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

Naz, Rajesh K.; Bradford, Carol R.; Menge, Alan C.

doi:10.3109/01485018408987528

Cited by 9 publications

(3 citation statements)

References 32 publications

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“…Forty-two days after the immunization, the rabbit sera recognized on average 12.6 Ϯ 1.2 bands by Western blot. This number is close to the number of alloantigens obtained (14 to 18) after immunization of gilts with pig membrane sperm protein (Haden et al, 2000), and superior to the number of bands (4 to 9) obtained after immunization of rabbits with sperm isoantigens extracted with several detergents (Naz et al, 1984). Although different detergents were used, it is probable that sperm heteroantigens are more numerous than isoantigens.…”

Section: Discussionsupporting

confidence: 66%

Partial Characterization of Antigenic Sperm Proteins in Foxes (Vulpes vulpes)

VERDIER,

ROUET,

ARTOIS

et al. 2002

Journal of Andrology

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The aim of this work was to identify antigenic proteins on fox spermatozoa. Fox spermatozoa proteins were injected into 3 female rabbits and into 3 male and 3 female foxes. In rabbits, a rapid humoral response was observed. Using rabbit sera for Western blotting, 23 fox sperm protein bands were recognized between 10 and 110 kd. In foxes, the time course of antibody response was studied in the same manner. The number of recognized bands was maximal on day 75 for 2 foxes, on day 90 for 3 foxes, and on day 120 for 1 fox. Western blot patterns varied from one fox to another. On the whole, 25 protein bands between 10 and 110 kd were recognized. Using fluorescein isothiocyanate (FITC) labeling on fox spermatozoa with rabbit and fox sera, we showed that several antigens recognized by the antisera were located at or near the surface of the spermatozoa. By two‐dimensional electrophoresis and gel‐purification, we have selected 6 highly antigenic proteins with molecular weights of 11.4, 14.7, 16.4, 16.4, 16.8, and 16.9 kd, and isoelectric points of 6.0, 6.0, 6.2, 5.5, 5.3, and 5.8, respectively, and one antigenic protein at 97 kd with an isoelectric point of 4.3 to 4.6. The results of this study can be used to characterize these 7 antigens selected more precisely by microsequencing or mass spectrometry.

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Section: Discussionsupporting

confidence: 66%

Partial Characterization of Antigenic Sperm Proteins in Foxes (Vulpes vulpes)

VERDIER,

ROUET,

ARTOIS

et al. 2002

Journal of Andrology

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“…The testes tissue, homogenized in 0.05 M Tris-HCl, was solubilized with detergent sodium deoxycholate (DOC) or with a milder chaotropic agent, lithium diiodosalicylate (LIS) (9 The development and characterization of the monoclonal antibody MA-24 has been reported (8). The IgG fraction of ascites fluid, prepared either by stepwise precipitation with sodium sulfate (10) or by using a protein A-Sepharose 4B column (Pharmacia), was coupled to CNBr-activated Sepharose 4B (11).…”

mentioning

confidence: 99%

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

Naz¹,

Phillips²,

Rosenblum³

1986

Proc. Natl. Acad. Sci. U.S.A.

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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-...

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“…The presence and titers of antibodies in the sera or uterine fluids were detected by the enzyme-linked immunosorbent assay (ELISA) using GA-1 as the antigen coated on plates as described earlier (Naz et al, 1983), the gelatin agglutination technique (Kibrick et al, 1952), the sperm immobilization technique (Isojima et al, 1968), indirect immunofluorescence technique (Menge and Peegel, 1980) and Western blot enzyme immunobinding procedure (Naz et al, 1985). Antisera with ELISA titers of __< 1600 were examined for their effect on in vitro development of early embryos and in artificial insemination of rabbits.…”

Section: Analyszs Of Seramentioning

confidence: 99%

Reduction of fertility in female rabbits and mice actively immunized with a germ cell antigen (GA-1) from the rabbit

Naz

Poffenberger

Menge

1986

Journal of Reproductive Immunology

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Female rabbits and mice were actively immunized against germ cell antigen (GA-1) of 63 kDa molecular mass isolated from rabbit sperm and testis. There was a significant (P less than 0.05) reduction of fertility in rabbits actively immunized with GA-1 as compared to controls, as seen by the percentage of 9-day implants/corpora lutea ratio (GA-1, 36.3%; controls, 85.7%). In mice, there was again a significant (P less than 0.01) reduction in fertility as seen by mean 7-9 day implants +/- S.D. per mated mouse actively immunized with GA-1 whether through the intraperitoneal route (GA-1, 1.2 +/- 1.6; controls, 8.0 +/- 3.4) or through the subcutaneous/intramuscular route (GA-1, 3.8 +/- 3.4; controls, 10.1 +/- 3.9). The antisera from these actively immunized animals were negative for sperm agglutinating and immobilizing antibodies. In the Western blot enzyme-immunobinding procedure, the antisera showed specific binding to a single protein of 63 kDa. The incidence of fertilization of eggs recovered from rabbits inseminated with anti-GA-1 antibodies-treated sperm was not significantly different from control rabbits. The percentage of fertilized eggs obtained from rabbits inseminated with anti-GA-1 antibodies-treated sperm that reached the blastocyst stage upon in vitro incubation, however, was significantly less than that for embryos obtained from rabbits inseminated with control serum-treated sperm. Incubation of normal fertilized eggs in vitro with the antibodies did not affect development. Neither antiserum nor immune uterine fluid reacted with 4-day blastocysts in the indirect immunofluorescence technique. It is concluded that active immunization with GA-1 results in post-fertilization reduction of fertility in rabbits and mice by inhibiting early embryonic development.

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

Cited by 9 publications

References 32 publications

Partial Characterization of Antigenic Sperm Proteins in Foxes (Vulpes vulpes)

Partial Characterization of Antigenic Sperm Proteins in Foxes (Vulpes vulpes)

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

Reduction of fertility in female rabbits and mice actively immunized with a germ cell antigen (GA-1) from the rabbit

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