg/kg), BN 50730 (10 mg/kg), or dexamethasone (1 mg/kg) before injection of 20 3 10210 moles PSP-I/PSP-II/
cavity. Neutrophil counts were evaluated 4 h after the spermadhesin injections. Control animals (Sal) were injected with 1 ml of sterile saline.
Values are reported as mean 6 SEM of five animals. *P , 0.05 compared
with Sal group; #P , 0.05 compared with animals injected with PSP-I/
PSP-II without previous treatment.
20 3 10210 moles of PSP-I/PSP-II. Treatment of the animals with indomethacin (5 mg/kg, s.c.), BN 50730 (10 mg/
kg, s.c.), or MK886 (1 mg/kg, oral) did not modify the
neutrophil migration induced by the spermadhesin heterodimer. On the other hand, dexamethasone (1 mg/kg, s.c.)
injected 1 h previous to the spermadhesin administration
inhibited by 71% the PSP-I/PSPII-induced neutrophil migration to the peritoneal cavity of rats.
Effect of Proteolysis on PSP-I/PSP-II-Induced
Neutrophil Migration
This approach was undertaken in order to investigate
whether the spermadhesin PSP-I/PSP-II-induced neutrophil
migration was due to the native protein conformation. PSPI/PSP-II heterodimer (20 3 10210 moles), intact or after
trypsin digestion, were injected i.p. in 1 ml of sterile saline,
and neutrophil migration was evaluated 4 h later using sterile saline-injected animals as controls. Trypsin treatment
did not impair the neutrophil migration-inducing ability of
the spermadhesin (data not shown), suggesting that a linear
epitope (a polypeptide stretch or a carbohydrate structure)
may retain this biological activity.
Mannose-6-Phosphate Inhibited the Neutrophil Migration
Induced by PSP-II but Not by PSP-I
The i.p. administration of 40 3 10210 moles of PSP-II
coincubated with 20 3 10210 moles of its specific binding
sugar mannose-6-phosphate resulted in a 52% reduction of
the neutrophil migration compared with that induced by
PSP-II alone. The chemotactic effect of PSP-I (40 3 10210
DISCUSSION
This study shows that the porcine spermadhesin PSP-I/
PSP-II and its subunits, PSP-I and PSP-II, display in vitro
PMN chemotactic activity. In addition, the time-course effect exerted by PSP-I/PSP-II on neutrophil and mononuclear cell recruitment into the peritoneal cavity of rats showed
the characteristic pattern of an inflammatory reaction. The
PSP-I/PSP-II proinflammatory effect could be confirmed in
two different models of inflammation: the rat paw edema,
evaluated 4 h after injection of 20 3 10210 moles PSP-I/
PSP-II/paw, and in the air pouch, which was evaluated 6 h
after subcutaneous administration of 20 3 10210 moles of
the spermadhesin complex (data not shown). The proinflammatory effect of the PSP-I subunit seems to involve a
direct interaction with neutrophils, whereas the PSP-I/PSPII and PSP-II neutrophil migration activity appears to be
potentiated by resident macrophages, which may release a
neutrophil chemotactic factor, possibly a cytokine. The
pharmacologic modulation experiments showing that dexamethasone, a cytokine antagonist, but not inhibitors of cyclooxygenase (indomethacin), lipoxygenase (MK886), and
PAF (BN 50730), altered the stimulatory effect of these
spermadhesins support this hypothesis. Moreover, the neutrophil migration effect induced by PSP-I/PSP-II and PSPII was potentiated upon mast cell depletion. However, this
approach could not modify the neutrophil migration response induced by PSP-I and by direct neutrophil chemoattractants such as fMLP, further indicating that the PSPI subunit may act directly on neutrophils. The role of mast
cells in the neutrophil migration activity of PSP-I/PSP-II
and PSP-II deserves further investigation. These cells could
be releasing inhibitory neutrophil chemotactic factors. The
role of inhibitory cytokines such as IL-4 and IL-10 controlling the immune and inflammatory response has been
strongly suggested [23–27]. The mechanisms underlying
the neutrophil migration activity of spermadhesin PSP-I/
PSP-II and its subunits are not clear. The results showing
that proteolysis did not impair the ability of the spermadhesin peptide mixture to promote neutrophil migration
suggest that a linear epitope (a discrete peptide sequence
or a carbohydrate structure) rather than the intact conformation of the full-length proteins may retain this biological
activity. Involvement of a glycan seems unlikely because
each subunit of the PSP-I/PSP-II complex contains a single
glycosylated asparagine residue, which exhibits large site
glycan heterogeneity and shares the same glycans but differs in their relative molar ratios [11]. On the other hand,
the demonstration that PSP-II possesses a mannose-6-phosphate binding site that is cryptic in the PSP-I/PSP-II heterodimer [8] and our present observation that mannose-6phosphate inhibits the neutrophil migration activity of PSPII but not that of PSP-I or PSP-I/PSP-II (not shown) indicate that the proinflammatory and mannose-6-phosphate
binding activities of PSP-II could be linked.
In conclusion, our data show a proinflammatory effect
of spermadhesin PSP-I/PSP-II heterodimer and that the two
subunits are involved in neutrophil activation via different
mechanisms. PSP-I and PSP-II [4] represent more than 50%
of the total boar seminal plasma proteins. During natural
PROINFLAMMATORY EFFECT INDUCED BY PSP-I/PSP-II
mating in the pig, the first spermatozoa reach the uterotubal
junction and the adjacent first portion of the oviductal isthmus within minutes [28], where a preovulatory sperm reservoir (of the order of 105) sufficient for full fertilization is
established [29, 30]. The remaining spermatozoa (10–50 3
109) is either voided by retrograde reflux (about 35%) or
eliminated by phagocytosis by uterine PMNs that traverse
the uterine lumen within 3–6 h postmating [31]. Several
factors have been suggested as mediators for PMN recruitment, such as fluid distention [32], spermatozoa [33], and
seminal plasma [34]. The results presented here support a
role for PSP-I/PSP-II as a postmating inflammation mediator. Ongoing investigations in our laboratories show that
the initial fraction of the boar ejaculate, which contains the
sperm population that eventually colonizes the oviductal
isthmus, is devoid of spermadhesins. The absence of the
inflammation mediator PSP-I/PSP-II in this sperm fraction
may provide a window of opportunity for spermatozoa to
reach the sperm reservoir before the PMNs reach the lumen.
Leshin et al. [10] have shown that PSP-I and PSP-II are
immunostimulatory proteins that modulate pig lymphocyte
activity in vitro. Besides, lymphocyte binding of PSP-I was
recently demonstrated by histochemical techniques [9].
These findings are in line with our results and as a whole
indicate that the PSP proteins may modulate immune responses in the porcine uterine environment that may contribute to the reproductive success of the species.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENTS
The authors wish to thank Ana Kátia dos Santos, Giuliana Bertozi
Francisco, and Fabı́ola Leslie Antunes Cardoso Maestriner (USP-Ribeirão
Preto, Brazil) for excellent technical assistance. The authors are grateful
to Prof. Heriberto Rodrı́guez-Martı́nez (Uppsala, Sweden) for critical reading of the manuscript and many helpful discussions.
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