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From the Hybridoma Division, National Institute for Research in Reproductive Health, Mumbai, India.
| Correspondence to: Vrinda V. Khole, Hybridoma Division, National Institute for Research in Reproductive Health, J. M. Street, Parel, Mumbai, 400 012 (fax: 22-2413-9412; e-mail: hybridoma{at}rediffmail.com). |
| Received for publication October 8, 2002; accepted for publication January 27, 2003. |
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Abstract |
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Key words: Neonatal tolerization, epididymis, monoclonal antibodies
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.
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Materials and Methods |
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Reagents
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 x 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 x
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 x 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.
Immunization
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.
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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 x 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
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).
IIF
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 x 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.
Immunohistochemistry (IHC)
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.
Agglutination
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 x 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 5x dark-field microscope.
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Results |
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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.
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IIF
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.
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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).
ELISA
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).
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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).
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IHC
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.
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Agglutination
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.
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Discussion |
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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.
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Acknowledgments |
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Footnotes |
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