Monoclonal Antibody to a Human Germ Cell Membrane Glycoprotein That Inhibits Fertilization

Abstract

Immunization of male or female animals of different species with homogenates of mature sperm, testis, or their extracts results in infertility. Antigenicity of sperm also has been implicated in the involuntary infertility of humans. The advent of hybridoma technology has made it possible to identify and purify sperm-specific antigens that are involved in fertility and infertility. The present authors selected one (MA-24) of 40 putative clones, secreting antibodies to sperm, for detailed analysis. This clone was determined to be of the 2a subclass of immunoglobulin G (lgG2). It was negative in the agglutination and immobilization assays, two methods that were used to test for antibodies to sperm in the serum of humans with involuntary infertility. MA-24 was germ cell-specific and did not bind to lymphocytes, erythrocytes, fibroblasts, lymphoblastoid cells, K562 leukemia cells, or other somatic cells by immunofluorescence. The MA-24 clone showed binding to the postacrosome, midpiece, and tail of viable and methanol-fixed human spermatozoa by immunofluorescence. It reacted with a single 23-kD protein band when a detergent-solubilized membrane preparation of human testis was used. The tissue-specific, but not species-specific, MA-24 antibody to the 23-kD protein completely blocked binding and penetration of zona-free hamster ova by human sperm. MA-24 also significantly inhibited fertilization in vitro of mouse eggs by murine sperm. The antigen (FA-1) to which the MA-24 antibody is directed has been isolated from human ejaculated sperm and testis by immunoaffinity chromatography. Preliminary data implicated FA-1 in human involuntary immunoinfertility. Sera from infertile patients with immunological involvement showed a strong binding to the purified antigen in the enzyme-linked immunosorbent assay. The binding pattern was comparable to that seen with the 0.3 M lithium diiodosalicylate extract of human sperm, a soluble preparation reported to contain surface antigen acting in fertilization. This finding indicates that the FA-1 antigen is immunogenic in humans. The mechanism or mechanisms by which MA-24 inhibits fertilization are not clear. Since both binding and penetration are inhibited in fertilization systems in vitro, these antibodies may hinder interactions between sperm and ovum or receptor recognition. The block is not due to agglutination or immobilization of sperm. MA-24 did not bind to ova, which shows the sperm specificity of the antibody. In the murine fertilization assay performed in vitro, MA-24 inhibited fertilization only in ova with intact zonae, and not when zonae were removed, which indicates that the inhibition occurs during the interaction between sperm and zona. In the heterogametic assay, however, MA-24 completely blocked binding and penetration of human sperm in zona-free hamster ova, an assay that has been reported to measure capacitation indirectly. These antibodies may be directed to a sperm component (enzymatic or nonenzymatic) that is vital for capacitation.