Journal of Reproductive Immunology, 22 (1992) 281-298
281
Elsevier ScientificPublishers Ireland Ltd. JRI 00787
Antifertility effects of antisperm cell-mediated immunity in mice Florina Haimovici, Kazue Takahashi
and Deborah
J. A n d e r s o n
Fearing Research Laboratory, Harvard Medical School, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital. 250 Longwood A venue, Boston, MA 02115 (USA)
(Accepted for publication 18 May 1992)
Summary C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u, protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u, sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a signifiCorrespondence to: Deborah J. Anderson, Ph.D., Fearing Research Laboratory, Brigham and Women's Hospital, 250 Longwood Avenue, Boston, MA 02115, USA.
0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
282
cant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.
Key words: pregnancy; infertility; abortion; sperm; cell-mediated immunity
Introduction
Immunization of female mice with sperm, sperm antigens or testis homogenates has produced decreased fertility in a number of studies (McLaren, 1964, 1966; Edwards, 1964; Bell, 1969; Tung et al., 1979; Wheat and Goldberg, 1979; Kasai et al., 1987). The degree of immunological response and anti-fertility effect can be influenced by the frequency of the immunizations (Snell and Poucher, 1991), the strain of mice (Madrigal et al., 1986; Kasai et al., 1987) and the route of immunization (Bell, 1969; Allardyce, 1984). Associations between antisperm antibody titer and reduced fertility have been varied. McLaren (1966) reported a significant association between sperm agglutination titer and reduced litter size in mice. However, Edwards (1964) and Bell (1969) using different immunization procedures, found no relationship between serum titer and fertilization rate. Testicular germ cells and spermatozoa can stimulate cell-mediated immunity in addition to humoral immune responses (EI-Alfi and Bassili, 1970; Mancini and Andrada, 1971; Nagarkatti and Rav, 1976; Anderson et al., 1982; McShane et al., 1985; Shelton and Goldberg, 1985; Naz and Mehta, 1989). Furthermore, various cytokines produced by activated lymphocytes and macrophages during cellular immune responses have been shown to affect reproductive functions adversely, including sperm motility (Hill et al., 1987; Eisermann et al., 1989), sperm-egg interaction (Maruyama et al., 1985; Hill et al., 1989), embryo viability (Hill et al., 1987; Tartakovsky and Ben-Yair, 1991), mouse trophoblast outgrowth (Haimovici et al., 1991) and human trophoblast (choriocarcinoma) cell proliferation in vitro (Berkowitz et al., 1988). It is possible, therefore, that intrauterine antisperm cell-mediated immune responses significantly contribute to the antifertility effects observed following immunization to sperm. Subsets of lymphocytes home to mucosa-associated lymphoid tissue through interactions between a distinct class of adhesion molecules expressed on the lymphocyte surface (homing receptors) and tissue-specific vascular
283
adhesion molecules expressed on the surface of high endothelial venules (Berg et al., 1989). Recent studies indicate that circulating memory/activated T lymphocytes and in situ mucosal lymphocytes which are negative for the peripheral-lymph-node (PLN) -homing receptor and positive for mucosallymphocyte-associated (MLA) -receptor show preferential binding to mucosal high endothelial venules and preferentially home to mucosal sites. Studies performed by McDermott et al. (1979, 1989) have provided evidence that the female reproductive tract is a part of the general mucosal immune system and that mucosal-derived B and T lymphocytes can specifically migrate to genital tissues. However, little is yet known about the mechanisms governing mucosal immune responses in the female reproductive tract and their role in infertility. In this study we explored various spermimmunization protocols to induce intrauterine immunological responses and infertility in mice and used a T cell passive transfer approach to study homing of mucosal primed T cells to the uterus and to determine the effects of sperm-sensitized T cells on fertility and pregnancy. Materials and Methods
Preparation of cells to be used as antigens Sperm. DBA/2 retired breeder males (Jackson Laboratories, Bar Harbor, ME) were killed by cervical dislocation and the cauda epididymides were removed and minced with fine scissors. Sperm were suspended in KrebsRinger solution, passed through a fine screen and collected from the cell pellet following centrifugation at 400 x g for 5 min. Motile sperm were further isolated from immotile sperm and somatic cells by centrifugation through a discontinuous gradient of Percoll (Pharrnacia, Piscataway, N J) (47%/90%) for 30 min at 500 x g. The cell pellet containing motile sperm was washed 3 times and resuspended at a final concentration of 1 x 107/ml in saline. The cell suspension was mixed 1:1 with CFA or IFA to form a homogenous emulsion, aliquoted and stored at -70°C until use. Spleen mononuclear leukocytes. Splenic mononuclear leukocytes from DBA/2 retired breeders were isolated by centrifugation through LympholyteM (Accurate Chemical & Scientific Corp., Westbury, NY) for 30 min at 500 x g. Leukocytes removed from the interface above the Lymphocyte-M were washed 3 times, resuspended at a concentration of 1 x 10 7 in saline, mixed 1:1 with CFA or IFA to form a homogenous emulsion and aliquoted and stored at -70°C until use. Red blood cells. Human red blood cells were isolated from heparinized peripheral blood by centrifugation through Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc., Piscataway, N J) for 30 min at 500 x g. The buffy coat on the red blood cell pellet containing granulocytes was discarded. The
284
bottom layer containing < 99% red blood cells (confirmed by microscopic evaluation) was collected, washed, resuspended in saline at a concentration of 1 x 107/ml, mixed 1:1 with adjuvant, aliquoted and stored at -70°C. Active immunization protocols C57BL/6 female mice (Jackson Laboratories, Bar Harbor, ME) maintained on a 12-h light/dark cycle were immunized with 1 x 10 7 allogeneic (DBA/2) sperm, in Complete Freund's Adjuvant (CFA; Cappel, Organon Teknika Corp., West Chester, PA). Control female mice of the same age and strain were injected with: (1) saline (0.9% NaCI in distilled H20) as a control for stress, (2) saline-plus-CFA as a control for the effects of the CFA alone, and (3) human red blood cells and (4) paternal (DBA/2) mouse lymphocytes in CFA to assess the sperm-specificity of the antifertility effect. Booster injections of 1 x 10 7 sperm or control cells, or saline alone in Incomplete Freund's Adjuvant (IFA; Cappel, Organon Teknika Corp.) were given on day 14 and 21. Injections (0.1 ml) were administered: (1) subcutaneously into the neck (s.c.), (2) intraperitoneally (i.p.), or (3) intrauterine (i.u.) through the cervix which was dilated by injection 12 h earlier with 1 IU of Pregnant Mare's Serum (PMS; Sigma Chemical Co., St. Louis, MO). For intrauterine injections, mice were anesthetized with 0.2 ml of Advertine solution (2.5 ml tert-amyl alcohol (Fisher Labs, Medford, MA), 1.25 g 2,2,2,tribromoethanol 99% (Aldrich Chemical Company, Milwaukee, WI) in 100 ml distilled water) and the cervix was visualized through a glass speculum. A catheter fitted with a 1-ml syringe and a 27 1/2-gauge needle (Becton Dickinson, Rutherford, N J) was threaded through the cervical opening and 0.1 ml of cells or saline emulsified with adjuvant was introduced into the uterine lumen. Twelve hours before the final booster injection, mice in all immunization groups were primed with 1 IU of PMS to cycle the animals but not to induce hyperstimulation. Thirty-six hours after the booster mice were injected with 1 IU human chorionic gonadotrophin (hCG; Sigma) and placed with breeder males from our stud colony; one female per male in a single cage. Half of the mice were assessed for number of ova and 2-cell embryos 40 h later and the other half were killed at day 15 of pregnancy and the numbers of viable fetuses, non-viable fetuses and resorption sites were assessed. Blood was drawn by retroorbital puncture for assessment of antisperm antibodies and uteri were snap frozen and stored at -70°C for subsequent immunohistologic examination. Each experimental group consisted of 10 mice and each experiment was repeated at least three times. Passive immunization studies Spleens and lymph nodes were harvested from s.c./s.c./i.u, immunized
285
C57BL/6 female mice one week after final immunization and from age and strain-matched non-immunized mice. The lymphoid organs were minced with fine scissors and cells were passed through a fine nylon screen. Red blood cells were lysed according to Maruyama et al. (1985) and mononuclear leukocytes were separated by gradient centrifugation on Lympholyte-M at 500 × g for 30 min and washed three times in RPMI-1640 medium (GIBCO, Grand Island, NY). T lymphocytes were obtained by nylon wool separation following a standard procedure (Trizio and Cudgowicz, 1974). Non-adherent cells (T cell enriched) were resuspended in RPMI 1640 at 10 x 107/ml and 0.1 ml was injected intravenously into the tail vein of C57BL/6 female mice. PMS (1 IU) was injected i.p. at the same time. Forty-eight hours later, mice were given 1 IU hCG i.p. and mated. On day 15 of pregnancy, mice were killed by cervical dislocation and the numbers of viable fetuses, non-viable fetuses and resorption sites were recorded. Uteri were frozen in OCT gel (Baxter McGaw, Park, IL) on dry ice and stored at -70°C for subsequent immunohistological assessment. Blood was collected by retroorbital puncture prior to termination and serum was stored at -70°C for antisperm antibody assay.
Immunohistologic staining of mononuclear cells T lymphocytes used for the passive transfer study were suspended in PBS at a concentration of 1 x 107; 5 #1 of the cell suspension was applied to individual spots of teflon-coated, eight-spot slides, air dried, fixed in acetone for 10 min and stored at -70°C until use. Uteri from actively immunized, passively immunized and non-immunized mice were embedded in groups of 5 in OCT gel on dry ice and stored at -70°C until use. Before sectioning the uteri were equilibrated in the cryostat for 1 h to reach -20°C. Tissue slices (4-6/~m) were placed on 3-spot teflon slides (Roboz Surgical Instrument Co., Washington, DC) and were air dried overnight and fixed in 2% paraformaldehyde. Macrophages and lymphocyte subpopulations were detected in cell smears and tissue sections by immunoperoxidase technique. A prewash in 0.02 N sodium azide/phosphatebuffered saline (PBS) solution was performed to destroy endogenous peroxidase before the primary rat anti-mouse monoclonal antibodies were applied (Helomy et al., 1988). All antibodies were diluted to optimal working concentration in PBS/I% BSA. For the monoclonal antibodies Thy 1 (1:20), L3T4 (1:20), B (1:20) and M1/70, 15 (1:50) (Sera Lab, Accurate Chemical Scientific Corp., Westbury, NY), the second immunoperoxidase-labelled antibody was a peroxidase-labelled F(ab')2 fragment mouse anti-rat IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA)diluted 1:100 in PBS containing 1% BSA. For the slides stained with biotinylated Lyt 2
286
antibody (1:20) (Becton Dickinson, Mountain View, CA), peroxidase labelled streptavidin (Kirkegaard & Perry Laboratories Inc., Gaithesburg, MD) was used diluted 1:200 in PBS/I% BSA. The substrate was 0.2 g of 3amino-9-ethyl carbasol (AEC) dissolved in 50 ml dimethyl formamyl and diluted in acetate buffer (pH 4.9). The slides were counterstained with hematoxylin and mounted with an aqueous mounting solution (GVA, Zymed Laboratories, South San Francisco, CA). Spleen was used as a positive tissue control and PBS instead of the first antibody as a negative control.
Evaluation of immunoperoxidase-staineduterine sections For each group of mice (5 uteri), three cross-sections from different areas of the block were evaluated. Localization of macrophages and lymphocyte subpopulations was noted descriptively. For quantitation, positive cells were counted in five random fields from each section using a light microscope ( x 500 magnification). The mean numbers and standard deviations of leukocyte subpopulations per mm 2 was calculated. For statistical evaluation, Student's t-test was used.
Detection of antisperm antibodies by indirect immunofluorescence The method used has been described in detail elsewhere (Madrigal et al., 1986). Epididymal sperm from DBA/2 retired breeders was washed and resuspended in PBS to 1 x 108 cells/ml and 5 #1 of this suspension was applied to each spot of teflon-coated, eight-spot microscope slides (diameter 8 ram, Roboz Surgical). After drying, the slides were fixed in acetone for 15 min and stored frozen at -70°C until use. On the day of assay, slides were thawed, rinsed in PBS, then sera from immunized and non-immunized mice were added in four-fold serial dilutions and incubated for 1 h. Following a wash step, fluorescein conjugated F(ab)2 fragment rabbit anti mouse IgG (Capell) diluted 1:40 was applied for 30 rain. Following a final series of washes, coverslips were mounted on the slides with a solution of pheneylene diamine/glycerol and fluorescence patterns were analyzed on a Zeiss fluorescence microscope. Results
Comparison of active immunization protocols Different routes of immunization were compared to determine the most effective way to induce immunologic infertility with sperm antigens. No significant difference was found between saline and sperm-immunized mice for any fertility parameter when either s.c. or i.u. immunization approaches were used exclusively (Table 1). Intraperitoneal immunizations were discontinued
76 (n = 17) 0.7 -~ 1.2 (n = 10) 3.1 ± 1.5 (n = 10) 9.16 ± 2.3 (n = 20)
0.3 ± 0.5 (n = 20)
77 (n = 18) 0.7 ± 1.1 (n = 10) 4.3 ± 2.1 (n = 10) 7.8 ± 4.2 (n = 20)
0.6 ± 0.8 (n = 20)
0.5 ± 0.7 (n = 18)
94 (n = 17) 1.0 + 1.3 (n = 10) 4.5 ± 2.0 (n = 10) 6.8 ± 2.2 (n = 18) 0.6 ± 0.1 (n = 20)
75 (n = 20) 1.7 + 2.0 (n = 10) 4.8 ± 2.2 (n = 10) 8.7 ± 4.2 (n = 20)
aBoth saline and sperm immunizations were performed with F r e u n d ' s adjuvant. bFertility rate = (No. of pregnant mice/Total No. of mice) %. *P < 0.01, **P < 0.005, ***P < 0.001.
Fertility rate b (%) Unfertilized oocytes 2-Cell embryos Fetuses per pregnant mouse Resorptions per pregnant mouse
Sperm
0.1 ± 0.4 (n = 30)
80 (n = 30) 0.3 + 0.8 (n = 30) 6.0 ± 3.2 (n = 30) 8.7 ± 4.6 (n = 30)
Saline
Sperm
4.2 ± 3.5* (n = 30)
78 (n = 30) 1.3 ± 1.0" (n = 30) 3.6 ± 2.7*** (n = 30) 1.81 ± 2.6** (n = 30)
2.6 ± 2.0*** (n = 28)
6.2 ± 4.0 (n = 27)
4.4 ± 2.2** (n = 28)
NA
NA
0.4 ± 0.1 (n = 27)
55 (n = 28) NA
Sperm
86 (n = 27) NA
Saline
Saline
Saline
Sperm
T-cell transfer from s.c./s.c./i.u, group
i.u./i.u./i.u,
s.c./s.c./s.c,
s.c./s.c./i.u.
Passive immunization
Active immunization a
Effects of different sperm immunization protocols on fertility parameters.
TABLE 1
288
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E
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E
m
D
2 ¸
6 Z
0-
m o
E
G
c
Q. C 0
Z
Fig. 1. Results of a single representative s.c./s.c./i.u, active immunization experiment with various control groups indicating reduced fertility and increased fetal resorptions in sperm-immunized mice. Immunization groups: wA, non-immunized; I~1, saline + adjuvant; II, DBA/2 lymphocytes + adjuvant; [], human RBC + adjuvant; m, DBA/2 sperm + adjuvant.
after the first experiment because Freund's adjuvant alone produced inflammation and adhesions that affected fertility. A combination of systemic (s.c.) and local (i.u.) immunizations with sperm in adjuvant did not cause a significant decrease in the percentage of mice that become pregnant as compared with the adjuvant control group (Table 1). However, a number of individual fertility parameters were reduced in s.c./s.c./i.u, sperm-immunized animals as
289
compared to adjuvant controls: on day 3 after mating the number of unfertilized oocytes per pregnant mouse was significantly increased (P < 0.01) and the number of 2-cell embryos per mouse was significantly decreased (P < 0.001); on day 15 of pregnancy the number of viable fetuses per pregnant mouse was significantly reduced (P < 0.005) and the number of fetal resorption sites was significantly increased (P < 0.01) (Table 1, Fig. 1).
12
o
Q
lO
E E
~
8
~
6
-~
4
~
2
Z
O to
O
E 6 E C
II
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~,4 ct. O to
"6
2
Z
Fig. 2. Effects of passive transfer of T lymphocytes from s.c./s.c./i.u, immunized mice (from experiment depicted in Fig. 1), on fertility of recipient mice. T lymphocytes from: gl, non-immunized; Irl, saline+adjuvant; I , DBA/2 lymphocytes+adjuvant; [], human RBC+adjuvant; II, DBA/2 sperm + adjuvant.
290 2000
@ E E u 0 .E Q.
1000
E
g Z
2000
®
E E 0 u 0 tO.
E
1000
F+ a
u 0 Z
Fig. 3. Mean numbers ± S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after s.c./s.c./i.u, immunizations (from experiment depicted in Fig. 1). Immunization groups: M, nonimmunized; B, saline in adjuvant; IXl,human RBCs in adjuvant; I~1, DBA/2 lymphocytes in adjuvant; II, DBA/2 sperm in adjuvant.
Passive immunization study In the passive immunization study, mice were administered T cell-enriched lymphoid cells from sperm-immunized, non-immunized and controlimmunized syngeneic mice via the tail vein. The transferred cells were primarily CD4+ lymphocytes; less than 15% of the cells were macrophages or CD8+ T cells. Numbers of viable fetuses and resorption sites were scored on
291 3000
© E E
2000
u
E ÷
1ooo 6
Z
0
3000
E E
@
2000
2 E "6
1000
Z
0
Fig. 3 (continued).
day 15 of pregnancy. There was no significant difference in any of the fertility parameters between mice receiving T cells from non-immunized mice and control immunized mice (Fig. 2). However, the fertility rate was reduced in mice receiving lymphocytes from sperm-immunized animals (Table 1). Furthermore, the number of fetuses per pregnant mouse was significantly decreased in the group that received T cells from sperm-immunized mice (P < 0.001) and the number of resorption sites was significantly increased (P < 0.005) (Table 1).
292 3000
E E -.~
Q
2000
0 0 0 e., ID.
E I-
"6
1000
Z
3000
® E E -.~ 2000 t~ 0
E I'÷
Q
1000
Z
Fig. 4. Mean numbers ± S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after passive transfer of T lymphocytes from s.c./s.c./i.u, immunized mice (same experiment as depicted in Figs. 1-3). Immunization groups: i , non-immunized; El, saline in adjuvant; [], human RBCs in adjuvant; El, DBA/2 lymphocytes in adjuvant; II, DBA/2 sperm in adjuvant.
Leukocyte subpopulations in uteri from actively and passively immunized mice Uteri from pregnant and non-pregnant cycled animals (day 15 after hCG and mating) were studied. Sections of uteri from pregnant animals in both sperm-immunized and control groups contained numerous lymphocytes and
293 2000
© % I= o 0 t,,t,,n
t=
1000
I"-" ÷
¢,,,, ¢J
"6 5 Z
0 6000
@ N
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3000 I:: 0
5 Z
Fig. 4 (continued).
macrophages and the patterns were indistinguishable from each other. In non-pregnant s.c./s.c./i.u, sperm-immunized mice, the number of T lymphocytes/mm 2 of uterine section was significantly higher than that observed in any control group (P < 0.001). Stage of estrus was not correlated with numbers of T cells in uterine horns of control mice or of sperm-immunized mice. Both CD4+ and CD8+ subpopulations were observed in uterine tissue from sperm-immunized mice, but CD8+ cells predominated. Most of the CD8+ cells were located in the periglandular space, while CD4+ cells were evenly spread throughout the epithelium and periglandular region. In uteri from sperm-immunized mice, macrophages were increased four-fold (P
281
Elsevier ScientificPublishers Ireland Ltd. JRI 00787
Antifertility effects of antisperm cell-mediated immunity in mice Florina Haimovici, Kazue Takahashi
and Deborah
J. A n d e r s o n
Fearing Research Laboratory, Harvard Medical School, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital. 250 Longwood A venue, Boston, MA 02115 (USA)
(Accepted for publication 18 May 1992)
Summary C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u, protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u, sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a signifiCorrespondence to: Deborah J. Anderson, Ph.D., Fearing Research Laboratory, Brigham and Women's Hospital, 250 Longwood Avenue, Boston, MA 02115, USA.
0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
282
cant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.
Key words: pregnancy; infertility; abortion; sperm; cell-mediated immunity
Introduction
Immunization of female mice with sperm, sperm antigens or testis homogenates has produced decreased fertility in a number of studies (McLaren, 1964, 1966; Edwards, 1964; Bell, 1969; Tung et al., 1979; Wheat and Goldberg, 1979; Kasai et al., 1987). The degree of immunological response and anti-fertility effect can be influenced by the frequency of the immunizations (Snell and Poucher, 1991), the strain of mice (Madrigal et al., 1986; Kasai et al., 1987) and the route of immunization (Bell, 1969; Allardyce, 1984). Associations between antisperm antibody titer and reduced fertility have been varied. McLaren (1966) reported a significant association between sperm agglutination titer and reduced litter size in mice. However, Edwards (1964) and Bell (1969) using different immunization procedures, found no relationship between serum titer and fertilization rate. Testicular germ cells and spermatozoa can stimulate cell-mediated immunity in addition to humoral immune responses (EI-Alfi and Bassili, 1970; Mancini and Andrada, 1971; Nagarkatti and Rav, 1976; Anderson et al., 1982; McShane et al., 1985; Shelton and Goldberg, 1985; Naz and Mehta, 1989). Furthermore, various cytokines produced by activated lymphocytes and macrophages during cellular immune responses have been shown to affect reproductive functions adversely, including sperm motility (Hill et al., 1987; Eisermann et al., 1989), sperm-egg interaction (Maruyama et al., 1985; Hill et al., 1989), embryo viability (Hill et al., 1987; Tartakovsky and Ben-Yair, 1991), mouse trophoblast outgrowth (Haimovici et al., 1991) and human trophoblast (choriocarcinoma) cell proliferation in vitro (Berkowitz et al., 1988). It is possible, therefore, that intrauterine antisperm cell-mediated immune responses significantly contribute to the antifertility effects observed following immunization to sperm. Subsets of lymphocytes home to mucosa-associated lymphoid tissue through interactions between a distinct class of adhesion molecules expressed on the lymphocyte surface (homing receptors) and tissue-specific vascular
283
adhesion molecules expressed on the surface of high endothelial venules (Berg et al., 1989). Recent studies indicate that circulating memory/activated T lymphocytes and in situ mucosal lymphocytes which are negative for the peripheral-lymph-node (PLN) -homing receptor and positive for mucosallymphocyte-associated (MLA) -receptor show preferential binding to mucosal high endothelial venules and preferentially home to mucosal sites. Studies performed by McDermott et al. (1979, 1989) have provided evidence that the female reproductive tract is a part of the general mucosal immune system and that mucosal-derived B and T lymphocytes can specifically migrate to genital tissues. However, little is yet known about the mechanisms governing mucosal immune responses in the female reproductive tract and their role in infertility. In this study we explored various spermimmunization protocols to induce intrauterine immunological responses and infertility in mice and used a T cell passive transfer approach to study homing of mucosal primed T cells to the uterus and to determine the effects of sperm-sensitized T cells on fertility and pregnancy. Materials and Methods
Preparation of cells to be used as antigens Sperm. DBA/2 retired breeder males (Jackson Laboratories, Bar Harbor, ME) were killed by cervical dislocation and the cauda epididymides were removed and minced with fine scissors. Sperm were suspended in KrebsRinger solution, passed through a fine screen and collected from the cell pellet following centrifugation at 400 x g for 5 min. Motile sperm were further isolated from immotile sperm and somatic cells by centrifugation through a discontinuous gradient of Percoll (Pharrnacia, Piscataway, N J) (47%/90%) for 30 min at 500 x g. The cell pellet containing motile sperm was washed 3 times and resuspended at a final concentration of 1 x 107/ml in saline. The cell suspension was mixed 1:1 with CFA or IFA to form a homogenous emulsion, aliquoted and stored at -70°C until use. Spleen mononuclear leukocytes. Splenic mononuclear leukocytes from DBA/2 retired breeders were isolated by centrifugation through LympholyteM (Accurate Chemical & Scientific Corp., Westbury, NY) for 30 min at 500 x g. Leukocytes removed from the interface above the Lymphocyte-M were washed 3 times, resuspended at a concentration of 1 x 10 7 in saline, mixed 1:1 with CFA or IFA to form a homogenous emulsion and aliquoted and stored at -70°C until use. Red blood cells. Human red blood cells were isolated from heparinized peripheral blood by centrifugation through Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc., Piscataway, N J) for 30 min at 500 x g. The buffy coat on the red blood cell pellet containing granulocytes was discarded. The
284
bottom layer containing < 99% red blood cells (confirmed by microscopic evaluation) was collected, washed, resuspended in saline at a concentration of 1 x 107/ml, mixed 1:1 with adjuvant, aliquoted and stored at -70°C. Active immunization protocols C57BL/6 female mice (Jackson Laboratories, Bar Harbor, ME) maintained on a 12-h light/dark cycle were immunized with 1 x 10 7 allogeneic (DBA/2) sperm, in Complete Freund's Adjuvant (CFA; Cappel, Organon Teknika Corp., West Chester, PA). Control female mice of the same age and strain were injected with: (1) saline (0.9% NaCI in distilled H20) as a control for stress, (2) saline-plus-CFA as a control for the effects of the CFA alone, and (3) human red blood cells and (4) paternal (DBA/2) mouse lymphocytes in CFA to assess the sperm-specificity of the antifertility effect. Booster injections of 1 x 10 7 sperm or control cells, or saline alone in Incomplete Freund's Adjuvant (IFA; Cappel, Organon Teknika Corp.) were given on day 14 and 21. Injections (0.1 ml) were administered: (1) subcutaneously into the neck (s.c.), (2) intraperitoneally (i.p.), or (3) intrauterine (i.u.) through the cervix which was dilated by injection 12 h earlier with 1 IU of Pregnant Mare's Serum (PMS; Sigma Chemical Co., St. Louis, MO). For intrauterine injections, mice were anesthetized with 0.2 ml of Advertine solution (2.5 ml tert-amyl alcohol (Fisher Labs, Medford, MA), 1.25 g 2,2,2,tribromoethanol 99% (Aldrich Chemical Company, Milwaukee, WI) in 100 ml distilled water) and the cervix was visualized through a glass speculum. A catheter fitted with a 1-ml syringe and a 27 1/2-gauge needle (Becton Dickinson, Rutherford, N J) was threaded through the cervical opening and 0.1 ml of cells or saline emulsified with adjuvant was introduced into the uterine lumen. Twelve hours before the final booster injection, mice in all immunization groups were primed with 1 IU of PMS to cycle the animals but not to induce hyperstimulation. Thirty-six hours after the booster mice were injected with 1 IU human chorionic gonadotrophin (hCG; Sigma) and placed with breeder males from our stud colony; one female per male in a single cage. Half of the mice were assessed for number of ova and 2-cell embryos 40 h later and the other half were killed at day 15 of pregnancy and the numbers of viable fetuses, non-viable fetuses and resorption sites were assessed. Blood was drawn by retroorbital puncture for assessment of antisperm antibodies and uteri were snap frozen and stored at -70°C for subsequent immunohistologic examination. Each experimental group consisted of 10 mice and each experiment was repeated at least three times. Passive immunization studies Spleens and lymph nodes were harvested from s.c./s.c./i.u, immunized
285
C57BL/6 female mice one week after final immunization and from age and strain-matched non-immunized mice. The lymphoid organs were minced with fine scissors and cells were passed through a fine nylon screen. Red blood cells were lysed according to Maruyama et al. (1985) and mononuclear leukocytes were separated by gradient centrifugation on Lympholyte-M at 500 × g for 30 min and washed three times in RPMI-1640 medium (GIBCO, Grand Island, NY). T lymphocytes were obtained by nylon wool separation following a standard procedure (Trizio and Cudgowicz, 1974). Non-adherent cells (T cell enriched) were resuspended in RPMI 1640 at 10 x 107/ml and 0.1 ml was injected intravenously into the tail vein of C57BL/6 female mice. PMS (1 IU) was injected i.p. at the same time. Forty-eight hours later, mice were given 1 IU hCG i.p. and mated. On day 15 of pregnancy, mice were killed by cervical dislocation and the numbers of viable fetuses, non-viable fetuses and resorption sites were recorded. Uteri were frozen in OCT gel (Baxter McGaw, Park, IL) on dry ice and stored at -70°C for subsequent immunohistological assessment. Blood was collected by retroorbital puncture prior to termination and serum was stored at -70°C for antisperm antibody assay.
Immunohistologic staining of mononuclear cells T lymphocytes used for the passive transfer study were suspended in PBS at a concentration of 1 x 107; 5 #1 of the cell suspension was applied to individual spots of teflon-coated, eight-spot slides, air dried, fixed in acetone for 10 min and stored at -70°C until use. Uteri from actively immunized, passively immunized and non-immunized mice were embedded in groups of 5 in OCT gel on dry ice and stored at -70°C until use. Before sectioning the uteri were equilibrated in the cryostat for 1 h to reach -20°C. Tissue slices (4-6/~m) were placed on 3-spot teflon slides (Roboz Surgical Instrument Co., Washington, DC) and were air dried overnight and fixed in 2% paraformaldehyde. Macrophages and lymphocyte subpopulations were detected in cell smears and tissue sections by immunoperoxidase technique. A prewash in 0.02 N sodium azide/phosphatebuffered saline (PBS) solution was performed to destroy endogenous peroxidase before the primary rat anti-mouse monoclonal antibodies were applied (Helomy et al., 1988). All antibodies were diluted to optimal working concentration in PBS/I% BSA. For the monoclonal antibodies Thy 1 (1:20), L3T4 (1:20), B (1:20) and M1/70, 15 (1:50) (Sera Lab, Accurate Chemical Scientific Corp., Westbury, NY), the second immunoperoxidase-labelled antibody was a peroxidase-labelled F(ab')2 fragment mouse anti-rat IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA)diluted 1:100 in PBS containing 1% BSA. For the slides stained with biotinylated Lyt 2
286
antibody (1:20) (Becton Dickinson, Mountain View, CA), peroxidase labelled streptavidin (Kirkegaard & Perry Laboratories Inc., Gaithesburg, MD) was used diluted 1:200 in PBS/I% BSA. The substrate was 0.2 g of 3amino-9-ethyl carbasol (AEC) dissolved in 50 ml dimethyl formamyl and diluted in acetate buffer (pH 4.9). The slides were counterstained with hematoxylin and mounted with an aqueous mounting solution (GVA, Zymed Laboratories, South San Francisco, CA). Spleen was used as a positive tissue control and PBS instead of the first antibody as a negative control.
Evaluation of immunoperoxidase-staineduterine sections For each group of mice (5 uteri), three cross-sections from different areas of the block were evaluated. Localization of macrophages and lymphocyte subpopulations was noted descriptively. For quantitation, positive cells were counted in five random fields from each section using a light microscope ( x 500 magnification). The mean numbers and standard deviations of leukocyte subpopulations per mm 2 was calculated. For statistical evaluation, Student's t-test was used.
Detection of antisperm antibodies by indirect immunofluorescence The method used has been described in detail elsewhere (Madrigal et al., 1986). Epididymal sperm from DBA/2 retired breeders was washed and resuspended in PBS to 1 x 108 cells/ml and 5 #1 of this suspension was applied to each spot of teflon-coated, eight-spot microscope slides (diameter 8 ram, Roboz Surgical). After drying, the slides were fixed in acetone for 15 min and stored frozen at -70°C until use. On the day of assay, slides were thawed, rinsed in PBS, then sera from immunized and non-immunized mice were added in four-fold serial dilutions and incubated for 1 h. Following a wash step, fluorescein conjugated F(ab)2 fragment rabbit anti mouse IgG (Capell) diluted 1:40 was applied for 30 rain. Following a final series of washes, coverslips were mounted on the slides with a solution of pheneylene diamine/glycerol and fluorescence patterns were analyzed on a Zeiss fluorescence microscope. Results
Comparison of active immunization protocols Different routes of immunization were compared to determine the most effective way to induce immunologic infertility with sperm antigens. No significant difference was found between saline and sperm-immunized mice for any fertility parameter when either s.c. or i.u. immunization approaches were used exclusively (Table 1). Intraperitoneal immunizations were discontinued
76 (n = 17) 0.7 -~ 1.2 (n = 10) 3.1 ± 1.5 (n = 10) 9.16 ± 2.3 (n = 20)
0.3 ± 0.5 (n = 20)
77 (n = 18) 0.7 ± 1.1 (n = 10) 4.3 ± 2.1 (n = 10) 7.8 ± 4.2 (n = 20)
0.6 ± 0.8 (n = 20)
0.5 ± 0.7 (n = 18)
94 (n = 17) 1.0 + 1.3 (n = 10) 4.5 ± 2.0 (n = 10) 6.8 ± 2.2 (n = 18) 0.6 ± 0.1 (n = 20)
75 (n = 20) 1.7 + 2.0 (n = 10) 4.8 ± 2.2 (n = 10) 8.7 ± 4.2 (n = 20)
aBoth saline and sperm immunizations were performed with F r e u n d ' s adjuvant. bFertility rate = (No. of pregnant mice/Total No. of mice) %. *P < 0.01, **P < 0.005, ***P < 0.001.
Fertility rate b (%) Unfertilized oocytes 2-Cell embryos Fetuses per pregnant mouse Resorptions per pregnant mouse
Sperm
0.1 ± 0.4 (n = 30)
80 (n = 30) 0.3 + 0.8 (n = 30) 6.0 ± 3.2 (n = 30) 8.7 ± 4.6 (n = 30)
Saline
Sperm
4.2 ± 3.5* (n = 30)
78 (n = 30) 1.3 ± 1.0" (n = 30) 3.6 ± 2.7*** (n = 30) 1.81 ± 2.6** (n = 30)
2.6 ± 2.0*** (n = 28)
6.2 ± 4.0 (n = 27)
4.4 ± 2.2** (n = 28)
NA
NA
0.4 ± 0.1 (n = 27)
55 (n = 28) NA
Sperm
86 (n = 27) NA
Saline
Saline
Saline
Sperm
T-cell transfer from s.c./s.c./i.u, group
i.u./i.u./i.u,
s.c./s.c./s.c,
s.c./s.c./i.u.
Passive immunization
Active immunization a
Effects of different sperm immunization protocols on fertility parameters.
TABLE 1
288
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Fig. 1. Results of a single representative s.c./s.c./i.u, active immunization experiment with various control groups indicating reduced fertility and increased fetal resorptions in sperm-immunized mice. Immunization groups: wA, non-immunized; I~1, saline + adjuvant; II, DBA/2 lymphocytes + adjuvant; [], human RBC + adjuvant; m, DBA/2 sperm + adjuvant.
after the first experiment because Freund's adjuvant alone produced inflammation and adhesions that affected fertility. A combination of systemic (s.c.) and local (i.u.) immunizations with sperm in adjuvant did not cause a significant decrease in the percentage of mice that become pregnant as compared with the adjuvant control group (Table 1). However, a number of individual fertility parameters were reduced in s.c./s.c./i.u, sperm-immunized animals as
289
compared to adjuvant controls: on day 3 after mating the number of unfertilized oocytes per pregnant mouse was significantly increased (P < 0.01) and the number of 2-cell embryos per mouse was significantly decreased (P < 0.001); on day 15 of pregnancy the number of viable fetuses per pregnant mouse was significantly reduced (P < 0.005) and the number of fetal resorption sites was significantly increased (P < 0.01) (Table 1, Fig. 1).
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Fig. 3. Mean numbers ± S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after s.c./s.c./i.u, immunizations (from experiment depicted in Fig. 1). Immunization groups: M, nonimmunized; B, saline in adjuvant; IXl,human RBCs in adjuvant; I~1, DBA/2 lymphocytes in adjuvant; II, DBA/2 sperm in adjuvant.
Passive immunization study In the passive immunization study, mice were administered T cell-enriched lymphoid cells from sperm-immunized, non-immunized and controlimmunized syngeneic mice via the tail vein. The transferred cells were primarily CD4+ lymphocytes; less than 15% of the cells were macrophages or CD8+ T cells. Numbers of viable fetuses and resorption sites were scored on
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day 15 of pregnancy. There was no significant difference in any of the fertility parameters between mice receiving T cells from non-immunized mice and control immunized mice (Fig. 2). However, the fertility rate was reduced in mice receiving lymphocytes from sperm-immunized animals (Table 1). Furthermore, the number of fetuses per pregnant mouse was significantly decreased in the group that received T cells from sperm-immunized mice (P < 0.001) and the number of resorption sites was significantly increased (P < 0.005) (Table 1).
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Fig. 4. Mean numbers ± S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after passive transfer of T lymphocytes from s.c./s.c./i.u, immunized mice (same experiment as depicted in Figs. 1-3). Immunization groups: i , non-immunized; El, saline in adjuvant; [], human RBCs in adjuvant; El, DBA/2 lymphocytes in adjuvant; II, DBA/2 sperm in adjuvant.
Leukocyte subpopulations in uteri from actively and passively immunized mice Uteri from pregnant and non-pregnant cycled animals (day 15 after hCG and mating) were studied. Sections of uteri from pregnant animals in both sperm-immunized and control groups contained numerous lymphocytes and
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macrophages and the patterns were indistinguishable from each other. In non-pregnant s.c./s.c./i.u, sperm-immunized mice, the number of T lymphocytes/mm 2 of uterine section was significantly higher than that observed in any control group (P < 0.001). Stage of estrus was not correlated with numbers of T cells in uterine horns of control mice or of sperm-immunized mice. Both CD4+ and CD8+ subpopulations were observed in uterine tissue from sperm-immunized mice, but CD8+ cells predominated. Most of the CD8+ cells were located in the periglandular space, while CD4+ cells were evenly spread throughout the epithelium and periglandular region. In uteri from sperm-immunized mice, macrophages were increased four-fold (P
