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Biology of Reproduction 60, 1285-1291 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Effects of Experimentally Generated Bull Antisperm Antibodies on In Vitro Fertilization1

C.A. Kima, J.J. Parrishb, H.W. Momonta, and D.P. Lunn2,a

a Department of Medical Sciences, School of Veterinary Medicine, and b Department of Animal Sciences, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only.

Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subfertility or infertility in bulls can have profound negative consequences on the cattle industry. This is particularly important because of the role of artificial insemination for the maintenance and improvement of productivity [1]. Possible causes for subfertility or infertility in bulls as in other species are poorly defined, but one important category may be immunological disease [2, 3]. Autoimmunity to sperm can occur because sperm cell antigens are first expressed during sexual maturation, long after the perinatal period when immunologic self-tolerance is induced [3]. Normally, spermatids and spermatozoa are isolated from the immune system within the seminiferous tubules. The seminiferous tubules are lined by Sertoli cells, which form tight junctions on the perimeter, separating germ cells from circulating leukocytes. Because of the presence of this blood-testis barrier, these cells are immunoprivileged. When this barrier is disrupted by disease, animals can be auto-immunized with previously sequestered sperm and testicular antigens. Subsequently, infertility can result from antibodies directly binding sperm, or from aspermatogenesis due to allergic orchitis [2]. A similar phenomenon occurs in vasectomized laboratory rodents and humans. A high percentage of these individuals develop epididymal sperm granulomas and testicular degeneration associated with the formation of antisperm antibodies [4, 5].

The significance of antisperm antibodies is controversial. Naturally occurring antisperm antibodies have been recognized in many species including horses, dogs, humans, and cows [6–9]. In humans, many different sperm antigens have been recognized by antibodies in the serum and seminal plasma of both infertile and fertile men, indicating that the functional effects of these antisperm antibodies are not well defined [10, 11]. A variety of tests have been performed with human antisperm antibodies, including hemizona assays, in vitro fertilization, and sperm motility assays to define their functional significance [12–14]. These tests have demonstrated that antisperm antibodies cause changes in sperm motility, decrease adherence of sperm to the zona pellucida, and decrease fertilization in vitro, establishing the potential of antisperm antibodies to cause infertility in vivo.

In cattle, experimentally induced antisperm antibodies were shown to cause infertility in heifers [15], although naturally occurring antisperm antibodies in cows did not cause infertility [9]. In bulls, antisperm antibodies have been experimentally generated by immunization with sperm or testicular homogenates in Freund's complete adjuvant [16, 17], but studies of the characteristics of these antibodies or their effects on fertilization have been extremely limited [18]. The aims of the present study were 1) to generate antisperm antibodies in bulls by auto-immunization without the use of adjuvants, 2) to determine whether similar antisperm antibodies occurred naturally in a bull stud population, 3) to determine whether these antibodies could reduce penetration rates in in vitro fertilization experiments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals

Three 13-mo-old Holstein bulls were used for antisperm antibody generation. The bulls were housed and fed in accordance with animal care guidelines of the University of Wisconsin Research and Animal Resources Committee, and the investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals, issued by the Society for the Study of Reproduction. Three age- and breed-matched controls were held at a commercial semen collection facility (ABS Global, Deforest, WI) under similar management conditions. Semen samples were collected from bulls using an artificial vagina. Semen evaluations and breeding soundness examinations were performed before, during, and after the immunization regimen to characterize changes in the quality of the ejaculates and reproductive health of the bulls. Approximately 7 mo after the last immunization, the bulls were castrated, and the testes were examined for gross and histological abnormalities. Tissue blocks were formalin-fixed and paraffin-embedded; sections were stained with hematoxylin and eosin.

Antisperm Antibody Generation

To generate antisperm antibodies, semen was collected from each bull, washed, and resuspended in PBS. Sperm were washed by diluting semen in PBS, centrifuging at 1000 x g for 15 min, and resuspending the pellet in PBS. This was repeated two or more times until the supernatant became clear. Sperm cells were counted with a hemocytometer, and immunizations were performed by resuspending 1 x 109 sperm in 5 ml of PBS. This dose was split into two 2.5-ml injections given s.c. behind the shoulder and i.m. into the muscles of the caudal thigh. Each test bull was immunized with his own sperm three times at approximately 3-wk intervals. Blood and semen were collected from test bulls for the preparation of serum and seminal plasma samples before, during, and after the immunization protocol and stored at -20°C. Similar samples were taken from control bulls at the same time as from test bulls.

ELISA

Seminal plasma was prepared by diluting semen samples 1:5 in PBS and centrifuging at 1500 x g for 30 min and collecting the supernatant. Serum and seminal plasma were tested for antisperm antibodies by an ELISA. Semen samples from 2 bulls from a local bull stud (ABS Global) were combined in proportions so as to contain equal numbers of sperm from each bull and washed as described above, and sperm were resuspended at a concentration of 1 x 107 to 5 x 107 sperm/ml in PBS. Polystyrene 96-well plates (Becton Dickinson, Lincoln Park, NJ) were coated with poly-L-lysine (50 µg/ml) for 40 min. The wells were washed twice with PBS, and the sperm suspension was applied to the coated wells. The ELISA plates were centrifuged at 450 x g for 5 min and then incubated at room temperature for 45 min. The plates were washed 3 times with PBS and examined for consistency of sperm adherence using an inverted microscope and phase contrast illumination.

Sperm were fixed in ELISA plates by incubation with 0.1% glutaraldehyde in PBS for 3 min and washed 3 times with PBS. Plates were incubated overnight at 4°C in blocking solution (PBS plus 1% Teleostean gelatin [Sigma, St. Louis, MO]) and washed twice with ELISA wash (PBS plus 0.05% v:v Tween 20 [Fisher, Fair Lawn, NJ]). The plates were used immediately or refilled with ELISA wash and stored at 4°C. A single separate batch of plates, prepared from a single pool of sperm, was used for each of the ELISA procedures, i.e., the analysis of isotype responses in the immunized bulls, and the study of a group of bulls and heifers described below.

For the isotype-specific ELISA, bull serum and seminal plasma samples were collected over a 140-day period starting before the first immunization and ending approximately 3 mo after the last immunization. These samples were diluted in blocking solution (serum at 1/500 and seminal plasma at 1/50) and incubated in ELISA plates. Plates were washed before incubation with murine monoclonal antibody (mAb) supernatants specific for the individual bovine immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA from the respective hybridoma cell lines M23, M3-7, M33, M67, which were generously provided by Dr. Klaus Nielson, Agriculture Canada [19]). After a further wash, peroxidase-conjugated, goat anti-mouse IgG, and IgM (heavy and light chain [H+L]; Affinipure; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied to the plates, and color was developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The reaction was stopped by addition of H3PO4. The absorbance at 450 nm of each well was determined spectrophotometrically with an ELISA reader (EL 311SX Microplate Autoreader; Bio-Tek Instruments, Winooski, VT). Heifer serum was used as a negative control for serum assays, while blocking solution was used as a negative control for seminal plasma assays. All samples were assayed in triplicate, all incubations were at 37°C for 90 min, and all washes were performed three times with ELISA wash.

A screening ELISA was developed to rapidly test for antisperm antibodies in a large group of serum and seminal plasma samples. These ELISAs were performed by incubating plates with diluted test serum (1/2000) or seminal plasma (1/50) followed by peroxidase-conjugated, goat anti-bovine IgG (H+L) polyclonal immunoglobulin (Affinipure; Jackson ImmunoResearch). TMB substrate was used for color development. Serial dilutions of a post-immunization bull serum sample were used to prepare a standard curve to which the samples were compared. Heifer serum was the negative control for serum assays, while blocking solution was the negative control for seminal plasma assays.

Antigen Characterization

Bovine IgG1 and IgG2 were isolated from pre- and post-immunization serum from the test bull with the highest ELISA serum antisperm antibody level (bull #377) by immunoaffinity chromatography and used in Western blots to characterize the sperm antigens recognized.

Isolation of IgG1 and IgG2 Ascites containing mAbs specific for bovine IgG1 and IgG2 were produced by conventional methods in BALB/c mice from the hybridoma cell lines M23 and M3–7, respectively. Antibovine IgG1 and IgG2 mAbs were purified from ascites by ammonium sulfate precipitation and dialyzed against PBS. Protein concentrations were estimated using a Bradford dye-binding procedure (Bio-Rad, Hercules, CA).

Two immunoaffinity columns were prepared by coupling the anti-IgG1 and anti-IgG2 mAbs to N-hydroxysuccinimide-activated agarose beads (Affi-gel 10; Bio-Rad) in a sodium phosphate conjugation buffer (10 mM NaH2PO4 and 150 mM NaCl; pH 7.5). For each column, 8 mg of antibody was bound to each milliliter of beads with a binding efficiency of approximately 85%. Five milliliters of beads were used in each column. Ethanolamine (1 M, pH 8.0) was added to block remaining active sites on the beads. The beads were placed in a 1-cm internal diameter glass column (Bio-Rad) and washed with PBS followed by elution buffer (1 M NaCl plus 100 mM glycine HCl, pH 2.5) to remove unbound protein. The columns were stored in PBS with 0.2% azide at 4°C.

Pre- and post-immunization serum samples from one of the test bulls were heat-inactivated at 56°C for 30–60 min and centrifuged at 2000 x g for 10 min. The supernatant was diluted 1:1 with PBS and filtered through a 0.22-µm filter before loading on the affinity columns. The columns were subsequently washed extensively with PBS, and affinity-purified bovine IgG1 or IgG2 was eluted by reverse flow application of elution buffer. Eluted protein was detected by UV absorbency at 280 nm and collected into 1/20 volume of 1 M sodium phosphate (pH 8) to prevent denaturation of the protein in acidic conditions. Purified immunoglobulins were subsequently dialyzed against PBS for 48 h at 4°C. The purity of the samples were assessed by SDS-PAGE analysis. In each instance, two bands were identified consistent with the heavy and light chains of the relevant IgG molecule.

Immunoblotting Semen from two normal bulls was combined, washed in PBS, and resuspended in lysing solution (pH 8; 2% NP40, 150 mM NaCl, 1 mM MgCl2, 20 mM Tris HCl, 1 mM PMSF) at a concentration of 1 x 108 sperm/ml. After a 30-min incubation at 4°C, the lysed cells were centrifuged at 1500 x g for 15 min, and the supernatant (sperm lysate) was collected.

The sperm lysate was subjected to SDS-PAGE in 12% or 13.2% gels under reducing conditions (Mini-PROTEAN II system; Bio-Rad). The sperm proteins were transferred to a nitrocellulose sheet using a semi-dry electrophoretic transfer apparatus (Trans-Blot SD; Bio-Rad). The nitrocellulose sheet was washed 3 times with wash solution (PBS plus 0.05% v:v Tween 20) and blocked with 3% type A pig skin gelatin (Sigma) in Tris-buffered saline (TBS) overnight at room temperature. All antibodies were diluted in 1% pig skin gelatin in TTB (0.05% Tween 20 in TBS), and incubations were performed for 2 h on a rocker at room temperature. The nitrocellulose sheet was washed three times and placed in a miniblotter (Pharmacia, Piscataway, NJ). Separate lanes were incubated with pre- and post-immunization IgG1 and IgG2 at a concentration of approximately 0.07 µg protein/ml of PBS. The lanes were washed, and peroxidase-conjugated, goat anti-bovine IgG, F(ab')2 fragment specific immunoglobulin (Affinipure; Jackson ImmunoResearch) was incubated in each lane. The nitrocellulose sheet was removed from the blotter and washed. An enhanced chemiluminescence (ECL, Amersham International plc, Buckinghamshire, England) detection system was used for development of the blot. Estimates of molecular weights were made by comparison to high- and low-range molecular weight standards (Bio-Rad).

In Vitro Fertilization Experiment

An in vitro fertilization experiment was conducted to test the effects of antisperm antibodies on penetration rates of in vitro-matured oocytes by sperm. Each of the three test bulls was randomly paired with a control bull. A series of experiments (trials) were conducted in which serum samples from one of these bull pairs were incubated with a sperm sample from one of two randomly selected bulls from a local bull stud (ABS Global). Each of the three test /control bull trials was repeated once (with each sperm sample) for an overall total of six trials. In each of these trials, there were six treatments. The first treatment was sperm incubated with no serum. The second treatment was sperm incubated with serum from an unbred heifer. The third and fourth treatments were sperm incubated with serum of an age- and breed-matched control bull collected at approximately the same times as the test bull sera. The fifth and sixth treatments were sperm incubated with pre- and post-immunization serum, respectively, from one test bull. All sera were heat-inactivated at 56°C for 30 min to inactivate complement.

Frozen semen was thawed in a 35°C water bath for 1 min. Live sperm cells were separated from dead cells and extender by centrifugation through a percol gradient [20]. Sperm were resuspended in bovine gamete medium 3 (BGM-3), which contains 2 mM CaCl2, 3.1 mM KCl, 0.3 mM NaH2PO4, 87 mM NaCl, 0.4 mM MgCl·6H2O, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 10 mM NaHCO3, 40 mM HEPES, and 6 mg/ml fraction V BSA [21]. Aliquots of live sperm (25 x 106 sperm/ml) were diluted 1:1 with BGM-3 or neat serum depending on the treatment and were incubated at room temperature for 30 min.

In vitro fertilization experiments were conducted according to Parrish et al. [22] with some alterations. Briefly, cumulus-oocyte complexes were aspirated from ovaries retrieved from slaughterhouse cows. The cumulus-oocyte complexes were washed with low bicarbonate-TALP (2 mM CaCl2, 3.2 mM KCl, 0.4 mM NaH2PO4, 114 mM NaCl, 0.5 mM MgCl·6H2O, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 2 mM NaHCO3, 10 mM HEPES, and 3 mg/ml fraction V BSA), and 10 cumulus-oocyte complexes were incubated in 50-µl drops of maturation medium (TC-199 Earle's salts plus 0.25 mM pyruvate, 10% fetal calf serum, 0.012 U/ml NIH-oLH, 0.01 U/ml NIH-oFSH, and 1 µg/ml estradiol-17ß) under oil in a Petri dish at 39°C in a humidified atmosphere containing 5% CO2 for 24 h. The cumulus-oocyte complexes were removed from the drops, and cumulus cells were removed from the oocytes by vortexing the complexes in a 1.5-ml centrifuge tube for 3 min. After the oocytes were washed three times in low-bicarbonate-TALP, six oocytes were placed into each of the five 48-µl drops of fertilization medium (2 mM CaCl2, 3.2 mM KCl, 0.4 mM NaH2PO4, 114 mM NaCl, 0.5 mM MgCl·6H2O, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 25 mM NaHCO3, 6 mg/ml fatty acid free BSA, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine, and 2 µg/ml of heparin) for a total of 30 oocytes per treatment. Sperm were added to each drop to achieve a final concentration of 1 x 106 sperm/ml.

After a 16-h incubation under oil at 39°C in a humidified atmosphere containing 5% CO2, loosely bound sperm were removed by passing the oocytes in and out of a narrow-bore glass pipette. The oocytes were mounted, fixed in a 1:3 acetic acid-ethanol solution for approximately 3 h, and stained with 1% (w:v) aceto-orcein. The intact oocytes were examined for sperm penetration with phase contrast microscopy at x500 magnification. Penetration was determined by the presence of two or more pronuclei, or one pronucleus and one or more sperm in the ooplasm. Oocytes in metaphase II were considered unpenetrated.

Antisperm Antibodies in a Group of Bulls

Serum from unbred heifers (n = 20) from a local farm and serum from a group of bulls (n = 90) from a commercial semen collection facility (ABS Global) were tested for the presence of antisperm antibodies by the screening ELISA. This required four ELISA plates, and each plate included 5 heifer samples and 22–23 bull samples, plus a positive control serum sample (bull #377).

Statistical Analyses

The in vitro fertilization study was designed as a randomized complete block to account for the use of different sperm and for the different days on which the six trials were conducted. The data were analyzed by ANOVA after arcsine transformation [23]. Linear contrasts and an LSD test ({alpha} = 0.05) were subsequently conducted to determine differences among the six treatments. In the study of antisperm antibody levels, differences between bull and heifer samples were analyzed by ANOVA, using the individual ELISA plates as a blocking factor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental Animals

No abnormalities were identified in breeding soundness examinations and semen evaluations of test bulls before, during, or after immunization. Gross and histologic examination revealed no testicular abnormalities that could be attributed to immunologic disease. Bull #376 had evidence of a recent scrotal hernia or seroma within 2 wk of the time of castration. This was associated with degeneration of the seminiferous tubules, histologically manifested by the lack of maturing and mature sperm cells.

Antisperm Antibody Generation

After immunizations, all test bulls had a transient rise in the level of antisperm IgM and persistently elevated levels of antisperm IgG1 and IgG2 in the serum (Fig. 1). Serum antisperm IgG1 levels rose rapidly after the first immunization, peaked shortly after the third immunization, and subsequently decreased over the following 3 mo for which they were measured. Serum antisperm IgG2 levels followed a similar pattern except in one bull in which serum antisperm IgG2 level peaked after the second immunization. No antisperm IgA could be detected in serum samples, and no antisperm antibodies of any isotype could be detected in seminal plasma samples.



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  		FIG. 1. Serum IgG1 and IgG2 antisperm antibody responses in bulls to immunization as measured by absorbance at 450 nm: triangles represent days of immunization; squares, diamonds, and circles represent bull 376, bull 377, and bull 477, respectively. O.D., optical density.

Antigen Characterization

When sperm proteins were separated by SDS-PAGE in 12% gels, the immunoblot revealed that both post-immunization IgG1 and IgG2 showed strong reactivity with a 45-kDa antigen (Fig. 2). In addition, the post-immunization IgG1 recognized two other antigens of 31 and 27 kDa with a very weak signal. When sperm proteins were separated by SDS-PAGE 13.2% gels, post-immunization IgG2 recognized a 4-kDa antigen.



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  		FIG. 2. Western blot displaying reactivity of purified bovine serum immunoglobulins with bovine sperm proteins. Lanes were probed with purified IgG1 pre (lane B)- and post (lane C)-immunizations and IgG2 pre (lane D)- and post (lane E)-immunizations. The control lane (lane A) was treated with the diluent (1% pig skin gelatin and 0.05% Tween 20 in TBS). The ECL detection system was employed. Molecular standards (Mr x 10-3) are on the left.

In Vitro Fertilization Experiments

The mean percentage of oocytes penetrated by sperm for each treatment is shown in Figure 3. The sperm incubated with post-immunization antisperm antibody-positive serum (treatment 6) had a lower penetration rate (p = 0.01) than the sperm incubated with no serum (treatment 1) and the sperm incubated with antisperm antibody-free serum (treatments 2–5). Surprisingly, sperm that were incubated with antisperm antibody-free serum (treatments 2–5) had a higher penetration rate (p < 0.05) than the sperm incubated with no serum (treatment 1).



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  		FIG. 3. Effect of antisperm antibodies on in vitro fertilization. Mean percentage of oocyte penetration after sperm were incubated with the sera from different treatments or the control diluent (BGM-3). The letters represent treatments with significant differences (p < 0.05). Lines above the bars represents the upper limit of the 95% confidence interval.

Antisperm Antibodies in a Group of Bulls

Serum antisperm antibody levels were higher (p < 0.01) in the bull sera than in the unbred heifer sera (Table 1); both bull and heifer values were considerably lower than that of the positive control serum from an immunized bull (#377) used as a standard (mean absorbance 0.583 at the same serum dilution). On the basis of this result, additional serum (n = 204) and seminal plasma (n = 46) samples from a group of bulls (ABS Global) were screened for the presence of antisperm antibodies by ELISA. The six serum samples with highest values in the screening ELISA were retested using the previously described isotype-specific ELISA. The isotype of the antisperm antibodies detected in the serum of all six animals was IgM. The three bulls with the highest ELISA values were resampled and retested approximately one year later and were negative for serum antisperm antibodies of all isotypes. No antisperm antibodies were detected in any of the seminal plasma samples tested.


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  		TABLE 1. Comparison of antisperm antibody levels in bulls and heifers by ELISA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Auto-immunization of bulls with sperm generated serum antisperm antibodies of IgG1 and IgG2 isotypes, and these antibodies blocked fertilization in vitro. The immunization regime was designed as a model of the antisperm autoimmune responses that could result from testicular disease in bulls. For this reason, bulls were auto-immunized without any adjuvant, in an attempt to mimic a natural immune response to previously sequestered sperm cells. While it is not certain that major histocompatibility complex antigens are normally expressed on the surface of sperm [24, 25], auto-immunization avoided the possibility of allotypic immune responses. Natural causes of testicular disease, including trauma and infection, can break down the blood-testis barrier and lead to exposure of sperm to leukocytes, with the subsequent formation of antisperm antibodies. The presence of antisperm antibodies in seminal plasma has been documented in cases of blood-testis barrier breakdown resulting from testicular injury [6], and serum antisperm antibodies can be seen as a result of bilateral or unilateral vasectomy [26]. Vasectomies are commonly performed in men as a means of contraception [27], and experimentally in other animals including rats, guinea pigs, and rams [26, 28, 29]. The occlusion of the ductus deferens results in epithelial rupture, formation of sperm microgranulomas, exposure of sperm antigens to the immune system, and generation of antisperm antibodies [26].

Previous experimental studies in bulls have generated antisperm antibodies by iso- and auto-immunization with sperm, testicular homogenates, or epididymal homogenates combined with Freund's adjuvant [16, 17]. In these studies, histologic testicular changes varied ranging from normal morphology to complete degeneration of the seminiferous tubules, with occasional leukocytes seen in seminiferous tubules of a few bulls. Some animals in these studies had limited evidence of anti-sperm antibodies in ejaculates [16]. The lack of testicular damage or pathology in our model may account for the absence of antisperm antibodies in the seminal plasma of test bulls. Alternatively, antibodies may have been present at levels below the limits of detection, or seminal plasma may be capable of blocking recognition in the ELISA system. It is also possible the route of immunization in our model was incapable of promoting a mucosal IgA immune response that might have been more likely to result in the presence of seminal plasma antisperm antibodies. The antibody isotype of antisperm antibodies varies according to the origin of the immune response. In man, vasovasostomy was associated with an IgA response that markedly reduced conception rate [30]. In a vasectomy model in rams, antisperm antibody responses were solely of the IgG and IgM isotypes, and IgA responses were absent [26]. Antisperm antibody responses associated with genital tract infections have been reported to be principally of the IgA isotype, while intake of spermatozoa via the alimentary canal in homosexual men results in IgG and IgM responses [31, 32].

While antibody responses in our model were limited to circulating IgG responses, these antibodies were of functional significance in that they blocked fertilization in vitro, a phenomenon not previously reported in cattle to our knowledge. There are a number of possible mechanisms that could explain the capacity of bull antisperm antibodies to reduce oocyte penetration by sperm [33], each of which would require further studies in order to define its role in reducing fertilization. Mechanisms could include alterations in sperm motility [34], sperm capacitation, sperm capability of binding to the zona pellucida [12], the acrosome reaction [35], and sperm penetration into the ooplasm [31]. An unanticipated result of the in vitro fertilization study was the observation that heat-inactivated normal bovine serum (antibody-free) increased penetration of oocytes by sperm. Several in vitro fertilization experiments have been performed with sperm incubated with heat-inactivated serum. However, these previous experiments determined that incubation with serum either had no effect in humans [36] or a negative effect on fertilization rates in mice [37]. This observation may have practical implications for improving bovine in vitro fertilization efficiency.

Identifying the antigen recognized by antisperm antibodies would help to explain the functional effects of these antibodies. In other species, antisperm antibodies have recognized antigens of 40 and 44 kDa in mice [37]; 62 kDa in horses [38]; and 50, 55, 57, 62, and 72 kDa in infertile humans [8]. Both immunopurified post-immunization bovine IgG1 and IgG2 from one of the test bulls recognized a 45-kDa sperm antigen. The identity of this bovine sperm antigen has not been determined, although the result of the in vitro fertilization study suggests that oocyte fertilization may depend on it. A panel of monoclonal antibodies against a range of 40- to 200-kDa bovine sperm antigens has been described, but none of these antibodies affected sperm-oocyte interaction [39]. Two previous reports describe monoclonal antibodies recognizing human sperm protein which are also capable of reducing in vitro fertilization in the bovine species [35, 40]. One of these antibodies recognizes the 37-kDa intra-acrosomal SP-10 sperm protein and acts by reducing the motility of capacitated sperm and their tight binding to the zona pellucida [35]. The second antibody recognizes the 53-kDa FA-1 antigen present in the post-acrosomal region. While neither of these antigens has obvious homology to the antigens recognized by the bovine antisperm antisera in this study, these monoclonal reagents may still prove useful for better defining the antigen specificity of bovine antisperm antibodies.

The comparison of antisperm antibody levels between a bull and a heifer population demonstrated significantly higher levels in bulls, although the total antibody concentration was considerably lower than that detected in the immunized experimental bulls. A possible explanation for this finding is that these antibodies were generated by rectal insemination in the group-housed bulls, since in group-housing, homosexual behavior is quite common. Antisperm antibodies may form as a result of exposure of sperm antigens to the rectal mucosa, and they have been detected in the sera of a high percentage of homosexual men [41]. These human antisperm antibodies are mostly of the IgG and IgM isotypes. The significance of the antisperm IgM in these bulls is unknown, although a recent human study correlates the presence of antisperm IgM on the sperm head or the tip of the tail with reduced in vitro fertilization rates in infertile couples [42].

In this study, s.c. and i.m. immunization with sperm without any adjuvant generated serum IgG1 and IgG2 antisperm antibodies. These antibodies were capable of reducing in vitro fertilization rates. Further studies with hemizona assays, acrosome staining, and in vitro fertilization with zona-free oocytes are needed to determine the mechanism by which in vitro fertilization rates are reduced. Nevertheless, these experiments demonstrate that auto-immunization with sperm can lead to the generation of circulating IgG antisperm antibodies that have the capacity to interfere with fertilization. This study did not identify similar antibodies in a provisional study of a population of young bulls, but it did identify low antibody levels, principally of the IgM isotype. Further studies are necessary to determine whether testicular pathology resulting from trauma or infectious disease can generate antisperm antibodies with the same functional effects as those generated by sperm auto-immunizations.


   ACKNOWLEDGMENTS
 
The authors would like to express their gratitude to Dr. Rick Nordheim for his help with the statistical design of the experiments. They would also like to thank Dr. Charles Brown and ABS Global for the generous donation of bull serum and semen samples.


   FOOTNOTES
 
1 This research was supported by the Food Animal Fund, University of Wisconsin, School of Veterinary Medicine and the College of Agriculture and Life Sciences. Back

2 Correspondence: D. Paul Lunn, Department of Medical Sciences, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706. FAX: 608 265 8020; lunnp{at}svm.vetmed.wisc.edu Back

Accepted: January 5, 1999.

Received: December 16, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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