Inhibition of Fertility in Rabbits by Monoclonal Antibodies Against Sperm

Abstract

Monoclonal antibodies were prepared against rabbit sperm antigens by fusing P3-X63-Ag8-653 mouse myeloma cells with lymphocytes from Balb/c mice immunized with Tergitol NP-40 detergent-solubilized rabbit epididymal sperm. Ascites fluid from mice injected with two of these hybridomas (8C4.1 and 8C10.5) was negative in immobilization and agglutination methods, however, acrosome positive on methanol fixed sperm and plasma membrane positive on unfixed sperm in indirect immunofluorescence. Insemination of female rabbits with the sperm treated with either of these monoclonal antibodies resulted in significant reduction in fertility as seen by the percentage of 9-day implants/corpora lutea ratio (8C4.1, 25.7%; 8C10.5, 1.9%; and control, 64.7%). Though the antibodies inhibited in vitro binding of the rabbit sperm with zonae pellucidae of rat ova, fertilization in vivo was not affected significantly. The antibodies did not demonstrate antiblastocyst activity by immunofluorescence. Both of these monoclonal antibodies appeared to recognize the same antigen by the SDS-PAGE/Protein Blot enzyme immunobinding procedure. The antigen was of testicular origin and had a molecular weight of approximately 63,000 daltons. It is concluded that these monoclonal antibodies which were organ specific, block post-fertilization fertility by inhibiting some step necessary for viable embryo formation.

Isoantigenicity of Rabbit Sperm, Testis, and Their Extracts as Demonstrated by Western Blot Enzyme Immunobinding Procedure

“…For each antiserum, all the detergent extracts were tried; those showing the best results were selected for use. Why certain antisera do not react with a particular detergent extract is not clear; e.g., ATA and ASA react very weakly if at all with blots of DOC, LIS, or SDS testis extracts; in contrast, the monoclonal antibodies (8C4.1 and 8C10.5) react quite well with DOC testis extract, weakly with LIS extract, and not at all with NP-40 extract [22]. animals, but we did not find any significant variation within our small groups (n = 2-5).…”

“…We have demonstrated the feasibility of t h s procedure for antigenic analysis [22]. The objectives of the study reported here were to use the Western blot procedure (1) to study the antigenicity of the whole sperm, testicular preparation, LIS extract of sperm, and NP-40 extract of the sperm pellet and (2) t o discover the antigens of sperm, testis, and conceptus against which the immune response in the genital tract is generated after local immunization.…”

Involvement of fertilization antigen (FA-1) in involuntary immunoinfertility in humans.

“…12 of these sera were collected from seven infertile couples admitted to Western blot enzyme immunobinding procedure. Antigenic specificities ofthe antisera and monoclonal antibodies were evaluated by the Western blot enzyme immunobinding procedure described earlier (15,16). Briefly a -1-2.5 mg protein solution of DOC (15 mM in 0.05 M Tris-HCl, pH 8.0)-solubilized washed ejaculated human sperm membrane preparation (pooled from three or more donors) was run under nonreducing conditions in the slab SDS-PAGE (10% polyacrylamide gel) according to the procedure described by Laemmli (17).…”

“…Immediately after electrophoresis, the protein bands were electrophoretically transferred from the slab gel to nitrocellulose paper by the method of Towbin et al (18). The reacted antigens were localized by incubating the strips of blotted nitrocellulose paper with sera (1:50) diluted in incubation buffer (50 mM Tris HC1, 150 mM NaCl, 5 mM EDTA, 0.25% gelatin, 0.05% NP-40, pH 7.4), goat anti-rabbit or goat antimouse or goat anti-human gamma immunoglobulins conjugated to peroxidase (1:800), and finally with the substrate solution (0.001% p-chloro-l-naphthol in 0.05 M Tris HC1, pH 7.4, containing 0.03% H202) as described earlier (15,16).…”

Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine.

“…They were also investigated for their immunoblot reaction with DOC-solubilized murine testes. The immunoblot procedure (21) and the immunodetection of antigen bands followed the procedure described before (22). The (1986) (300 units/ml) and counting the ratio of fertilized ova to total ova recovered.…”