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a Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well known that mammalian spermatozoa acquire their fertilizing ability during their passage through the epididymis (for reviews see [3–5]). Evidence from several laboratories has demonstrated that androgen-dependent epididymal secretory proteins associate with the surface of spermatozoa during maturation and participate in the development of the ability of sperm to interact with both the zona pellucida [6–9] and the oolemma [10, 11]. Therefore, deliberate induction of antibodies against epididymal proteins might either interfere with their sperm surface association or mask these antigens on the sperm surface in both the male and female tract. Furthermore, while immunization of males with sperm proteins raises the possibility of autoimmune disruption of testicular function and thereby compromising hormone synthesis and/or spermatogenesis, the use of specific epididymal proteins acquired during epididymal transit represents an attractive mode of fertility regulation.
Rat epididymal glycoprotein DE (37 kDa), first described by our laboratory [12], is synthesized in an androgen-dependent manner by the epithelium of the proximal segments of the epididymis and associates with the sperm surface during epididymal transit [13, 14]. DE, originally localized on the dorsal region of the sperm head, migrates to the equatorial segment concomitantly with the occurrence of the acrosome reaction (AR) [15] and participates in the sperm-egg fusion process through complementary sites localized on the egg surface [11, 16].
Previous results have indicated that active immunization of male and female Lewis and Wistar rats with purified DE raised auto- and iso-antibodies, respectively, against the protein in over 90% of the animals, producing a significant and reversible inhibition of fertility in both sexes [17]. Subsequent in vitro studies in which tissues and sperm from untreated fertile males were exposed to the immune sera demonstrated that the antibodies specifically recognized DE in epididymal tissue, as well as on the surface of both fresh and capacitated spermatozoa, and produced a significant inhibition in the fertilizing ability of sperm artificially inseminated in the uterus of normal females [18].
Although these in vitro results support the participation of the anti-DE antibodies as mediators of the antifertility effect, several questions arise regarding the in vivo mechanism responsible for this effect. In the present study, we addressed these questions in the male by studying the effects of immunization with epididymal protein DE on the reproductive organs and spermatozoa recovered directly from DE-immunized animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Young adult male (70–80 days old) and both adult (90 days old) and prepubertal (28–30 days old) female Wistar rats were used. Animals were maintained with food and water ad libitum in a temperature-controlled room with a 12L:12D cycle. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Mating
Male fertility before and after immunization was evaluated by natural mating. Each male was housed with three females of proven fertility for 7 days. Females were examined thereafter, and those showing evidence of pregnancy were removed from the cage and allowed to deliver in a separate cage. The fertility of each male in each mating is expressed as the number of pregnant females/total number of females in the cage (three) x 100. Only males that had previously impregnated at least two of the three females in 1 wk were used for subsequent immunization.
Immunization
The immunization schedule is indicated in Figure 1. Males received 4 injections of 100 µg of purified protein DE [19, 20] at 3- to 5-wk intervals. The first injection consisted of 300 µl of antigen emulsified with 300 µl of Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO). For subsequent injections, Freund's incomplete adjuvant (Sigma) was used. Each animal received 300 µl of the emulsion i.m. in a rear leg and 300 µl s.c. in the back. A total of 15 male Wistar animals were immunized with protein DE (DE-immunized group). The same number of animals were injected as described above but received an antigen-free preparation (control group).
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Collection and Storage of Sera
Animals were anesthetized with ketamine hydrochloride (10 mg/100 g BW) and bled from the jugular vein at various times during the study (Fig. 1). Samples were incubated for 30 min at 37°C, incubated overnight at 4°C, and then centrifuged for 15 min at 1750 rpm. Sera were separated from the pellet and stored at -20°C until use.
ELISA
Purified native DE diluted in 0.3 M NaHCO3, pH 9.6 (0.05 µg/well), was coated onto a 96-well microtiter plate (Nunc A/S, Roskilde, Denmark) and incubated overnight at 4°C. After nonspecific binding sites were blocked for 90 min at room temperature (RT) with a solution of 20 mg/ml powdered skim milk in PBS, each serum diluted 1:100 in PBS containing 1% BSA (PBS-BSA 1%), or each fluid diluted 1:10 in PBS-BSA 1%, was placed in duplicate wells and incubated for 90 min at 37°C. Wells were then washed and incubated for 1 h at 37°C with biotin-conjugated anti-rat IgG antibody (Sigma; 1:500 in PBS-BSA 1%). After washing, ExtrAvidin-alkaline phosphatase (Sigma; 1:1000 in PBS-BSA 1%) was added, and incubation was continued for 30 min at RT. Wells were washed, and color reaction was allowed to develop by addition of p-nitro-phenyl-phosphate (1 mg/ml in 1 M diethanolamine, 0.5 mM MgCl2, pH 9.8). The absorbance (abs) at 405 nm was determined with a microplate reader (Cambridge Technology, Inc., Watertown, MA).
For those cases in which the amount of DE on the sperm surface was evaluated, sperm were recovered from various sections of the epididymis, and 50 µl of each suspension (1 x 105 cells/ml) was placed in 96-well microtiter plates. The plates were centrifuged for 10 min at 2000 rpm, and 2% glutaraldehyde in PBS was added to each well for 10 min. Wells were then washed and stored overnight at 4°C in PBS. Incubations with the blocking solution, antibodies, and alkaline phosphatase were performed as indicated above for purified DE, except that rabbit antibody against DE (anti-DE) diluted 1:200 in PBS-BSA 1% was used as the first antibody, followed by biotin-conjugated goat anti-rabbit IgG (Sigma) at a 1:500 dilution as a second antibody. Color reaction was allowed to develop as described above. Rabbit anti-DE antibody specifically recognizes protein DE in Western blots of both rat epididymal cytosol and rat epididymal sperm extracts. Other characteristics of the antibody have been previously reported [21].
Histology
A total of five DE-immunized and five control animals were used for histological examination. One animal was examined after the first mating (Week 17), one after the second mating (Week 22), and three after the last mating (Week 35). Males were anesthetized, and the testes, epididymides, and vas deferens from DE-immunized and control animals were removed and fixed for at least 24 h by immersion in Bouin's solution diluted 1:1 with deionized water. Tissues were then processed for paraffin embedding and sectioning by routine methods. Sections were stained with hematoxylin and eosin solutions and examined under light microscopy. Six serial sections from three different regions of the testes, as well as six serial sections from each epididymal segment and vas deferens, were examined.
Indirect Immunofluorescence (IIF)
Fresh and capacitated sperm were washed with PBS supplemented with 4 mg/ml of BSA and resuspended in the same buffer. Sperm were then fixed for 10 min in 2% p-formaldehyde in PBS at RT and air-dried onto polylysine-coated slides (0.1 mg/ml). After washing with PBS, sperm were incubated for 30 min with normal goat serum (5% in PBS) at 37°C and then exposed overnight at 4°C to anti-DE (1:100). Slides were then washed with PBS, incubated 30 min at 37°C with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma; 1:100 in PBS), washed, mounted in 90% glycerol in PBS, and examined in a Nikon (Garden City, NY) Optiphot microscope equipped with epifluorescent optics.
For immunohistochemistry, tissue sections obtained from DE-immunized and control animals were deparaffinized, rehydrated, and subjected to IIF as described above.
Superovulation and Egg Preparation
Prepubertal female rats were superovulated by an injection of 20 IU (i.p.) of eCG (Sigma) followed 48–72 h later by an injection of 25 IU (s.c.) of hCG (Sigma). Eggs in cumulus were recovered by puncturing the oviductal ampullae of superovulated females 12–15 h after hCG injection. Cumulus cells were removed by incubating the egg-cumulus complex for 3 min in capacitating medium [22] containing 0.1% hyaluronidase (type IV; Sigma). The zona pellucida was removed from the cumulus-free eggs by treatment of eggs for 10–20 sec with acid Tyrode's solution (pH 2.5). Zona-free eggs were thoroughly washed in capacitation medium, pooled, and placed in a Petri dish containing 100 µl of capacitating medium under paraffin oil (Fisher Scientific Co., Pittsburgh, PA; saybolt viscosity 125/135).
In Vitro Sperm Capacitation
Spermatozoa were collected by puncturing the cauda epididymidis in capacitating medium at 37°C and were placed in a tissue culture dish (35 x 10 mm; Falcon Plastics, Los Angeles, CA). Spermatozoa were allowed to disperse, and aliquots of the suspension were diluted in fresh capacitation medium at a final concentration of 2 x 105 spermatozoa/ml. Tissue culture wells (16 mm; Costar, Cambridge, MA) containing 500 µl of the sperm suspension were then incubated for 5 h under paraffin oil at 37°C in an atmosphere of 5% CO2 in air.
Sperm-Egg Fusion Assay
Capacitated sperm were added (final concentration 0.5–2.0 x 105 cells/ml) to zona-free eggs, and the gametes were coincubated for 2–3 h under the conditions described above. Eggs were then washed in fresh medium to remove loosely adherent sperm, mounted on slides, and evaluated for sperm penetration under phase-contrast microscopy (x400). Eggs were considered penetrated if two pronuclei and a sperm tail were present.
Collection of the Reproductive Fluids
Control and DE-immunized animals were anesthetized and exsanguinated by perfusion of PBS through the left ventricle to avoid blood contamination when the fluids were recovered. The epididymis and vas deferens from one side were removed and carefully dissected. The contralateral epididymides were used for sperm function studies. After extensive cleaning and rinsing, the epididymis was cut into caput, corpus, and cauda. Cauda epididymal fluid was collected by making a single puncture in one tubule and allowing the release of the luminal content into a microtube. The content from the vas deferens was recovered by gently squeezing the tubule. For the caput and corpus regions, several incisions were made, and the luminal content was also recovered by squeezing the tissues. The fluids obtained were centrifuged at 500 x g for 10 min, and the supernatants were stored at -20°C. In all cases the volume of the supernatants was determined for subsequent calculation of dilutions.
Sperm Motility and Viability
For motility assessment, aliquots of sperm suspension were placed on prewarmed slides, and the percentages of motile sperm were determined subjectively under the light microscope. For viability assessment, 40 µl of motile sperm suspensions was stained with prewarmed eosin 0.5% (yellowish; Sigma) in saline solution, and the incorporation of the dye was evaluated by light microscopy. The percentage of viability was calculated as the number of sperm that did not incorporate the dye over the total number of sperm counted.
Statistical Analysis
Results are expressed as mean values ± SD for each series of experiments. Statistical significance data were analyzed by the Mann-Whitney test. The association between absorbance values and animal fertility was analyzed by the G-test of independence and subsequent calculation of the coefficient of association (
coefficient). Values with a confidence level of p < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male rats were immunized with purified DE, and antibody levels and animal fertility were both evaluated at various intervals according to the schedule shown in Figure 1.
In an attempt to correlate the immunological response and animal fertility among DE-immunized animals, the absorbance values and the corresponding fertility for each individual male were analyzed. Comparison of these two parameters is presented in Figure 2 and indicates that whereas most (91%) of the animals with absorbance values lower than 0.5 presented high fertility rates (66–100%), the majority (67%) of those with absorbance values higher than 0.5 (0.5–1.5) presented the lowest fertility rates (0–33%).
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A total of nine animals presenting the highest reduction in fertility (0%) and absorbance values higher than 0.5 were chosen for all the subsequent studies aimed at elucidating the mechanism of fertility inhibition.
Histological Examination
In order to exclude the possibility that the reduction in male fertility was due to a deleterious effect of the antibodies on the reproductive organs, the testes, epididymides, and vas deferens of DE-immunized and control animals were subjected to histological examination.
Results indicated that tissues from all the DE-immunized animals examined presented no differences from the corresponding controls. Testis sections from DE-immunized animals exhibited no signs of infiltration and showed normal seminiferous tubule diameter and abundant spermatids. The epididymal tubules from the successive regions of the organ as well as the vas deferens from DE-immunized animals showed no evidence of leukocyte infiltration and contained morphologically mature sperm in their lumen (data not shown).
Entry of Antibodies into the Epididymis
Once any possible deleterious effect of the antibodies on the reproductive tissues was excluded, the inhibition of fertility observed in DE-immunized males might be explained by the entry of the antibodies into the epididymis and either their effect on the synthesis, secretion, or association of DE with the sperm surface or, alternatively, their binding to protein DE already present on the sperm surface.
To investigate the entry of anti-DE antibodies into the epididymis, fluids recovered from successive regions of the organ as well as from the vas deferens of DE-immunized and control animals were used as first antibodies in ELISA. Results indicated that the absorbance values corresponding to the reproductive fluids from DE-immunized animals were significantly higher than those corresponding to controls (Fig. 3).
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If immunization had interfered with the synthesis or association of DE with the sperm, we should detect a lower amount of DE on the sperm surface. This possibility was examined by both IIF and ELISA. Histological sections from the proximal regions of the epididymides of DE-immunized and control animals were subjected to IIF using rabbit anti-DE antibody and FITC-anti-rabbit IgG as first and second antibodies, respectively. The results showed no difference in the immunofluorescent pattern observed for DE-immunized and control animals (Fig. 4). In both cases, an intense fluorescent labeling was observed in the apical regions of principal cells as well as on the sperm in the lumen, suggesting that sperm from the two groups of animals had comparable amounts of DE on their surface. To confirm these results, sperm from DE-immunized and control animals were recovered from the successive regions of the epididymis, and the DE protein present on the sperm surface was quantified by ELISA. Results, shown in Figure 5, revealed no significant difference in the amount of protein detected for the two groups of animals.
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Antibody Effects on Sperm Function
The results described above indicated that the anti-DE antibodies would enter the epididymis without affecting the final association of DE with the sperm surface. This left the possibility that the antibodies had interfered with sperm function by association with the DE protein present on the sperm surface. In order to examine this possibility, several functional parameters were analyzed.
a) Sperm Motility and Viability
Sperm motility was evaluated by light microscopy of sperm recovered from successive regions of the epididymis. Results indicated no significant difference in the motility of sperm from DE-immunized animals and controls (Fig. 6A). Neither were the percentages of viable sperm from DE-immunized animals significantly different from those in the controls (Fig. 6B).
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b) Sperm Capacitation and AR
Since protein DE migrates from the dorsal to the equatorial segment of the acrosome concomitantly with the occurrence of the AR, the ability of the cells to undergo capacitation and AR was evaluated according to the percentage of cells showing DE on the equatorial segment by IIF. There were no significant differences in this respect between the two groups (52% immune vs. 53% control).
c) Sperm Fertilizing Ability
Taking into account the observation that protein DE has a demonstrated role in the sperm-egg fusion process [11,16], the likely possibility that the anti-DE antibodies had interfered with this specific event was evaluated. For this purpose, sperm from DE-immunized and control animals were exposed to zona-free rat eggs in vitro, and the percentage of penetrated eggs for each group was determined 2–3 h later. Since gamete fusion is preceded by a first step of sperm binding to the oolemma, the ability of sperm to bind to the egg plasma membrane was also evaluated.
The results shown in Figure 7 indicate that while no differences were observed in the percentage of oocytes with bound sperm or in the number of sperm bound per oocyte, the percentage of penetrated zona-free eggs was significantly (p < 0.05) lower for DE-immunized than for control animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In some cases, animals with high absorbance values and no inhibition of fertility were observed, suggesting that a certain level of circulating IgG antibodies might be required (abs > 0.5), but not be sufficient, to affect fertility. In this regard, the possibility that other immunoglobulins could participate in the inhibition of fertility should not be excluded. IgAs are present in several organs of the male reproductive tract [23, 24] including the epididymis [25, 26]. In humans, although the level of IgA antibodies does not appear as critical as IgG antibodies for fertilization suppression, a combination of IgA on sperm and high serum anti-sperm antibody levels reduces the probability of fertilization [25, 27, 28]. In addition, immunologic cytokines released by activated lymphocytes, macrophages, and other white blood cell products might also be involved in fertility inhibition, since evidence to date indicates that these soluble factors can adversely affect several aspects of sperm function (i.e., motility, AR, fusion ability) [29–31].
Although Western blot experiments indicated that anti-DE sera specifically recognize DE in epididymal tissue and epididymal sperm [18], a detrimental effect caused by the immune response on the epididymis, as well as upon other reproductive tissues, could not be ruled out. Histological studies here revealed, however, no testicular, epididymal, or vas deferential pathology in DE-immunized animals.
Several reports have demonstrated the entrance of IgGs into the epididymis to different extents, as well as their association to sperm in several species, including man [32–37]. These results, together with our observations indicating the ability of the immune sera to recognize DE in epididymal tissue and sperm [18], led us to explore the possibility that anti-DE antibodies entered the epididymis and interfered either with the synthesis or secretion of DE by the epithelial cells or, alternatively, with the association of the protein with the sperm surface. Results obtained by ELISA revealed significantly higher levels of anti-DE antibodies in epididymal fluids recovered from DE-immunized than from control animals, representing approximately 1% of sera absorbance values. Although the possibility of blood contamination cannot be excluded, the prior perfusion of the animals, the careful dissection of the organs, and the fact that no erythrocytes were observed in the microscopic examination of the fluids support the conclusion that anti-DE antibodies found in the fluids resulted from transudation of sera antibodies into the reproductive tract. This finding is in agreement with the described entrance of immunoglobulins into the epididymis as reported by other groups, and it supports the concept that the hemato-epididymal barrier is much more permeable than the blood-testis barrier, especially for molecules the size of IgG [38].
IIF and ELISA studies revealed no difference in the amount of DE on epididymal tissue and epididymal sperm from DE-immunized and control animals, indicating that the presence of anti-DE antibodies in the epididymis would not affect the production of DE or its association with the sperm surface during maturation. These results, and the ability of the immune sera to bind to DE on the sperm surface in vitro [18], suggested the possibility that the antibodies had interacted with DE already present on sperm, affecting their fusion ability. Although previous results indicated that the immune sera do not affect sperm motility, viability, or ability to undergo capacitation and/or AR, under in vitro conditions [18], the possibility exists that other factors appearing after active immunization of the animals could have affected these aspects of sperm function. The results indicated no differences in the numbers of viable and motile epididymal sperm directly recovered from immunized and control animals. Neither the ability of these sperm to undergo capacitation nor the AR was affected, as judged by the percentage undergoing redistribution of DE from the dorsal to the equatorial segment of the acrosome. However, when exposed to zona-free rat oocytes, epididymal sperm recovered from DE-immunized males exhibited a significantly lower ability to penetrate the eggs. This inhibition in egg penetration was not due to the inability of sperm to bind to the egg surface, since no differences were observed in the percentage of oocytes with bound sperm or in the number of sperm bound per egg—confirming that inhibition of fertilization involved gamete fusion. This finding is consistent with the demonstrated functional role of DE [11,16].
The results obtained in this study, together with the observation that the immune sera are able to inhibit sperm-egg fusion in vitro [17], suggest that male fertility inhibition after immunization with DE might involve the entry of anti-DE antibodies into the epididymis and their specific interference with the ability of sperm to fuse with the egg. However, we could not detect association of the antibodies with the sperm surface using only the second antibody in IIF experiments. This observation is consistent with the results from previous experiments in which sperm that were recovered from cauda epididymal tubules and cultured for 24 h in the presence of rabbit anti-DE, exhibited a very low level of staining when incubated with second antibody (8%), in spite of presenting a significantly reduced fertilizing ability after uterine insemination [39]. A failure to detect anti-sperm antibodies by immunofluorescence in patients showing positive anti-sperm antibodies by other techniques has also been reported [40, 41]. It is possible, therefore, that although not sufficient to be detected by IIF, the levels of antibody bound to sperm are capable of affecting the ability of sperm to fuse with the egg. We cannot, however, exclude the possibility that additional immunological factors are operative in the reduction in fertilizing ability of epididymal sperm in DE-immunized males.
The present results provide essential information for a better understanding of the molecular mechanisms underlying immunological infertility, as well as for the development of new and safe contraceptives in humans. The relevance of these findings is further supported by recent reports from two independent groups describing the existence of a human homologue of protein DE [42, 43]. The functional role of this epididymal protein is currently under investigation in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Diego Ellerman, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490 (1428), Buenos Aires, Argentina. FAX: 541 786 2564; ellerman{at}proteus.dna.uba.ar
Accepted: June 15, 1998.
Received: January 27, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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