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

Abstract

A fertilization antigen, FA-1, was purified from either deoxycholate-or lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography using a monoclonal antibody, MA-24, which inhibited fertilization in vitro. The FA-1 was recovered at high (11.4) or low (2.8) pH using stepwise elution procedures of the deoxycholate or lithium diiodosalicylate extracts, respectively. Both of these fractions showed a single band of 47 kDa when analyzed by NaDodSO4/PAGE and silver staining. Following removal of the detergent and extensive dialysis at pH 5.8 or treatment with 0.15 M NaCl, even in the presence of detergent, a monomer of 23 kDa was detected. Two-dimensional PAGE of FA-1 showed, four or five polypeptides in the 47-kDa or 23-kDa range. The dialyzed FA-1 contained a major 23-kDa and a minor 48-kDa band when separated on both sucrose and cesium chloride gradients. High performance size-exclusion chromatography showed a major peak at 23 kDa and a minor peak at 50 kDa. Further analysis of the 23-kDa peak by reverse-phase chromatography resolved the antigen into three peaks, which gave similar two-dimensional gel patterns as the native FA-1. Lectin affinity chromatography on a lens culinaris column demonstrated that a part of the antigen was bound to the lectin while the rest was not. The FA-1 revealed a positive reaction with periodic-Schiff reagent and contained glucose and mannose, which together constituted 18.8% of the total antigen mass. Amino acid analysis showed a high percentage of aspartic acid, glutamic acid, serine, and glycine. As a single injection of MA-24 significantly reduced fertilization rates in vivo, the purified FA-1 is an attractive candidate for the development of contraceptive vaccine.Immunization of male and female animals of various species with extracts of sperm or mature testes results in a significant inhibition of fertility (1, 2). The whole sperm or testes cannot be used for immunoregulation of fertility due to the presence of numerous antigens on the germ-cell surface that could be shared with other somatic tissues (3,4). The utility of any antigen as a contraceptive vaccine is contingent upon its tissue-specificity, its involvement in fertilization and fertility, and its homogeneity. A few sperm-specific antigens have been purified to homogeneity and characterized, and some of them are relevant to fertility. Immunization with lactate dehydrogenase-X (LDH-C4) resulted in inhibition of fertility (5, 6), and antiserum to rabbit sperm autoantigen (RSA-1) also showed a reduction in fertility (7).We reported a monoclonal antibody, MA-24, developed against human spermatozoa that inhibited in vitro fertilization of murine oocytes by mouse sperm and zona-free hamster ova penetration by human sperm without agglutinating or immobilizing spermatozoa (8). MA-24 was directed against the 23-kDa antigen of human germ-cell plasma membranes, and the fertilization antigen FA-1 was isolated using immunoaffinity chromatography from human testes and sperm. Interestingly, FA-1 cross-...

Antiidiotypic antibodies to sperm in sera of fertile women that neutralize antisperm antibodies.

“…1 and 6) or infertile women (nos. 27-29) were incubated (overnight at 40C) with either the antiFab' or control Fab' CNBr-activated Sepharose 4B immunoadsorbent columns (7,8). After washing the column at pH 8 (0.1 M phosphate buffer), the bound Fab's were eluted at pH 2.8 (0.1 M glycine, HC1 buffer) (7,8).…”

“…FA-I was purified from lithium diiodosalicylate (LIS)-solubilized human testes by immunoaffinity chromatography using MA-24 monoclonal antibody (7,8). Each batch of FA-I was tested for its homogeneity.…”

“…The hybridomas secreting FA-l monoclonal antibody (MA-24) were injected into pristane-sensitized BALB/c mice to obtain ascites fluid (7,8). The monovalent (Fab') antibodies were prepared from the ascites fluid (as described below), affinity purified using protein A-Sepharose 4B column, and then immunoaffinity purified using FA-l antigen in an immunocolumn (described below).…”

“…The anti-FA-l Fab' and control Fab' were covalently coupled to CNBr-activated Sepharose-4B beads (Sigma Chemical Co.) by a procedure described elsewhere (7,8).…”

“…We have isolated and characterized a sperm-specific glycoprotein, the fertilization antigen (FA-l ),' from human and murine male germ cells that seems to have a role in human fertilization (7)(8)(9). Interestingly, sera from infertile women and not from fertile women react strongly with FA-1, indicating its involvement in involuntary infertility in humans (10,11 ).…”