Suppression of rat epididymal sperm immunogenicity by a seminal vesicle secretory protein and transglutaminase both in vivo and in vitro

BIOLOGY OF REPRODUCTION 50, 593-602 (1994)

Suppression of Rat Epididymal Sperm Immunogenicity by a Seminal Vesicle SecretoryProtein and Transglutaminase Both In Vivo and In Vitro'

G. PELUSO,3 R PORTA,2 '4 C. ESPOSITO,4 M.A. TUFANO,5 R TORALDO, 6 M.L. WVUOT , 7 G. RAVAGNAN, 8

and S. METAFORA9

Institute of Protein Biochemistry and Enzymology,2 National Research Council, Arco Felice, Naples, ItalyDepartment of Biochemistry and Biophysics,4 University of Naples, Naples, Italy

Institute of Microbiology,5 2nd University of Naples, Naples, ItalyDepartment of Pediatrics,6 University of Naples, Naples, Italy

Institute of General Pathology and Oncology,7 2nd University of Naples, Naples, ItalyInstitute of Experimental Medicine,8 National Research Council, Rome, Italy

International Institute of Genetics and Biophysics,9 National Research Council, Naples, Italy

ABSTRACT

The pretreatment of epididymal spermatozoa with SV-IV, one of the major secretory protein produced by the epithelium ofadult rat seminal vesicles, was found to markedly decrease their ability to induce in vivo peritoneal macrophage activation,measured as class II major histocompatibility complex surface antigen expression, superoxide anion production, phagocytic ac-tivity, and antigen presentation. In addition, the treatment of spermatozoa with SV-IV produced a significant decrease of theirimmunogenicity evaluated in vitro by [3 H]thymidine incorporation in splenocyte/spermatozoon co-culture. The concurrent pres-ence of SV-IV and transglutaminase, an enzyme secreted in large amounts from the rat anterior prostate, amplified these phe-nomena. The suppression of the epididymal sperm immunogenicity is suggested to be of crucial importance for the preventionof the immune response to the sperm introduced in the immunocompetent female genital tract during coitus.

INTRODUCTION

The antigenic properties of the male gamete have beenwell established [1-3]. The strong immunogenicity of mam-malian epididymal spermatozoa is due to the presence ontheir surface of a mosaic of different types of nonself-spe-cific, highly immunogenic auto- and alloantigens includingthose of the major histocompatibility system [1-5]. It hasalso been demonstrated that the female reproductive tractis fully capable of cell-mediated and humoral immune re-sponses [5-7]. Therefore, the reaction evoked in the femalegenital tract by sperm alloantigens should produce an im-munologic sensitization of the female to sperm antigens as-sociated with a severe immunologic damage of spermato-zoa. It is well known, in fact, that epididymal spermatozoanot yet mixed with the secretion of the male accessory sex-ual glands are remarkably effective in sensitizing femalesto sperm antigens [7,8]. Nature, however, has evolvedmechanisms able to prevent the immune reaction to spermantigens, so that spermatozoa can survive until fertilizationtakes place. We have experimental evidence, in fact, that theimmunogenicity of ejaculated spermatozoa is very weak, thatthe immunologic sensitization of inseminated females is veryrare, and that detectable transplantation immunity is absent

Accepted October 11, 1993.Received June 7, 1993.'This work was supported by grants from CNR Target Project on "Biotechnology

and Bioinstrumentation," Progetto finalizzato "Ingegneria Genetica" and from III andIV Progetto AIDS of the I.S.

2Correspondence: Prof. Raffaele Porta, Department of Biochemistry and Bio-physics, Via Costantinopoli 16, 80138 Napoli, Italy. FAX: (39-81) 5665863.

in inseminated females [3,5, 9, 10]. The mechanism of spermimmunoprotection in the female genital tract is not wellunderstood. It has been suggested that an immunosup-pressive event induced by seminal plasma (SP) and/oruterine factors could play an important role in this process[3,7,9, 11]. This hypothesis is supported by several studiesdemonstrating that mammalian SP contains high and lowmolecular weight factors (proteins, peptides, prostaglan-dins, polyamines, etc.) endowed with immunosuppressiveproperties that can minimize sensitization and allow spermsurvival in the immunocompetent female genital tract [3, 11].These factors might inhibit immune responses by maskingsperm cell antigenic determinants and/or by directly in-terfering with immunocompetent cells [3, 11]. Among them,two homologous steroid-dependent proteins, uteroglobin(UG; Mr = 15 000) and SV-IV (seminal vesicle protein 4,according to its electrophoretic mobility in SDS-PAGE; Mr= 9758], have been purified, and their biochemical and bi-ological characteristics have been studied in detail [12-16].These proteins, present in large amounts in uterine andseminal vesicle secretions of rabbits and rats, respectively,possess powerful nonspecies-specific immunosuppressive,anti-inflammatory, antichemotactic, and antiphagocyticproperties [12-16]. The ability of UG and SV-IV to inhibitphospholipase A2 activity may explain their anti-inflamma-tory properties [12-14,17]. Macrophages and T lympho-cytes are the possible targets of the immunosuppressive ef-fects of these proteins [14,18]. It has been found that UGin the presence of transglutaminase (TGase; E.C. 2.3.2.13),an enzyme able to cross-link specific proteins by E(y-glu-

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tamyl)lysine isopeptide bonds [19], dramatically decreasesthe immunogenicity of both rabbit blastocysts [20] andspermatozoa [2] in vitro. We also have experimental evi-dence that the protein SV-IV is able to bind to the plasmamembrane of rat epididymal spermatozoa in both mono-meric and TGase-induced polymeric form [4, 21].

On the basis of all these findings, we found it of interestto investigate the ability of SV-IV, in the presence or ab-sence of TGase, to influence the immunogenicity of rat ep-ididymal spermatozoa, both in vivo and in vitro. In partic-ular, the in vivo experiments were designed to evaluate theeffect of rat epididymal spermatozoa, pretreated or not withSV-IV or SV-IV/TGase and then injected into the peritonealcavity of syngeneic animals, on the activation state of theperitoneal macrophages. The cellular activation was mea-sured by several parameters such as class II major histo-compatibility complex (MHC) surface antigen expression,superoxide anion production, phagocytic activity, and abil-ity to present antigens. The relevance of the i.p. immuni-zation model to mating derives from the following consid-erations: 1) both the peritoneal cavity and the female genitaltract are fully immunocompetent anatomic environments[1, 5, 7]; 2) after mating, live spermatozoa are detectable inthe peritoneal cavity [22]; and 3) i.p. injection of sperma-tozoa is frequently used in the medical practice of artificialinsemination with a high degree of success and without im-munologic side effects [23].

MATERIALS AND METHODS

Animals

Male and female Wistar rats and female Brown-Norwayrats were obtained from Charles River Co. (Como, Italy),and housed (four rats/cage) in self-contained laminar flowrooms. The rats weighed approximately 250 g.

SV-IV Purification

The protein SV-1V was purified to homogeneity from adultrat (Fisher-Wistar strain) seminal vesicle secretory fluid ac-cording to a published procedure [24]. The purity of theprotein was assessed by 15% PAGE in denaturing or non-denaturing conditions [25], by fingerprinting technique [26],by amino acid composition analysis [25], and by fast atombombardment mass spectrometry [27]. Protein concentra-tion was determined by the method of Lowry et al. [28].The preparations of SV-IV were shown to be completelyfree of lipopolysaccharide [29] and tumor necrosis factor[30] by specific biological assays.

Preparation of Peritoneal Exudate Cells (PEC) andEpididymal Spermatozoa from Rats

To obtain PEC, female rats were exsanguinated after etheranesthesia. Ten milliliters of RPMI 1640 medium containing100 U/ml penicillin, 100 RIg/ml streptomycin, and 2 U/ml

heparin were injected into the peritoneal cavity and im-mediately removed by a syringe. The cells were washedtwice in the same solution without heparin, counted, andresuspended in RPMI 1640 complete medium containing10% fetal calf serum (FCS), antibiotics, and glutamine (2mM). Epididymal spermatozoa (105), obtained from Wistarrats by conventional procedures, were suspended in 1 mlof Hanks' Balanced Salt Solution (HBSS) culture medium(without phenol red dye and modified by addition of 2.5mM CaCl2 and 3 mM dithiothreitol) and incubated for 30min at 370C with or without 5 M SV-IV, in the presenceor absence of 0.6 ,ug TGase, purified from guinea pig liver(Sigma Chemical Co., St. Louis, MO) according to Connel-lan et al. [31], and/or 2.5 mM EDTA. After this treatment,the sperm cells were washed three times with the samemedium and then were used for the experiments.

Interperitoneal Injection of Epididymal Spermatozoa

For the in vivo studies, we injected into the peritonealcavity of adult rats an appropriate number of sperm cellspretreated or not with SV-IV, in the presence or absenceof TGase, in order to determine possible changes in thecomposition of PEC populations and in the class II MHCsurface antigen expression, antigen presentation, superox-ide anion production, and phagocytic activity of peritonealmacrophages. Groups of 10 virgin female Wistar rats re-ceived injections of 1 ml of HBSS without spermatozoa(control animals, group A); 7 days later they received an-other injection of the same medium, and PEC were col-lected at different times after the second injection. Othergroups of 10 virgin female Wistar rats (experimental ani-mals) were separately injected, according to the timeschedule of the control animals, with 1 ml of completeFreund adjuvant (FA.; group B), and 1 ml HBSS containing105 spermatozoa previously incubated without SV-IV andTGase (group C) or with SV-IV in the absence (group D)or presence (group E) of the enzyme. The PEC from ex-perimental animals were also collected at different timesafter the second injection. The FA was used as a positivecontrol, because of its effectiveness in promoting a strongactivation of the immunocompetent cells [32]. The integrityof the cell structure and the viability of both macrophagesand epididymal spermatozoa were evaluated by phase con-trast microscopy and the trypan blue dye exclusion test.

Fluorescence-Activated Cell Sorter (FACS) Analysis

PEC were stained for immunofluorescence analysis byindirect labeling procedures. Unconjugated antibodies wereused as first reagent, and fluorescein isothiocyanate (FITC)-labeled xenogeneic polyspecific or IgG class-specific anti-bodies were used as second reagent. Controls, includingonly the second reagent without the first, were routinelycarried out in the indirect staining procedures. OX-41(monocyte/macrophages) and OX-6 (MHC class II) mouse

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anti-rat monoclonal antibodies (Techno-Genetics, Houston,TX) were used. After being immunostained, the cells werecounterstained with propidium iodide to allow exclusionof dead cells (brightly stained by the propidium iodide)from the immunofluorescence analysis.

Cell fluorescence was analyzed by a Becton Dickinsoncytofluorograph (FACScan, Mountain View, CA) with use ofthe 488-mm emission line of an argon laser. For each stainedcell sample, values of forward scatter (related to the cellsize), right-angle scatter (related to granularity), green flu-orescence (FITC), and red fluorescence (propidium io-dide) were obtained from 10 000 cells. Live cells were thosewithout bright propidium iodide staining and with suffi-cient forward scatter to exclude contaminating erythrocytesor cellular debris. Macrophages were identified and sepa-rately analyzed on the basis of their characteristic combi-nation of forward, right-angle scatter and their positive flu-orescence staining with the OX-41 monoclonal antibody.Control cell samples not stained with specific antibodiesallowed the definition of the proper FACScan gates used todistinguish the positive from the negative cells in eachstaining procedure. The percentages of fluorescence-posi-tive live cells and the mean fluorescence and scatter valuesof the fluorescence-positive cells were determined by aHewlett-Packard computer system. In sandwich stainingprocedures, the percentage of specifically stained cells wasdetermined by subtracting the percentage of positive cellspresent in the stained control (second layer only) from thatobtained after staining with both first- and second-layer an-tibodies.

Superoxide Anion (02 -) Quantification

Superoxide anion (0,2-) production by peritoneal mac-rophages was assessed by measuring the superoxide dis-mutase-inhibitable reduction of ferricytochrome C accord-ing to a published micromethod [33]. The amount ofsuperoxide anions released into the medium from the per-itoneal macrophages was calculated by the following for-mula: 02- nmol/well = absorbance (at 550 nm) x 15.87.The amount of peritoneal macrophage proteins was eval-uated by the method of Lowry et al. [28].

Splenocyte-Spermatozoon Coculture

Rat (Wistar) epididymal spermatozoa (105) suspended in1 ml of HBSS medium (modified by addition of 2.5 mMCaCl2 and 3 mM dithiothreitol) were pretreated for 2 h at37°C with or without SV-IV (5 iM), in the presence or ab-sence of TGase (0.6 ,ug/ml), purified from guinea pig liveraccording to Connellan et al. [31], and/or 2.5 mM EDTA.The sperm cells were washed three times with HBSS andthen were mixed with 4 x 105 rat (Brown-Norway) sple-nocytes. Complete HBSS medium, containing 10% FCS, wasused to culture the splenocyte-sperm mixture, in a final vol-ume of 200 $tl (in 96-well microtiter plates). Each culture

was in triplicate. After 5 days of incubation at 37°C in ahumidified CO2 (5%) incubator, 1 [,Ci of [3H]thymidine (2Ci/mmol, Amersham Corp., Amersham, UK) was added perwell, and the culture was continued for another 12 h. Thecells were then collected, washed, and filtered through glass-wool filters, and their radioactivity was measured in a Beck-man (Palo Alto, CA) scintillation counter after Instagel(Packard Instr., Groningen, The Netherlands) addition tothe dried filters. The viability of splenocytes was evaluatedby Con A stimulation and [3H]thymidine incorporation.

Antigen Presentation BioassayPEC collected from differently treated inbred C57B1/6

mice were suspended at various concentrations in Dulbec-co's modified Eagle's medium buffered with 10 mM HEPESand supplemented with 2 x 10- 3 M L-glutamine, 5 x 10 - 5

M 2-mercaptoethanol, and 5% heat-inactivated FCS. Onehundred microliters taken from each cell suspension wasplated in triplicate in 96-well tissue culture plates. The ad-herent cells were incubated with 80 RM of the horse apo-cytochrome c fragment 1-65 at 37°C in a CO2 (5%) incu-bator for 3 h. The cells were then washed and fixed at roomtemperature with 1% paraformaldehyde. One hundredthousand T hybridoma 3.7.III cells, recognizing the horseapocytochrome c fragment 1-65 and restricted to the I-Abmolecule, were added to each well, and the plates wereincubated at 37°C for 24 h. T-cell-specific activation was readout as interleukin-2 (IL-2) production by measuring the abilityof supernatants to support the proliferation of the IL-2-re-sponsive CTLL cell line. Aliquots (25 pl) of the supernatants(S) from cultures containing 105 T-cell hybridoma and var-ious number of adherent PEC, obtained from differentlytreated animals (S1 = 103 PEC; S2 = 10 4 PEC; S3 = 10 5

PEC; S4 = 5 x 105 PEC; S5 = 106 PEC; S6 = 1.5 x 106 PEC;S7 = 2 x 106 PEC) were added to the CTLL cultures (105cells/well) to assess the effect of the supernatant additionon the cell proliferation as measured by [3H]thymidine in-corporation into DNA (see Splenocyte-Spermatozoon Co-culture above). The variability (expressed as SEM) in thevalues of thymidine incorporation was about + 10%. TheT-cell hybridoma, CTLL cells, and the antigen were a gen-erous gift from Prof. G. Corradin, Lausanne University, Swit-zerland.

Statistical AnalysisSignificance of the results obtained in the different ex-

periments reported in this paper was assessed by Student'st-test, and a p value of less than 0.05 was considered sig-nificant.

Phase Contrast Light Microscopy and TransmionElectron Microscopy (TEM) Analysis of SpermPhagocytosis by Peritoneal Macrophages

Rat PEC were diluted to 4 x 106 cells/ml in RPMI 1640medium with 10% FCS, and then 1-ml aliquots were plated

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1 1 1 1 1 1 1 1 1 1 1 1

2 4 6 8 10 12 TIME (days)

FIG. 1. Kinetics of la induction in the rat peritoneal macrophage population after i.p. injection of lo5 differently treated rat epididymal spermatozoa (Spz). Ten thousand peritoneal macrophages obtained from animals of groups A (solid triangles; injected with HBSS culture medium), B (solid squares; injected with F.A.), C (solid circles; injected with Spz), D (open squares; injected with SV-IV-treated Spz), and E (open circles; injected with SV-IV + active-TGase-treated Spz) were stained with either the FITC-labeled monoclonal antibody anti-I-A (0x6) or an FlTCconjugated IgG2b isotype control, and analyzed for fluorescence intensity by flow cytometry. Each value represents the mean of six independent determinations performed in triplicate. Variability (expressed as SEMI in the percentages of I- A+ peritoneal macrophages was about + 8%. For experimental details see Materials and Methods.

in each well of flat-bottomed 6-well culture plates. After 3-h incubation at 37°C to allow for macrophage adherence, nonadherent cells were removed by four washes with pre- warmed HBSS containing 1% FCS. To triplicate wells was added either 1 ml of medium or 1 ml of medium containing 2 X lo5 rat epididymal spermatozoa (target cells). Twenty- four hours later the viability of either untreated or SV-N (5 pM) + TGase (0.6 pg/ml)-treated spermatozoa was evaluated by the trypan blue dye exclusion test, and the adherence of these cells to macrophages was determined by phase-contrast light microscopy. The ability of peritoneal

macrophages to phagocytose spermatozoa, either untreated or pretreated with SV-N and TGase, was assessed by TEM. The macrophages, after sperm phagocytosis, were washed in PBS and fixed with 2.5% glutaraldehyde in 0.1 M caco- dylate buffer, pH 7.5, for 60 min. The fixed macrophages were washed in PBS, postfixed for 60 min at room tem- perature with 1% 0 s 0 4 in PBS, washed again in PBS, de- hydrated in graded alcohols (from 35 to 75%), and embed- ded in L.R White (London Resin Co., London, England) at 55°C for 48 h. Ultrasections (100 nm) were cut with a dia- mond knife and collected on nickel grids. The sections were poststained with uranyl acetate and lead citrate before electron microscope observation.

RESULTS

PEC Analysis Following Injectwn of Dz@erently Treated Spemzatozoa in the Peritoneal Cavity

A variety of foreign molecules or cells injected in the rat peritoneal cavity caused the formation of an exudate rich in polymorphonuclear leukocytes (PMN), macrophages, and lymphocytes. On the basis of this consideration, we evaluated in different groups of animals (see Materiaks and Methods) the cell composition of the peritoneal leukocyte population at various times after the experimental treatment by both cytologic (Wright's Giemsa staining) and cy- tofluorometric (fluorescent monoclonal antibody labeling) techniques (see Materiaki and Methods). On Day 1 after the second injection, the peritoneal exudates of all differently treated animals contained a high percentage of PMN (about 70%), with 19% macrophages and 11% lymphocytes. On Day 3, this proportion was reversed: the exudates contained 74% macrophages, 25% lymphocytes, and 1% PMN. During the following days, the lymphocyte number grad- ually increased and the macrophage number decreased, so that by Day 12 they were both approximately 50%, about the same value found in the peritoneal resident exudate populations. The only difference was detected in the cell population profile of the FA-injected animals (group B), in which, on Day 1 after the second injection, a lower influx of PMN was found (33%), the percentage of macrophages and lymphocytes being about 48% and 19%, respectively. In contrast to the absence of significant changes in the peritoneal leukocyte subset population, marked differences in PEC numbers were observed among the differently treated animals. In particular, on Day 3 following the second injection of SV-IV-treated spermatozoa (group D), the PEC number was about 45% lower than that observed in group C animals (animals that had received injections of untreated spermatozoa), whereas the PEC number detected after the injection of spermatozoa treated with SV-N and guinea pig liver TGase (group E) was similar to that of the HBSS-injected rats (group A; about 60% lower than that observed in group C animals).


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FLUORESCENCE INTENSITYFIG. 2. Fluorescence intensity of peritoneal I-A+ macrophages obtained from the differently treated rats. Fluo-

rescence intensity of the cells isolated from individual animals was measured on Day 3 after the second injection.Spz = epididymal spermatozoa. Dotted line: control animals (group A; injected with HBSS culture medium). Solidline: experimental animals of group C, injected with untreated Spz (panel I); group B, injected with F.A. (panel II);group D, injected with SV-IV-treated Spz (panel III); and group E, injected with SV-IV+active-TGase-treated Spz (panelIV). Ordinate: cell number. Abscissa: fluorescence intensity. Data are representative of six different experiments. Forexperimental details see Materials and Methods.

Effect of SV-IV/TGase Spermatozoon Pretreatment onImmunoglobulin Antigen (la) Expression in thePeritoneal Macrophage Population after SpermatozoonInjection

Experiments were performed to test the effect of the in-jection of spermatozoa previously treated with SV-IV, in theabsence or presence of guinea pig liver TGase, on the in-duction of peritoneal macrophage Ia expression. The dose-response curve (not shown), obtained on Day 3 after thesecond injection, demonstrated that the largest Ia induction(I-A+ = 53%) was obtained with 105 spermatozoa previ-ously incubated in the absence of SV-IV and TGase. Thisvalue represents about a fourfold increase over either con-trols injected with HBSS (group A) or group E animals in-jected with 105 SV-IV + TGase-treated spermatozoa. On theother hand, as little as 104 untreated spermatozoa induceda twofold increase in the percentage of I-A+ cells. The pos-sibility that endotoxin contamination of spermatozoon sam-ples was at the basis of Ia induction was ruled out by theLimulus amebocyte lysate test [29]. In the group C (animalsinjected with spermatozoa previously incubated in the ab-sence of SV-IV and TGase), the maximum number of ex-udate cells and I-A+ macrophages was found to occur onDay 3 following the second injection (15.7 + 2.1 x 106 PEC

with 4.6 + 0.9 x 106 I-A+ macrophages). As reported inFigure 1, the percentage of I-A+ macrophages in the groupC animals progressively increased until Day 3, remainingat about the same level (50%) in the following 5 days. Incontrast, the spermatozoon treatment with SV-IV and TGase(group E) did not vary the percentage of I-A+ macrophagesin comparison with the HBSS-treatment (group A), whereasonly a slight increase was observed in the group of animalsinjected with spermatozoa treated with SV-IV alone (groupD). The difference observed between groups D and C, aswell as that between groups E and C, was found to be sta-tistically significant (Student's t-test, p < 0.01). In the ani-mals injected with FA (group B), the percentage of I-A+

macrophages was higher than in the animals of other groupsduring the entire time course.

The mean fluorescence intensity (FI) of the peritonealmacrophages stained for I-A increased until Day 3 only inrats injected with FA. (group B) or with untreated sper-matozoa (group C). Three days after the i.p. injection ofuntreated spermatozoa (group C), two main types of cellswere detectable in the macrophage population, namely cellswith a low I-A density (mean FI = 7) and cells with a highI-A density (mean FI = 70) (Fig. 2, panel I). The FI valuealso increased, from 6 on Day 1 to 70 on Day 3, in rats of

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SUPERNATANTSFIG. 3. Antigen presentation function of peritoneal macrophages obtained from differently treated animals. Su-

pernatant aliquots (25 p1) taken from cultures containing 105 T-cell hybridoma and various numbers of adherent PEC(S1 = 103 PEC; S2 = 104 PEC; S3 = 105 PEC; S4 = 5 x 105 PEC; S5 = 106 PEC; S6 = 1.5 x 106 PEC; S7 = 2 x 106PEC) obtained from differently treated animals. Spz = epididymal spermatozoa; open squares, group B (animals in-jected with F.A.); solid squares, group C (animals injected with untreated Spz); open circles, group D (animals injectedwith SV-IV-treated Spz); open triangles, group E (animals injected with SV-IV+active-TGase-treated Spz). Each valuerepresents the mean of six independent determinations performed in triplicate. Variability (expressed as SEM) in thevalues of 13H]thymidine incorporation was about 10%/. For experimental details see Materials and Methods.

group B (FA-injected animals; Fig. 2, panel II). These datareflect an increase of I-A expression on the peritoneal mac-rophage surface. In contrast, the macrophages elicited byspermatozoa treated with SV-IV (group D) showed only asmall increase in I-A expression on their surface (Fig. 2,

panel III), whereas the i.p. injection of spermatozoa treatedwith SV-IV in the presence of TGase (group E) did not pro-duce any change in the macrophage I-A expression in com-parison with the control HBSS- injected rats (group A; Fig.2, panel IV).

TABLE 1. Inhibitory effect of either SV-IV or SV-IV and active TGase on 02- production by rat peritonealmacrophages either resting (R) or sensitized in vivo with rat syngeneic epididymal spermatozoa (SES) and thenstimulated in vitro with either SES or Zymosan.a

Nanomoles 02- produced/120 min/mg protein (+SEM)b

Peritoneal macrophages

Stimuli R FAC SES SES + SV-IVd SES + SV-IV/T'

None 6 + 2 29 + 3 18 + 2 8± 1 9 + 2Zymosan + SODf 5 3 5 2 4 2 6 + 1 7 3Zymosan 280 + 20 488 + 15 406 + 21 290 + 16 195 + 18SES 4+ 6 180 4 90+ 4 42± +8 24 +-7SV-IV-treated SESg 26 + 5 22 + 2 30 + 3 20 + 2 19 + 2SV-IV/T-treated SESh 7 + 3 27 + 6 15 + 4 6 2 8 3

'For experimental details see Materials and Methods.bThe amount of superoxide anions released into the medium from the peritoneal macrophages was expressed asnmoles of 02- produced per 2 hr and per mg of peritoneal macrophage proteins ± standard Error of the Mean (SEM).Each value represents the mean + SEM of 10 independent determinations performed in triplicate.

CFA = rat peritoneal macrophages sensitized in vivo with complete Freund adjuvant.dSES + SV-IV = rat peritoneal macrophages sensitized in vivo with SV-IV (5 M)-treated SES.eSES + SV-IV/T = rat peritoneal macrophages sensitized in vivo with SV-IV (5 iM) and active TGase (0.6 ig/ml)-treated SES.

fSOD = superoxide dismutase (300 U/ml).0SV-IV-treated SES = SES treated with 5 pM SV-IV.hSV-IV/T-treated SES = SES treated with 5 p.M SV-IV and 0.6 pg/ml active TGase.

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FIG. 4. Ability of rat peritoneal macrophages to phagocytose epididymal spermatozoa. For experimental detailssee Materials and Methods. A) Untreated spermatozoa (phase contrast light microscopy; magnification x400). B) SV-IV (5 M) + active TGase (0.6 g/ml)-treated spermatozoa (phase contrast light microscopy; magnification x400). C)Untreated spermatozoa (TEM; magnification x10 000). D) SV-IV (5 L~M) + active TGase (0.6 pig/ml)-treated sperma-tozoa (TEM; magnification x10 000). In panel C, the triangular electron-dense structure present in the macrophagecytoplasm and indicated by the arrow represents an engulfed sperm head.

Inhibition of Peritoneal Macrophage Antigen Presentationafter Injection of SV-IV/TGase-Treated Spermatozoa

To determine whether the I-A+ macrophages induced byspermatozoa had an active function in the antigen presen-tation, we investigated the ability of murine antigen-pulsedperitoneal macrophages to induce T-cell activation in vitro.Macrophages from mice treated with HBSS (group A) orF.A. (group B), or with untreated (group C), SV-IV-treated(group D), or "SV-IV + TGase"-treated (group E) mouseepididymal spermatozoa (the experimental conditions de-fining these animal groups were identical to those abovereported for rats) were pulsed in vitro with the antigenapocytochrome c fragment 1-65, fixed with paraformal-dehyde, and then tested for their ability to induce IL-2 se-cretion from a T-cell hybridoma specific for the antigen andrestricted to I-Ab. The macrophages from groups B and Cwere markedly more active in presenting antigen in com-parison with macrophages obtained from groups D and E(Fig. 3;p < 0.01), the activity of the latter group being sim-ilar to that of group A cells. Thus, the spermatozoa injectedi.p. were able to induce Ia-rich macrophage populations

with the expected increase in antigen presentation func-tion, whereas their treatment with SV-IV, and even morewith SV-IV and TGase, inhibited this induction.

Inhibition of Superoxide Anion Production by PeritonealMacrophages after Injection of SV-NIV/TGase-TreatedSpermatozoa

In the complex of biochemical and biological events as-sociated with the process of macrophage activation, we in-vestigated the release of superoxide anions, as a marker forthe macrophage respiratory burst capacity. The superoxideanion release was measured after the in vitro exposure ofrat peritoneal macrophages to different stimuli (zymosan,rat syngeneic epididymal spermatozoa treated or not withSV-IV + TGase). The macrophages obtained from rat sper-matozoon-injected animals produced an amount of 02- sig-nificantly lower (p < 0.01) than that released from mac-rophages of F.A.-treated animals, but significantly higher (p< 0.01) than that released from macrophages of both con-trols and animals injected with spermatozoa treated withSV-IV, in the presence or absence of TGase (Table 1). In

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TABLE 2. Suppression of rat epididymal spermatozoon immunogenicityby either SV-IV or SV-IV and active TGase as measured by [3H]thymidineincorporation into rat splenocytes.'

[3H]Thymidine incorporation into

Treatment splenocytesbof sperm cpm % of control

None (control 8,860 - 570 100SV-IV 3,300 - 440c 37TGase 8,685 620 98TGase + SV-IV 1,110 - 140 c 12

aFor experimental details, see Materials and Methods.bEach value represents the mean SEM of 10 independent determinationsperformed in triplicate.

CSignificantly different from the control (p < 0.01).

particular, these experiments clearly showed that the treat-ment of spermatozoa with both SV-IV and TGase inducedthe most evident decrease in 02- release from macro-phages of the differently treated animals challenged in vitrowith sperm cells.

Pretreatment of Spermatozoa with SV-IV/TGaseDramatically Reduces Their Phagocytosis by PeritonealMacrophages

We tested the macrophage peritoneal populations for theirability to phagocytose the spermatozoon target cells. Ultra-structural studies revealed the entry of acrosome-intact spermheads into the macrophages through a process of plasmamembrane invagination associated with the formation ofphagocytic vacuoles only when the spermatozoa were notpreviously treated with SV-IV and TGase (Fig. 4).

Suppression of Spermatozoon Immunogenicity by SV-IV/TGase as Measured by Mixed Cell Culture

The suppressive effect of SV-IV on the strong immuno-genicity of epididymal spermatozoa was also shown in vitroby a mixed cell culture of rat splenocytes and spermatozoa.The splenocytes were used as responder cells that, whenstimulated by the surface antigens of the rat epididymalspermatozoa (H-2, H-3, and H-13 defined transplantationantigens, antigenic determinants of Ia type, differentiationantigens, etc.) [1,7,10,34], enlarge, synthesize DNA andproliferate, with the intensity of cell division proportionalto [3 H]thymidine incorporation [2]. The epididymal sper-matozoa were pretreated with SV-IV, TGase, or SV-IV + TGasefor in vitro evaluation of the ability of these seminal pro-teins to mask the sperm cell antigens and hence decreasetheir immunostimulatory properties. The data reported inTable 2 clearly demonstrate that the treatment of sperma-tozoa with either SV-IV (5 jIM) or SV-IV (5 ,uM) + TGase(0.6 jig/ml) produced about 60% and 90% inhibition ofthe epididymal sperm immunogenicity, respectively, asmeasured by [3 H]thymidine incorporation in a lymphocyteproliferation test.

DISCUSSION

Immunologic mechanisms have been invoked as a causeof infertility because spermatozoa express on their surfacedifferentiation and histocompatibility antigens that can berecognized as nonself by the female and male immune sys-tem. Immunization with spermatozoa produces infertility inexperimental animals, and antibodies directed against sper-matozoon antigens have been detected and associated withinfertility in animals and humans. Cell-mediated immunemechanisms are virtually unexplored in infertility, and it iswell known that the cellular mediators of cell-mediated im-mune responses, T lymphocytes and macrophages, arepresent in both female and male genital tracts. The datareported in this paper clearly demonstrate that the peri-toneal macrophages become strongly activated when chal-lenged either in vivo or in vitro with untreated epididymalspermatozoa. The pretreatment of epididymal spermatozoawith SV-IV significantly reduces several parameters of themacrophage activation, most probably as a consequence ofa marked decrease of sperm cell immunogenicity. In par-ticular, this effect was observed to be higher when the spermcells were pretreated with SV-IV and active TGase (in thepresence of Ca2+ and in the absence of EDTA), whereas theuse of EDTA abolished the TGase effect (data not shown).Moreover, no changes in the spermatozoon-elicited im-mune responses were detected when the spermatozoa weretreated with active TGase in the absence of SV-IV. The mo-lecular mechanism underlying the decrease of spermato-zoon immunogenicity following sperm treatment with SV-IV and active TGase is probably related to a phenomenonof membrane antigen masking due to the binding of theseproteins on the sperm cell surface, even though othermechanisms cannot be completely ruled out [2]. By eval-uating the activation level of rat peritoneal macrophageschallenged with i.p. injected syngeneic ejaculated sperma-tozoa, we demonstrated that these spermatozoa possess avery low immunogenicity (data not shown). Other animalstudies have also shown that in mice the ejaculated sper-matozoa, although bearing on their surface antigenic de-terminants pertaining not only to the MHC class I/H-2 (H-2D and H-2K) and class II/Ia systems but also to the minorH-3 and H-13 systems, are poorly immunogenic as a con-sequence of a possible masking event operated by specificproteins present in the seminal plasma [1, 2, 5, 7, 9-11]. Onthe basis of these data, we can speculate that the antigenmasking occurring on the epididymal spermatozoon sur-face under the influence of the SV-IV and active TGasetreatment could be the primary event that, by suppressingthe phagocytosis of epididymal spermatozoa by peritonealmacrophages, would markedly decrease the expression inmacrophages of the biological phenomena related to theprocess of cell activation triggered by allogeneic cells (in-crease in class II MHC surface antigen expression, super-oxide anion production, antigen presentation, etc.). It is

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worth noting that no suppressing effects on peritoneal mac-rophage activation in vivo or on [3H]thymidine incorpora-tion by rat splenocytes co-cultured in vitro with epididymalspermatozoa were observed when albumin, actin, or cy-tochrome c were substituted for SV-IV at similar concen-trations (data not shown).

The suppressive effect of SV-IV on the epididymal sper-matozoon immunogenicity was also demonstrated in vitroby a mixed culture of rat splenocytes and spermatozoatreated with SV-IV either in the absence or in the presenceof active TGase. The data obtained indicate that the treat-ment of spermatozoa with either SV-IV or SV-IV plus activeTGase produced about 60% and 90% reduction of the ep-ididymal sperm immunogenicity, respectively. In these ex-periments, the marked decrease of immunogenicity isprobably also related to the masking of the highly immu-nogenic antigens present on the sperm cell surface by theSV-IV binding on gamete plasma membrane.

The greater effect always observed when the sperm cellswere pretreated with SV-IV and active TGase, in compari-son with that obtained after treatment with SV-IV alone, mightbe explained by the fact that branched SV-IV homopoly-mers, resulting from the enzymatic action of TGase on SV-IV [27], bind to the surface of sperm cells in larger amountsthan the native monomeric SV-IV [21].

We have previously reported that a TGase activity, vari-able from individual to individual [35], and a family of pro-teins immunologically related to SV-IV [36] are present inhuman seminal fluid. Therefore, the experimental evidencethat epididymal spermatozoa activate peritoneal macro-phages, while their treatment with SV-IV and active TGasealmost abolishes this effect, might indeed have potentiallyimportant clinical implications. The cervix, uterus, and ovi-ducts, as well as the peritoneum, are immunologically dy-namic tissues, and the macrophages residing there can beactivated by a variety of nonself antigens [37, 38]. In womenwith immunologic reproductive failure, the spermatozoa maybe killed either as innocent bystanders, following localmacrophage activation in response to bacterial or viral an-tigens, or more specifically damaged after cell-mediatedimmune responses directed against sperm antigens [39]. Onthe other hand, it is well known that the i.p. injection ofejaculated spermatozoa in women triggers adverse immunereaction against spermatozoa in only a few cases [23]. Thisfact can be due to specific seminal proteins inducing a de-crease in the immunogenicity of the epididymal sperma-tozoa following ejaculation. In this respect, previous studieshave indicated that a wife's alloimmunity to the sperm ofher husband is associated with the autoimmunity of thehusband to his own sperm [40]. Therefore, our results sup-port the view that reduced levels of immunosuppressivefactor(s) in the seminal fluids and/or uterine secretions couldcause, in both male and female genital tracts, an adverseimmune reaction against spermatozoa that would severelyimpair their ability to survive and fertilize.

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Suppression of rat epididymal sperm immunogenicity by a seminal vesicle secretory protein and transglutaminase both in vivo and in vitro