- Top of page
- Materials and Methods
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.
- Top of page
- Materials and Methods
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.