Fertilization antigen-1: cDNA cloning, testis-specific expression, and immunocontraceptive effects

Abstract

cDNA encoding for a sperm antigen, designated fertilization antigen (FA-1), was cloned and sequenced from murine testis cDNA-lambdagt11 expression library using FA-1 mAb. Computer-generated translation analysis of 649-bp cDNA yielded an ORF of 164 amino acids with the first ATG Met start codon at nucleotide 81 and the stop codon TAA at nucleotide 577 and a polyadenylylation tail following the stop codon. The translated protein has a calculated molecular mass of 18.2 kDa and a potential N-linked glycosylation site at amino acids 158-160, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced amino acid sequence indicated it to be a membrane-anchored peptide. Extensive computer search in the GenBank, National Biomedical Research Foundation, and Swiss sequence banks did not identify any known nucleotide/amino acid sequence having homology with FA-1 cDNA or deduced amino acids, indicating it to be a novel protein. Northern blot analysis and reverse transcription-PCR indicated testis-specific expression of FA-1 antigen. The FA-1 cDNA was subcloned into pGEX-2T vector and expressed in glutathione S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with ZP3 of oocyte zona pellucida and its affinity-purified antibodies completely blocked sperm-zona pellucida interaction in mice. These findings suggest that the sperm-specific recombinant FA-1 antigen is an attractive candidate for the development of a contraceptive vaccine.

Figure 1

(A) Nucleotide (nt) and deduced amino acid (aa) sequences of FA-1 cDNA. The mouse testis cDNA-λgt11 expression library was screened with the FA-1 mAb. The positive immunoreactive clone containing 0.7-kb insert was eluted from λgt11 by EcoRI, subcloned into pBluescript II SK + at EcoRI site, and sequenced with dsDNA Cycle Sequencing System using T3 and T7 promoter primers. (B) Hydropathy plot of the deduced amino acid sequence of FA-1 cDNA. It was computed by using the Kyte–Doolittle (29) (solid line) and Engelman et al. (30) (dotted line) methods.

Figure 2

(A) Northern blot of various tissues mRNAs probed with FA-1 cDNA. Two micrograms of poly(A)⁺ RNA isolated from each tissue was separated on a 1.2% denaturing agarose/formaldehyde gel and transferred to nitrocellulose membrane. The membrane was prehybridized with QuickHyb solution, incubated (56°C, 2 hr) with ³²P-labeled FA-1 cDNA probe, washed, and exposed to x-ray film for 24 hr to 3 weeks. (B) After the membrane was stripped of the FA-1 probe, it was rehybridized (65°C, 2 hr) with ³²P-labeled β-actin probe, washed, and exposed as above.

Figure 3

RT–PCR pattern of various murine tissue RNAs. Two micrograms of whole RNA from each tissue, free of chromosomal DNA contamination, were reverse-transcribed in a total volume of 20 μl. Two microliters of the resulting cDNA was amplified by PCR for 30 cycles using the FA-1-specific or β-actin-specific primers. The FA-1-specific primer set was expected to amplify a 495-bp fragment. (A) Ethidium bromide-stained electrophoretic gel pattern of the PCR products amplified by using FA-1 primers. (B) Hybridization pattern of the amplified products with ³²P-labeled FA-1 cDNA on the Southern blot. The β-Actin-specific primer set was expected to amplify a 256-bp fragment. (C) Ethidium bromide-stained electrophoretic gel pattern of the PCR-amplified products. (D) Southern blot hybridization pattern with ³²P-labeled β-actin probe.

Figure 4

SDS/PAGE pattern of FA-1 recombinant protein and its immunoreactivity in the Western blot procedure. The subcloning of FA-1 cDNA into pGEX-2T vector at EcoRI site and the expression and purification of recombinant protein are described in Methods and Materials. Twenty to 40 μg of the nonreduced recombinant protein was run before (lane a) and after (lane b) treatment with 1.5 M NaCl (lane b) in SDS/PAGE and either stained with silver nitrate (A) or electrophoretically transferred to nitrocellulose membrane for Western blot analysis using FA-1 mAb (B) or myeloma control antibody (C). The glutathione S-transferase protein (20 μg) was used as control in these experiments (lane c).

Figure 5

Interaction between the recombinant FA-1 protein and ZP3 of oocyte zona pellucida. The solubilized-zona pellucida preparation from murine oocytes was separated in SDS/PAGE (5–15%), and then transferred to nitrocellulose membrane for Western blot procedure. The recombinant protein was biotinylated and separated from unbiotinylated protein following the manufacturer’s protocol. The Western blot was incubated with biotinylated protein (≈1.5 μg/ml), washed, allowed to react with streptavidin-horseradish peroxidase (1:1,500), washed again, incubated with substrate, and exposed to x-ray film for 1–5 min. The solubilized zona pellucida preparation showed three bands (ZP1, ZP2, and ZP3, respectively) in SDS/PAGE (lane a). The recombinant FA-1 protein specifically recognized ZP3 on the Western blot (lane b). The biotinylated BSA used as a control did not react with any band on the Western blot (lane c).