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BIOLOGY OF REPRODUCTION 55, 194-199 (1996) Effect of Boar Seminal Immunosuppressive Fraction on B Lymphocytes and on Primary Antibody Response' Leopold Veselsk ,2,3 Jaromir Dostl,4 Vladimir Holfii,3 Josef Soucek,5 and Blanka Zelezna 3 Institute of Molecular Genetics,3 Academy of Sciences of the Czech Republic, 166 37 Prague, Czech Republic Institute of Animal Physiology and Genetics, 4 Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic Institute of Haematology and Blood Transfusion,5 128 20 Prague, Czech Republic cytes [11]. In vitro immunosuppressive activity can be attributed to spermine [12], and, as has been shown, the in vivo suppression of T lymphocytes requires the presence of spermine or related enzymes locally [13]. Seminal plasma impairs the ability of macrophages and neutrophils to generate oxygen species after being triggered with opsonized zymosan and phagocyte opsonized bacteria [141. The physico-chemical characterization of seminal plasma has provided evidence that transglutaminase together with rat seminal immunosuppressive component, and prostaglandins are the principal molecules contributing to seminal plasma immunosuppression of macrophages and NK cells [15-17]. Kelly et al. [18] have demonstrated that prostaglandins of the E series are almost entirely responsible for the inhibition of immunosuppression of the NK cell system. Nevertheless, seminal plasma is a complex mixture, and there is evidence of other immunosuppressive components present in seminal secretions. A protein of high molecular mass, isolated from human seminal plasma and identified as transforming growth factor , inhibited both DNA synthesis and killing activity of interleukin 2-stimulated murine lymphocytes [19]. It has also been documented that extracellular organelles of prostate-origin prostasomes may play a complementary role to other immunosuppressive factors contained in human semen [20]. Recently we have investigated the absorption of the immunosuppressive component (immunosuppressive fraction, ISF) isolated from boar seminal vesicle secretion on the surface of white blood cells after its in vivo application to male BALB/c mice. It was demonstrated that seminal ISF caused a decrease in lymphocyte numbers in the blood of treated mice, but the concentration of the granulocytes was not changed [21]. To better understand the role of seminal immunosuppressor in the regulation of immune reactions in the organism, it is important to determine which cell subset of the immune system absorbs the immunosuppressor and which cells are inhibited after the in vivo application of ISF The aim of this study was to determine the direct immunosuppressive effect of seminal ISF administered in vivo on separated T and B lymphocytes. The ability of ISF to inhibit the primary antibody response to soluble or corpuscular antigens was also studied. ABSTRACT Repeated i.p. or rectal treatment of male and female mice with an immunosuppressive component isolated from boar seminal vesicle secretion reduced responses of B lymphocytes to mitogen as evaluated by [3 H]thymidine or bromo-deoxyuridine incorporation. The proliferative activity of T lymphocytes was not affected. By means of the immunofluorescence method, the seminal immunosuppressive component was detected on the membranes of B lymphocytes separated from the spleens of mice treated in vivo with immunosuppressor. An i.p. injection or rectal infusion of the immunosuppressive component also led to a suppression of primary antibody response to soluble and particulate antigens. These findings indicate that in vivo deposition of semen may compromise some aspects of the immune system and may be an important cofactor in the development of viral and bacterial infections in homosexual men. INTRODUCTION The female reproductive tract has the capacity to mount an immune response to environmental stimuli. Intravaginal immunization with antigens introduced into the female genital tract can elicit a specific humoral response, demonstrating that the female genital tract is not an immunoprivileged site. Antibodies have been induced by direct immunization of the vagina with protein antigens or as a result of natural or experimental infection of the lower reproductive tract with a variety of pathogens [1, 2]. In the male and female genital tracts, many cells belonging to the immune system are physiological residents [3]. However, insemination with histoincompatible sperm normally does not invoke an antisperm immune response. Seminal plasma may provide a physiological protective environment to prevent immune recognition of highly antigenic sperm. The abrogation of the immune response to sperm is important for successful conception [4], but at the same time other essential immunological events are suppressed. In vitro studies have demonstrated that seminal plasma components can impair the generation of cytotoxic T cells, the response of B cells to a variety of antigens [5, 6], and the cytotoxic effect of natural killer (NK) cells and of activated human antitumor effector cells [7, 8]. The inhibition of immunocompetent cell activity has been demonstrated in a number of species, but the effect of seminal immunosuppressors on macrophages and lymphocytes is not species-specific [9,10]. Human seminal plasma components can also decrease the antibacterial activity mediated by peripheral blood lympho- MATERIALS AND METHODS Isolation of ISF ISF was isolated from boar seminal vesicle secretion according to the procedure described by Dostal et al. [21]. Seminal vesicle secretion was precipitated in 8% ethanol (pH 7.2) at -2.5°C. A sample of 100 mg of the precipitate, which had been dialyzed and dissolved in PBS (pH 7.2), was applied to a Sephacryl S-200 column (2.4 x 63 cm; Pharmacia, Uppsala, Sweden) equilibrated with PBS (pH 7.2) at a flow rate of 4.2 ml per 15 min. The fractions with Accepted March 11, 1996. Received September 25, 1995. 'Supported by grant No. 310/93/0307 from the Grant Agency of the Czech Republic. 2Correspondence: Leopold Veselsky, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague 6, Czech Republic. FAX: 42224310 955. 194 EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES inhibitory activity on porcine and murine lymphocytes were pooled and run through a Sephadex G-75 column (1.4 x 94 cm; Pharmacia) equilibrated with PBS (pH 7.2) at a flow rate of 1.5 ml per 20 min. The active fraction was further purified by HPLC on a Vydac C-18 RPc column (Vydac, Hesperia, CA) in a gradient of 20-40% acetonitrile in 0.01% trifluoroacetic acid at a flow rate of 1 ml/min. The gradient was set according to a high-pressure LKB system (LKB, Bromma, Sweden) with two 2150 pumps and a 2152 controller. The yield of ISF after a final step of purification was 100-200 pxg/ml of boar seminal secretion. For the analysis of molecular mass, a 1 mM solution of ISF was prepared in 70% acetonitrile with 1% trichloroacetic acid. An aliquot of 0.2 1d (2 pmol ISF) of sample solution was mixed with 0.1 p1 of protein matrix (1 mM sinapinic acid) on a disposable sample slide. The droplet was allowed to dry, and the slide was loaded into a Lasermat Mass Analyzer (Finnigam MAT, San Jose, CA). Immunization The ability of ISF to suppress the primary antibody response to a soluble antigen keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) and sheep red blood cells (SRBC) was evaluated. Fifteen male and 15 female BALB/ c mice each received an i.p. injection of 0.2 mg ISF in 0.1 ml PBS, and another group of 15 male and 15 female mice each received a rectal infusion of 0.4 mg ISF in 0.1 ml saline, on Days 0 and 1. On Day 2, the mice were immunized i.p. with 250 jIg KLH. The same-day schedule of ISF rectal or i.p. treatment was used in mice immunized with SRBC (1 x 108 cells in 0.1 ml PBS). Saline instead of ISF was administered as a control, with the same immunization procedure. Antibody titers to KLH or SRBC were measured by ELISA in the blood serum of experimental and control mice on Day 10 after the immunization. The technique of rectal infusion of ISF used was described previously [21, 22]. Cell Preparation, and T and B Lymphocyte Separation The effect of ISF on B or T lymphocyte proliferation was evaluated by mitogen-induced lymphocyte proliferation and by an immunoassay system for detection of bromodeoxyuridine incorporation (Amersham kit; Amersham, Little Chalfont, UK). For i.p. application, 3 groups of 5 mice each received injections of 0.2 mg of ISF on Days 0 and 1. The fifth day after the first injection of ISE splenocytes were obtained from the treated mice. For rectal infusion, 3 groups of 5 mice each were anesthetized with ether and received infusions of 0.4 mg of ISF in 0.1 ml saline on Days 0 and 1. The ninth day after the first infusion, splenocytes were tested. Controls received saline instead of ISF Spleens of the treated mice were homogenized, and the cell suspension was passed through a 110-iLm mesh, washed three times by centrifugation at 400 X g for 5 min each time, and resuspended in RPMI 1640 culture medium. Purified T and B lymphocytes were prepared by a two-cycle passing procedure of spleen cells through nylon wool columns [23]. The proportion of T and B lymphocytes in T and B cellenriched cell populations was tested by a fluorescence activating cell sorter (FACS) analysis using anti-CD3 and anti-Ig antibodies, respectively. T cell-enriched cell populations contained 85-90% of T cells and less than 5% of Ig+ cells. B cell-enriched populations contained over 90% of Ig+ cells and less than 5% of T cells. Separated lym- 195 phocyte subsets were used for measurement of lymphocyte proliferation. The absorption of ISF in vivo on the surfaces of T and B lymphocytes was determined by an immunofluorescence assay. Measurement of Lymphocyte Proliferation Separated T and B lymphocyte suspensions from mice treated rectally or i.p. with ISF were adjusted to 2 x 106 cells ml in RPMI 1640 medium (Serva, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 IU/ml penicillin, and 0.1 g/ml streptomycin. The B lymphocytes, stimulated with 5 [Ig pokeweed mitogen (PWM; Sigma) per ml RPMI 1640 medium, and the T lymphocytes, stimulated with 5 ig concanavalin A (Con A; Serva, Heidelberg, Germany) per ml RPMI 1640 medium, were cultured in triplicate in culture microtiter plates. Nonseparated lymphocyte cultures were stimulated with 10 g phytohemagglutinin (PHA; Wellcome Research Laboratories, Dartford, UK) per milliliter of RPMI 1640 medium. The lymphocytes were cultured at 37°C for 72 h. After 48 h of culture, 37 kBq [3 H]thymidine (spec. act. 840 GBq/ml; Institute for Research, Production and Application of Radioisotopes, Prague, Czech Republic) per well was added. The cells were collected onto glassfiber discs by use of a semiautomatic harvester, and the radioactivity was measured according to a standard liquid scintillation technique. The viability of the lymphocytes was greater than 95% as determined by trypan blue dye exclusion. In the immunoassay system for detection of bromo-deoxyuridine incorporation, the proliferation of lymphocytes was accomplished according to instructions supplied with the cell proliferation assay kit (Amersham). Suspensions of T and B lymphocytes separated from rectally or i.p. treated mice were adjusted to 1.5 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FSC, L-glutamine, penicillin, and streptomycin. Triplicate cultures of B lymphocytes stimulated with PWM, of T lymphocytes stimulated with Con A, and of nonseparated lymphocytes stimulated with PHA were incubated in 100 p,1 of culture medium in culture microtiter plates at 37°C for 72 h. The cells were incubated during the last 2 h of culture with 100 1 of labeling medium containing a solution of 5-bromo-2'-deoxyuridine and 5-fluoro-2'-deoxyuridine in a ratio of 10:1 (v:v). The T-cell plates were centrifuged at 1500 X g for 10 min and dried at 37°C for 3 h after medium was removed. The B cells were washed with PBS. The B and T cells were fixed in a solution of ethanol:acetic acid:water (9:5:5 v:v:v) for 30 min and washed in 0.1% Tween 20 in PBS. The plates were blocked with 1% nonfat milk for 15 min at 22°C, and 50 l1 of anti-5-bromo-2'-deoxyuridine, containing the nuclease to denature DNA, was added into the wells. After the plates were washed, 50 p,1 of antimouse IgG2a serum conjugated with peroxidase was added. Bound peroxidase activity was detected by use of 2-2'azino-bis(3-ethylbenzthiazoline sulfonate) and H2 0 2 as substrate. Incorporation of 5-bromo-2'-deoxyuridine into replicating DNA was determined at 410 nm. The effects of ISF on the proliferation of normal human lymphocytes and on two human tumor cell lines, ML-1 (non-T, non-B cells) and K 562 (erythroleukemia) were also tested. The effect of ISF on proliferation of normal human lymphocytes was evaluated by mixed lymphocyte culture (MLC). Lymphocytes from two unrelated persons, isolated on a Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden), VESELSKY ET AL. 196 FIG. 1. Antibody response of male and female mice treated rectally or i.p. with ISF two days before KLH immunization. Antibodies to KLH were measured by ELISA on Day 10 after immunization. Values are expressed as mean + SD from three different experiments with 5 male or female mice. Comparable titer values were obtained when mice were immunized with SRBC. *Difference in suppression of primary antibody response to challenging antigen between ISF treated and control mice, p < 0.01. +Difference in suppression of primary antibody response to challenging antigen between ISF-treated male and female mice, p < 0.01. KLH rectal ISF KLH F 1~~~* rectal ISF KLHJ i. p. ISF KLH2 i, p. ISF KLHd g+ were mixed at a ratio of 1:1. The final lymphocyte concentration was adjusted to 1 x 106 cells/ml RPMI 1640 medium supplemented with 20% human AB serum and antibiotics. The effect of ISF on the growth of the two tumor cell lines was evaluated by inhibition of spontaneous cell proliferation. The tumor cell concentration was adjusted to 2 x 105 cells/ml RPMI 1640 medium supplemented with 10% FCS and antibiotics. All cell cultures (100 1I per well) were done in triplicate in microtiter plates. Seminal ISF (100 I 1 per well) was added at concentrations of 50, 100, and 150 l.ug into wells. The cells were cultured at 37C for 6 days. Four hours before the termination of incubation, the cells were pulsed with 37 kBq [3 H]thymidine. The values were measured by a standard liquid scintillation technique [24]. 10 Antibody Titer 3 (x 10 ) pensions (6 x 106 cells/ml) were incubated with an equal amount of LIS-4 (120 jig lyophilized antibody/ml PBS) at 22°C for 20 min, were thoroughly washed with 1% BSA in PBS and 1% non-fat milk in PBS, and were incubated at 22°C for 20 min with fluorescein isothiocyanate conjugated to swine anti-mouse immunoglobulin diluted 1:40, containing 0.02% sodium azide (USOL). A droplet of cell suspension was applied to a glass slide and examined under the Orthoplan fluorescence microscope (Leitz, Wetzlar, Germany) with an excitation filter of 450-490 nm and barrier filter of 515 nm. Statistical Analysis The significance of differences between experimental and control groups was analyzed by means of Student's ttest. ELISA The microtiter plates were coated with 1 g of KLH and incubated at 4°C for 18 h. After the plates were washed in PBS-Tween (PBS containing 0.1% Tween 20 and 1% BSA), serially diluted antisera to KLH from mice treated rectally or i.p. with ISF were added and incubated at 22°C for 2 h. The plates were washed, and porcine anti-mouse IgG serum conjugated with peroxidase (USOL, Prague, Czech Republic) diluted 1:3000 was added. Bound peroxidase activity was detected with o-phenylenediamine and H20 2 used as substrate. The absorbance was determined at 492 nm. For erythrocyte ELISA, the plates were incubated with 0.01% poly L-lysine (Sigma) in PBS at 22 0C for 1 h. After being washed, the plates were filled with 100 p.1 of SRBC (1 X 106 cells/ml) and incubated at 22°C for 1 h. After the plates were washed in PBS, serially diluted antisera to SRBC from mice treated rectally or i.p. with ISF were added and incubated at 22°C for 2 h. The same procedure was used as for detection of KLH antibodies in ELISA. Antisera of control mice immunized with KLH or SRBC but not treated with ISF served as positive controls. Normal sera of mice treated with saline alone were used as negative controls. Indirect Immunofluorescence Technique Indirect fluorescence was used in studies of ISF absorption on separated T and B lymphocytes isolated from ISFtreated mice 5 days after i.p. injection and 9 days after rectal infusion of ISE Production of the monoclonal antibody to ISE LIS-4, used in the immunofluorescence assay for detection of ISF absorption on lymphocytes was described previously [21]. Separated T and B lymphocyte sus- RESULTS Isolation of ISF The ISF separated from boar seminal vesicle secretion by gel filtration on a Sephadex G-75 column [10] was further purified by reversed-phase HPLC. The inhibitory activity was estimated by inhibition of mitogen-induced lymphocyte proliferation on porcine lymphocytes. One milliliter of seminal vesicle secretion yielded 100-200 g of ISE The molecular mass of ISF estimated on a Lasermat Mass Analyzer (Finningam-MAT; San Jose, CA) was 14 kDA [21]. Suppression of Primary Antibody Response We examined the ability of ISF to suppress the antibody response to soluble and particulate antigens. The i.p. or rectal administration of ISF two days before immunization of male or female mice significantly suppressed the primary antibody response to both KLH and SRBC (Fig. 1). In ISFtreated females immunized with KLH, the antibody response suppression was 81% after i.p. injection and 69% after rectal infusion of ISF (p - 0.01). In ISF-treated males immunized with KLH, the antibody response suppression was 98% after i.p. injection and 96% after rectal infusion of ISF (p 0.01). The difference in the suppression of primary antibody response to challenging antigens between ISF-treated male and female mice was statistically significant (p < 0.01). Comparable antibody titers were obtained when male and female mice were immunized with SRBC. Antibody titers to KLH and SRBC were measured by ELISA on Day 10 after immunization. EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES 197 TABLE 1. Effect of i.p. and rectal administration of ISF on T and B lymphocytes evaluated by mitogen-induced lymphocyte proliferation test.a Treatment B lymphocytes PWM 5 .g/ml % Inhibition T lymphocytes Con A 5 ig/ml % Inhibition Nonseparated lymphocytes PHA 10 g/ml % Inhibition Peritoneal Saline ISF 68 080 + 4272 10 104 t 2546 85* 44 840 ± 5240 45 971 + 4081 - 27 457 ± 3820 10 782 ± 1672 61* Rectal Saline ISF 72 936 + 5637 16 235 + 1206 78* 56 262 ± 2635 51 320 ± 3960 - 31 672 ± 2968 13 303 + 1012 58* Data represent the mean ± SD from 3 different experiments with 5 mice in each group. The lymphocyte proliferation was expressed as incorporation of [3H]thymidine. * p < 0.01. Suppression of the Proliferative Response of Lymphocytes To examine whether administration of ISF affected the mitogenic activity of T and B lymphocytes, mice were pretreated i.p. or rectally with ISF On Day 5 after i.p. injection and on Day 9 after rectal infusion, spleen cells from the treated mice were prepared and assayed for cell proliferation upon in vitro stimulation with mitogens. In the mitogen-induced proliferation test, the blastogenic response of B lymphocytes stimulated by PWM was significantly reduced (p - 0.01). The proliferative activity of T lymphocytes stimulated with Con A was not affected (Table 1). A similar inhibitory effect of ISF on B lymphocytes was observed in the immunoassay system for detection of bromodeoxyuridine incorporation. The proliferative activity of B lymphocytes was decreased by 76% after the rectal infusion and by 79% after the i.p. injection of ISE The proliferative activity of T lymphocytes was not affected (Table 2). Absorption of ISF on B Lymphocytes For the in vivo absorption of ISF on separated T or B lymphocytes of the treated mice, an indirect fluorescence method was used. The positive reaction with LIS-4 antibody was found on the membranes of 40-60% of the nonseparated lymphocytes and on membranes of 95-100% of the B lymphocytes separated from spleens of mice treated i.p. or rectally with ISF (Fig. 2, a and b). No positive reaction of LIS-4 with T lymphocytes separated from spleens of i.p. or rectally treated mice was found (Fig. 2c). The reaction of lymphocytes from control mice with LIS-4 was also negative. Negative results were also obtained when separated T lymphocytes were incubated in vitro with 100, 200, and 300 Rg of ISF per 1 ml media for 3 days at 37°C. Immunofluorescence of these ISF-treated T lymphocytes incubated with monoclonal antibody to ISF was equally as negative as the reaction shown in Figure 2c. Immunofluorescence of B lymphocytes was positive. Effect of ISF on Human Leukocytes The effect of ISF on human peripheral blood leukocytes and tumor cell lines ML-1 and K562 was evaluated by MLC and a spontaneous cell proliferation assay. The results from three separate experiments indicated that ISF suppressed only lymphocytes of healthy men. The proliferation of tumor cell lines was not affected (Fig. 3). DISCUSSION Seminal plasma suppresses a variety of immunological functions in vitro and in vivo [10, 25]. It has been concluded in studies of NK cell function that prostaglandins in primate semen are responsible for the inhibition of cell function [26]. Recent studies have indicated that rectal insemination of semen had several consequences. Significantly lower titers of IgM, IgG, and IgA antibodies to KLH were found in immune sera from KLH-immunized rabbits inseminated with homologous semen [27]. It was also demonstrated that cellular immune functions, including NK cell activity and mitogen stimulation of lymphocytes, were not reduced after a single rectal insemination of human seminal plasma into rhesus monkeys. However, metabolite values of blood plasma prostaglandin E2 did become elevated in the inseminated monkeys but not in controls [28]. Anal infusion of prostaglandin E2 or D 2 into male rats reduced in vivo response of T lymphocytes to PHA, but the T-cell response of female rats was not significantly changed [29]. TABLE 2. Effect of Intraperitioneal and intrarectal administration of ISF on T and B lymphocytes evaluated by an immunoassay system for detection of bromodeoxyuridine incorporation.a Treatment B cells PWM 5 g/ml % Inhibition T cells Con A 5 g/ml % Inhibition Nonseparated lymphocytes PHA 10 i.g/ml % Inhibition Peritoneal Saline ISF 0.235 0.049 0.049 ± 0.007 79* 0.296 ± 0.029 0.279 ± 0.049 - 0.286 ± 0.057 0.112 ± 0.021 61* Rectal Saline ISF 0.290 ± 0.052 0.074 ± 0.005 76* 0.317 ± 0.056 0.298 + 0.036 - 0.263 + 0.046 0.113 ± 0.027 57* Data represent the mean SD from 3 different experiments with 5 mice in each group. Lymphocyte proliferation was expressed as incorporation of 5-bromo-2'deoxyuridine into replicated DNA determined by ELISA using specific monoclonal antibody. * p < 0.01. 198 VESELSKY ET AL. CPI ISF pg/ml FIG. 3. Effect of seminal immunosuppressor on proliferation of healthy human lymphocytes (circles), tumor cell line K 562 (triangles), and tumor cell line ML-1 (squares). Lymphocyte proliferation was expressed as incorporation of [3H]thymidine - SD from three different experiments. FIG. 2. a) Immunofluorescent detection of immunosuppressive factor on membranes of nonseparated lymphocytes in mice infused rectally with immunosuppressor. b) Immunofluorescent detection of immunosuppressive factor on membranes of B lymphocytes in mice infused rectally with immunosuppressor. c) Nonpositive immunofluorescent reaction of LIS-4 antibody with T lymphocytes in mice infused rectally with immunosuppressor. a-c, x800. Nevertheless, it has been found that boar seminal plasma contains an extremely low concentration of prostaglandins (0.01 mg/ml) as compared with that in humans (0.578 mg/ ml) [30]. Seminal plasma expresses a number of semen-specific antigens that can elicit the immune response in sexual partners [31 ]. The lymphocyte function may be paralyzed by seminal plasma immunosuppressive factors, but semen macrophages can retain phagocytic, adherent, and motile functions [3]. No effect of boar ISF was found on cells involved in transplantation events including NK cell activity [32]. Moreover, seminal ISF did not affect the proliferation of human tumor cell lines ML-1 and K 562, but it inhibited the proliferation of MLC-stimulated normal human lymphocytes. It can be concluded that the effect of ISF on the immune system cells is not speciesspecific but that its antiproliferative activity depends on the presence of complementary receptors on the leukocyte membranes. The present study also demonstrated ISF on membranes of the B-cell lymphocyte subpopulation isolated from spleens of mice treated i.p. or rectally with ISE In vivo treatment with ISF significantly reduced the mitogenic activity of B lymphocytes stimulated with PWM. However, seminal immunosuppressor was not detected on T-cell membranes; likewise, T-cell proliferation was not abrogated by ISE The effect of ISF on white blood cells depends, we assume, on its half-life. In a preliminary study, we determined the duration of immune suppression induced by ISF treatment after primary and secondary immunizations with soluble and corpuscular antigens. The results indicated that the treatment with ISF leads to a prolonged immunosuppression but not to a permanent tolerance to antigens. These preliminary data suggest that prolonged immunosuppression is associated with the continued presence of ISF in blood serum. It can also be assumed that the capability of the receptors to bind ISF on the cell surface is renewed. The most important result of our study is the evidence that ISF inhibited the antibody response to challenging antigens. The finding of the primary antibody inhibition to antigens indicated the sex-related difference of the sensitivities to ISE Even when the capacity of ISF to suppress the antibody response in females was also significant, the antibody titers in the female sera were higher than those found in male sera. It seems reasonable to conclude that females are less sensitive to male seminal immunosuppressor. These data indicate that the boar seminal immunosuppressor might impede the development of humoral im- EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES mune response via reduction of lymphocyte proliferation and inhibition of antibody response, both in males and females, after its in vivo deposition. We do not suppose that rectal infusion of ISF traumatizes the mucosal barrier. If there were a breach of the intestinal wall after rectal infusion, the suppressive activity of ISF on white blood cells would be simultaneous with the i.p. injection. The results of our previous paper [21] indicated that the suppressive effect of ISF on white blood cells reached a maximum on Day 5 after the i.p. application. However, maximum reduction of the lymphocyte number was achieved on Day 9 after rectal infusion of ISE Also, inspection (after they were killed) of the mice treated rectally with ISF excluded physical damage of the colon, including the rectal colon. The finding that seminal ISF infused rectally inhibited the primary antibody response suggests that seminal immunosuppressive components may interfere with immunological functions associated with a number of pathological states including AIDS, and may be a factor that decreases an immune response. Excessive exposure to seminal plasma at sites other than the genital tract may have a systemic effect. Seminal components may gain access to the circulation if they are deposited in the gastrointestinal tract, especially if it has been traumatized [4]. We have postulated that seminal components deposited in the rectum may be a factor in the etiology of AIDS and other viral and bacterial infections in males and females. Immunosuppressive molecules might reduce the host defense mechanisms, especially after repeated exposure. ACKNOWLEDGMENTS The authors thank Mrs. M. Ho~kovd and Mrs. L. KoberovA for their excellent technical assistance. REFERENCES 1. Parr EL, Parr MB. A comparison of antibody titers in mouse uterine fluid after immunization by several routes and the effect of the uterus on antibody titers in vaginal fluids. J Reprod Fertil 1990; 89:619-625. 2. Stern JE, Nelson TS, Gibson SH, Colby E. Anti-sperm antibodies in female mice: responses following intrauterine immunization. Am J Reprod Immunol 1994; 31:211-218. 3. Anderson DJ, Hill JA. Immunological aspects of the reproductive organs and implications of intercourse. Curr Opin Immunol 1989; 1: 1119-1124. 4. James K, Hargreave TB. 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