Hybridoma Division, National Institute for Research in Reproductive Health, J. M. Street, Parel, Mumbai, 400 012 (fax: 22-2413-9412; e-mail: email@example.com).
ABSTRACT: Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.
Spermatozoa released from the testis are immature and undergo posttesticular maturation in the epididymis. Posttesticular maturation of spermatozoa is not intrinsic to sperm, but the epididymal epithelium plays an active role in it (Kirchhoff, 1999). It has been reported that the development of fertilizing ability is acquired by the sperm during its sojourn through the epididymis. There is continuous increase in the capacity of spermatozoa for sustained progressive motility as they approach the cauda epididymis (Moore, 1998).
There are a number of clinical evidences to show the correlation between abnormalities and disturbances in the epididymal secretions and infertility (Blaquier, 1987; Fichorova and Nakov, 1993; Fichorova et al, 1995). Vasectomy also has shown to cause irreversible damage to epididymis and hence could be one of the causes of infertility even after vasovasostomy (Guillemette et al, 1999; Turner et al, 2000). All of this evidence points to the important role of the epididymis in bestowing on the sperm motility and fertilizing ability.
Several proteins have been identified and studied for their contribution toward sperm maturation. However, many more still remain to be identified, and their functions need to be ascertained. Identification of newer epididymal proteins and annonating their function will help in understanding the mechanism of sperm maturation and the sequence of events therein. This will further help in selecting epididymal targets for contraception that will specifically alter the ability of the sperm to fertilize without any side effects.
Several different approaches have been exploited for the identification of epididymal proteins such as the use of lectins (Srivastav, 2000), subtractive screening of the epididymal cDNA library (Kirchhoff, 1998), the use of the expressed sequence tags (Holland and Nixon, 1998), proteomics (Syntin et al, 1996), and neonatal tolerization (Ensrud and Hamilton, 1991, Khole et al, 2000). Neonatal tolerization is a powerful tool for raising monoclonal antibodies (mabs) to rare or weakly immunogenic antigens. This has been used extensively in various fields for generating mabs to rare or less immunogenic antigens (Golumbski and Diamond, 1986; Hockfield 1987; Ou et al, 1991; Williams et al, 1992; Imam et al, 1994; Lebron et al, 1999; Sleister and Rao, 2001). In this approach, once a state of tolerance to an antigen is established, the tolerized animals could be subsequently immunized with a crude preparation of the desired antigen (immunogen). By inducing the immune tolerance to the tolerogen, the immune system will generate an immune response to only those desired epitopes not included in the tolerogen preparation. This approach increases the probability of obtaining antibodies to functionally significant components that may be weak immunogens.
We have standardized neonatal tolerization for identification of epididymal proteins. In the first step, the Balb/c neonates were tolerized to testicular sperm protein, and, in second step, starting on day 21, these animals were boosted with epididymal sperm proteins to induce an epididymis-specific immune response. The present article discusses the characterization of a panel of mabs specific to epididymal proteins generated using this approach.
Materials and Methods
All the animals used in the study were procured from the Animal House of the Institute and were inbred strains. The study was approved by the Ethics Committee for Care and Use of Laboratory Animals for Biomedical Research of the National Institute for Research in Reproductive Health, Mumbai, India. Balb/c mice were selected as a model for study because these animals were further used for Hybridoma technology to raise the mabs. The myeloma cell line from the Balb/c mice was procured from National Centre for Cell Sciences, Pune, India. Details of the animals of different age groups used for the present study were female Balb/c neonates aged day 0, female adult Balb/c aged 4 to 6 weeks and weighing 18 to 20 g, and male Holtzman rats aged 4 to 6 weeks and weighing 180 to 200 g.
Adjuvants were obtained from Sigma (St Louis, Mo). All the reagents for electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were obtained from SRL India Ltd (Mumbai, India). Nitrocellulose sheets were from Amersham (Buckinghamshire, England), conjugates were from Bangalore Genei India Ltd (Bangalore, India), and ELISA plates were from Nunc, (Denmark).
Preparation of Tolerogen (Testicular Protein)
Testicular proteins were used as tolerogen. Testicular protein extract was prepared according to the method described by Khole et al (2000). In brief, testes from four rats were teased in Ham's F10 medium and incubated at 37°C for 30 minutes, and the supernatant containing testicular sperm preparation was spun down at 500 × g for 20 minutes to get the pellet. The pellet was resuspended in 2 mL of red blood cell lysing solution for 5 minutes. The pellet was diluted 4 times in Ham's F10 medium and centrifuged at 500 × g for 20 minutes. The pellet was then resuspended in 2 mL of phosphate-buffered saline (PBS) and sonicated for 5 minutes. The solution was then centrifuged, and the supernatant was used as testicular protein.
Preparation of Immunogen (Epididymal Sperm Protein)
Rat cauda epididymal sperm proteins were used as the immunogen. Cauda epididymides from 4 rats were taken. The cauda epididymides were teased in Ham's F10 medium and kept at 37°C for 30 minutes. The supernatant was then collected and centrifuged at 500 × g for 20 minutes. The pellet was then washed twice with medium and then resuspended in PBS and sonicated for 5 minutes. After centrifugation, the supernatant was used as rat epididymal sperm protein or immunogen. Concentrations of both rat testicular protein and epididymal sperm protein were estimated according to the method of Lowry et al (1954).
Induction of Tolerance in the Neonates
Tolerization was carried out according to the method described by Khole et al (2000). In brief, 12 Balb/c neonates were injected intraperitoneally (IP) with 20 μg of tolerogen (rat testicular protein) in 50 μL of PBS within 24 hours of birth. On day 5, another injection of tolerogen (20 μg/50μL) was given IP. Mice were bled through the retro-orbital plexus to obtain the tolerization sera on day 21. For further experiments, only female mice were used.
On day 21, these tolerized animals were divided into 2 groups— group 1 and group 2. Each group contained 6 animals. Animals from group 1 were injected with immunogen (100 μg/100μL in PBS) emulsified in Freund's complete adjuvant (FCA) subcutaneously at multiple sites. Group 2 animals were immunized with tolerogen (100μg/100μL in PBS) emulsified with FCA. Two boosters were given at biweekly intervals and a week after the last booster, all the animals were bled through the retro-orbital plexus to obtain the tolerized and immunized (T-I) sera. The neonatal tolerization and immunization protocol is depicted in Figure 1.
Production of Mabs
Mabs were produced according the method of Kohler and Milstein (1975), using Balb/c mice that were neonatally tolerized and immunized as described above. The clones were selected by their ability to react specifically to epididymal proteins by ELISA. Culture supernatants were precipitated using 40% ammonium sulphate. In brief, ammonium sulphate—saturated deionized water was added dropwise to culture supernatant at a concentration of 40% and stirred for 30 minutes at 4°C. It was then centrifuged at 5000 × g for 20 min. The pellet was washed twice with 40% ammonium sulphate, and the final pellet was reconstituted in PBS and dialyzed against water. This purified antibody was concentrated by lyophilization.
Characterization of Mabs
Mabs were characterized using ELISA, indirect immunofluorescence (IIF), Western blot, immunohistochemistry (IHC), and agglutination.
ELISA was done using both rat testicular as well as epididymal sperm proteins. Both rat testicular and epididymal sperm proteins (10 μg/well) diluted in carbonate-bicarbonate buffer (pH 9.6) were coated onto flat-bottomed 96-well microtiter plates and incubated overnight at 4°C. Nonspecific binding sites were blocked by adding 300 μL of 2% nonfat dry milk (NFDM) to the wells and incubating for 1 hour at 37°C. The wells were washed 3 times for 5 minutes each with PBS that contained 0.05% Tween 20 (T-20). Wells were then incubated with 100 μL of immune sera serially diluted between 1: 100 and 1: 3200 in 0.2% NFDM in PBS for 1 hour at 37°C. The wells were then washed as described above and incubated with 1: 5000 dilution of rabbit anti-mouse IgG labeled to horseradish peroxidase (HRP) for 1 hour at 37°C. The wells were washed 3 times, as described earlier, and the immunoreactivity was visualized using 200 μL of substrate solution (8 mg o-phenylenediamine dihydrochloride plus 0.03% H2O2 in 0.1 M citric acid and 0.2 M disodium hydrogen orthophosphate). The readings were taken at 492 nm in a Titertek multiscan plate reader (Titertek).
Rat cauda epididymal sperm were released in Ham's F10 medium. The sperm count was adjusted to 106 cells/mL, and 200 μL of sperm were taken in a conical Eppendorf tube. Ten microliters of respective mabs were added to respective tubes, to a dilution of antibody of 1: 20. Tubes were incubated at 4°C for 1 hour. After the incubation, the spermatozoa were diluted with 1 mL of PBS that contained 2% bovine serum albumin (BSA), centrifuged at 150 × g for 10 minutes, and the supernatant discarded to remove unbound antibody. Similarly, 2 more washes were given. The final sperm pellet was then resuspended in 200 μL goat anti-mouse fluorescein isothiocyanate (1: 100 in 2% BSA) and incubated at 4 C for 1 hour. Spermatozoa were washed as described above and finally resuspended in 100 μL of anti-fading agent (10 mg of p-phenylenediamine dihydrochloride in 90% glycerol in PBS). Fluorescence was observed under epifluorescence microscope (Ziess, Gottingen, Germany). Fluorescence was also performed using a rat sperm—smeared glass slide. SP2/0 culture supernatant was used as the negative control.
Western Blot Analysis
Sodium dodecyl sulfate—polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970). Rat testicular and cauda epididymal sperm protein was loaded in each well (45 μg). Western blotting was carried out according to the procedure described by Towbin et al (1979). Nitrocellulose strips were blocked with 5% NFDM in PBS at room temperature for 1 hour on rocker. Strips were then incubated with mabs (1: 25 dilution in 0.5% NFDM) at 4°C overnight. Strips were given three separate washes of 10 minutes with 0.5% T-20 in PBS. The nitrocellulose strips were then incubated with rabbit anti-mouse HRP conjugate (1: 5000 dilution in 0.5% NFDM) for 1 hour at room temperature. Strips were again washed as described above. The blots were developed using 10 mg 3,3-diaminobenzidine in 10 mL of PBS containing 10 μL of 30% H2O2. SP2/0 culture supernatant was used as negative control.
Whole rat epididymis and testis were fixed in Bouin's fixative for 24 to 48 hours. The tissues were then processed for paraffin embedding and sectioning. Tissue sections were deparaffinized, rehydrated, and incubated for 30 minutes in 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Nonspecific reactivity was blocked with 2% BSA in PBS for 1 hour. The sections were incubated with the mabs (1: 25 dilution in 0.2% BSA) at 4°C overnight. Sections were washed 3 times for 5 minutes in PBS. Rabbit anti-mouse HRP (1: 250 dilution in 0.2% BSA) was applied to sections for 2.5 hours at room temperature. All the incubations were done in a humid chamber. The slides were then washed as described above. Color reaction was carried out using 3,3-diaminobenzidine (DAB) +H2O2 (10 mg DAB+ 10 μL H2O2 in 10 mL PBS). The slides were then counterstained with hematoxylin. The slides were dehydrated and mounted in DPX mount. For the control group, the sections were incubated with SP2/0 culture supernatant. SP2/0 culture supernatant was used as a negative control.
Cauda epididymides from one rat were taken. The cauda epididymides were teased in Ham's F-10 medium and kept at 37°C for 30 minutes. The supernatant was then collected and centrifuged at 520 × g for 20 minutes. The pellet was then washed twice and then resuspended in Ham's F10 medium. The count was adjusted to 106 sperm/mL. To 200 μL of the suspension, 10 μL of 2 mg/mL precipitated culture supernatant of different mabs were added and kept at 37°C for agglutination. After 30 minutes, 10 μL of the agglutination reaction mixture was taken on a glass slide and visualized under 5× dark-field microscope.
Mice were neonatally tolerized with testicular proteins at birth and immunized with epididymal sperm proteins, to induce a specific immune response to epididymal sperm proteins. Figure 1 is the pictorial representation of the protocol. Neonatal Balb/c mice were tolerized with rat testicular protein on days 0 and 5. Of these only female Balb/c mice were continued further in the study. On day 21, the sera from these animals were checked for their reactivity with both rat testicular and cauda epididymal sperm protein by ELISA (data not shown). The results indicated that there were no antibodies reacting to testicular proteins. This could either be because the mice were tolerized to testicular proteins or the testicular protein was not immunogenic in nature. To rule out the second possibility, the immunogenicity of testicular protein was confirmed by injecting adult Balb/c female mice. The neonatally tolerized mice were divided in 2 groups. Animals from group 1, which were injected with cauda epididymal sperm proteins, showed very significant immune response to epididymal sperm proteins and insignificant reactivity with testicular proteins. Group 2 animals, which continued to be immunized with the tolerogen (testicular proteins), did not show any response to the tolerogen. This confirmed that the neonates were completely tolerized and did not mount any response even on being challenged in adulthood.
Generation of Mabs
A week after the second booster, the animals from group 1 were bled. Serum samples from these animals were checked for antibody titer to epididymal proteins. The animal showing the highest titer was selected for fusion. Primary screening of the reacting hybrids was performed by ELISA (data not shown) using whole rat cauda epididymal sperm. Positive clones from the primary round were subjected to secondary screening using rat testicular and cauda epididymal sperm protein as antigen. Results from the secondary screening are depicted in Figure 2. Each circle represents one clone. It was seen that there were very few clones reactive with testicular sperm proteins and that the reactivity was also not significant (panel 1). On the other hand, a large number of clones showed high reactivity with epididymal sperm proteins (panel 2). At tertiary screening, 5 clones were selected for subcloning on the basis of their high reactivity with cauda epididymal sperm protein.
IIF was done with these 5 subcloned hybrids, to elucidate the regionalized distribution of antigen on rat sperm. Figure 3A illustrates characterization of clones by indirect immunofluorescence. Serum, which was used for fusion, from a T-I mouse bled a week after the last booster localized proteins on different regions of rat spermatozoa such as head and midpiece. Figure 3A1 represents the phase contrast of the above image.
Figure 3 B through D shows reactivity of different clones obtained after fusion using splenocytes from T-I mouse. It was seen that clones localized antigens on different regions of rat sperm. Antibody V3C8 and V3F4F4 localized antigens on the postacrosomal and equatorial region of the sperm (Figure 3B), whereas acrosomal cap localization was seen with monoclonal antibody V2C4E2 (Figure 3C). Midpiece localization was seen when monoclonal V1B8E10 and V3C10 were used (Figure 3D).
After clonal expansion of these hybrids in vitro, the culture supernatant was collected, and antibodies were partially purified by ammonium sulfate precipitation. Figure 4 illustrates the titration of precipitated culture supernatant of different mabs with rat testicular as well as epididymal sperm proteins. It was seen that the clones showed little reactivity with testicular sperm protein (Figure 4A), compared with that with epididymal sperm protein (Figure 4B).
Western Blot Analysis
Western blot analysis was done for determining the molecular weight of proteins identified by different mabs. Figure 5 shows that all the mabs identified proteins approximately in the range of 27 KDa: lane A, V1B8E10; lane B, V3F4F4; lane C, V3D7; lane D, V3C10; lane E, V3C8; and lane F, negative control (SP2/0 supernatant).
To study the regionalized distribution of proteins identified by different mabs, immunohistochemical localization study was done in sagittal section of rat epididymis. Figure 6 illustrates the immunohistochemical localization of antigens in the different regions of the epididymis using different mabs. Panels A through D represent the proximal caput, distal caput, corpus, and cauda region of epididymis. With all the antibodies, immunostaining appeared first in the supranuclear region of the epithelium lining of the distal caput. Distal caput sperm were, however, not immunostained. Intense immunostaining was seen both in the supranuclear and the microvilli region of the corpus epithelium as well as spermatozoa. In the caudal epithelium region, mabs V1B8E10, V3C10 and V3C8 showed immunostaining only in the epithelial microvilli, whereas V2C4E2 and V3F4F4 showed immunostaining in both the supranuclear region and microvillus. Caudal sperm showed staining with all the mabs tested.
Surface localization of antigens by all the mabs by indirect immunofluorescence led us to study the in vitro agglutination pattern of the rat sperm. Figure 7 shows representative agglutination pattern of rat sperm with different mabs. There was radial agglutination (head to head type) with monoclonals V3C8, V3F4F4, and V2C4E2 (Figure 7A). Clones V1B8E10 and V3C10 showed comet-shaped agglutination (Figure 7B). It was interesting to note that the agglutination pattern correlated with the IIF staining.
A highly regulated and complex series of events in the epididymis brings about the metamorphosis of an immature nonfunctional sperm into a mature sperm capable of progressive motility. Sperm maturation is brought about by an interaction of spermatozoa with proteins that are synthesized and secreted by the epithelium in different regions of the epididymis. The identification and characterization of such proteins of epididymal origin would enable us to understand the mechanism of sperm maturation and help us to identify precise targets for both infertility diagnosis and contraception.
Of the various approaches used for identification of sperm antigen, mabs have been very popular. They provide a powerful analytical tool that allows for the recognition of individual determinants in a complex antigenic structure and have been applied to studies in reproductive biology (Bellve and Moss, 1983). Sera from infertile males and females or vasectomized males have also proved to be a good source of anti-sperm antibody for characterization of sperm antigen involved in fertility. Using sera from infertile males, Poulton et al (1996) identified a 18-KDa sperm protein of epididymal origin and suggested that autoimmune infertility might represent a response to the epididymal rather than testicular sperm.
He further suggested that mabs raised to such unique and immunologically accessible sperm coating antigens in the epididymis rather than in the testis would seem to present a theoretical solution to the male infertility. Hamilton et al (1985) reported a mab, EC1, that was found to react with epididymal but not testicular sperm. Except for the few such reports, conventional immunization with whole spermatozoa followed by hybridoma technology has invariably resulted in the production of antibodies predominantly to testicular antigens, probably because of its strong immunogenicity. This has made it difficult to produce mabs to epididymis-specific antigens. Therefore, a unique approach of neonatal tolerization was tried by Ensrud and Hamilton (1991). These authors used the protocol similar to that described by Golumbski and Diamond (1986). They used caput sperm membrane preparation as the tolerogen and caudal sperm membrane preparation for immunization of the tolerized animals. By using this approach, they were successful in identifying a corpus epididymis—specific protein. However, we used testicular sperm proteins for tolerization. This was done so as not to miss any maturational proteins that may be present in the caput epididymis. Our results demonstrate that neonatal tolerization with testicular protein followed by immunization with epididymal sperm antigens enhances the production of antibodies to proteins of epididymal origin.
Neonatal tolerization, also called subtractive immunization (Williams et al, 1992), is a two-step process. The first step is a suppression step, in which a state of tolerance is induced in the immune system to a specific set of molecules (tolerogen). In the second step, the immunogen is introduced in the immune system. According to the suppressor cell mechanism for the induction of neonatal tolerization and maintenance of the T suppressor cell repertoire in the circulation, there is a need for the continuous presence of the tolerogen (Roser, 1989). In our study, the continuous presence of tolerogen was ensured, because the immunogen (epididymal sperm protein) was a mixture of testicular protein (tolerogen) as well as epididymal proteins.
In the present study, the fusion yielded a large number of hybridomas, the majority of which showed high reactivity with epididymal sperm protein, whereas a small number was found to react with testicular sperm protein, as seen in Figure 2. This indicates that neonates were successfully tolerized to testicular antigen and mounted an immune response to epididymis-specific proteins. Immunofluorescent localization using polyclonal serum from the T-I mouse used for fusion shows that it identifies antigens in different regions of the sperm, such as the acrosome, postacrosome, equator, midpiece, and tail. This indicates that epididymal proteins are located on different regions of the sperm and are likely to play domain-specific roles such as sperm-egg interaction, acrosome reaction, and motility, which are essential for fertilization. Similar observations have been made by various research groups (Orgebin-Crist, 1967; Horan and Bedford, 1972; Dyson and Orgebin-Crist, 1973; Olson et al, 1987; Mathieu et al, 1992; Haidl et al, 1993; Hayashi et al, 1996; Batova et al, 1998; Jaiswal and Majumder, 1998; Smithwick and Young, 1999). It was interesting to note that the pattern of IIF localization was identical in both gluteraldehyde-fixed spermatozoa smeared on glass slides and in spermatozoa in suspension. This observation, along with the agglutination pattern, indicates that the proteins identified by the mabs are on the surface of sperm. Surface localization of sperm antigens is one of the criteria for an ideal contraceptive target. Targeting the epididymis for contraception has some definite advantages. First, the onset of infertility (and its reversal) is far quicker than any agent attacking the testicular production of spermatozoa. Second, because maturing cells are targeted, damage to the genetic material, a possible sequelae of its effect on dividing germ cells, is avoided (Hinton, 1980).
Western blot analysis indicated that all of the mabs identified proteins in approximately the same range, 27 kDa. This points to the antibodies identifying either different epididymal antigens of an approximately similar molecular weight or different epitopes of the same antigen with different regional localization on sperm. Our data show that all the antigens identified by different mabs are synthesized in the supranuclear region of the distal caput, followed by maximum synthesis in the corpus epithelium. The immunohistochemical localization using different mabs indicates that the protein(s) are synthesized and secreted mainly in the corpus region. However, mabs V2C4F2 and V3F4F4 also localized proteins in the caudal epithelium. The presence of the protein on the spermatozoa in corpus and cauda may be due to its secretion mostly by the principal cells of the corpus and, to some extent, by the caudal epithelium. The principal cells of the corpus have a well-developed endoplasmic reticulum and Golgi apparatus, and these cells have been shown to be actively involved in protein synthesis (Flickinger, 1979, 1981) and physiological functions of the epididymis involving endocytosis (Hermo et al, 1998) and secretion (Legare et al, 1999). The coating nature of the antigens is further substantiated by the surface immunofluorescent localization on the sperm and the agglutinating nature of the mabs. Similar immunoreactivity has been seen in case of MEP7, AEG, protein D and E, and PES (Rankin et al, 1992). Looking at its region-specific synthesis and localization on caudal sperm, we feel that this protein may have some role in the posttesticular maturation of spermatozoa.
In conclusion, neonatal tolerization followed by hybridoma has definitely increased the chance by many fold for obtaining mabs to epididymis-specific proteins. All of the mabs identified proteins of a similar molecular weight, as seen by Western blot analysis; however, IIF localization showed regional variations. We also saw regional differences in IHC localization that used different mabs. Surface localization of the antigens and epididymal specificity indicates that these proteins are likely to play a major role in sperm maturation and certainly need to be pursued in greater details. We have, therefore, undertaken a study on epitope analysis using the different mabs, as well as 2-dimensional Western blot using these mabs. Further microsequencing of the proteins identified by each of the mabs would enable us to delineate the similarity/dissimilarity of these proteins.
S.A.J. thanks the Board for Research in Nuclear Sciences (Department of Atomic Energy) for a Senior Research Fellowship during the tenure of this work. We thank Dr C. P. Puri, Director, National Institute for research in Reproductive Health for his encouragement and interest in the work.
Supported by grant 99/37/19/BRNS cell from the Board for Research in Nuclear Sciences (Dept of Atomic Energy), Government of India.