Isolation and characterization of a human sperm antigen gene h-Sp-1.
We isolated and characterized a human sperm antigen gene (h-Sp-1) from human testis complementary DNA using antiserum against the human sperm membrane. Northern blot analysis detected two transcripts (2.3 and 1.1 kb) of the h-Sp-1 gene. The 2.3-kb transcript is ubiquitous, whereas the 1.1-kb transcript is specific to the human testis with a high level of expression. Determination of the base sequence of h-Sp-1 showed a size of 2170 bp and 43.4% homology with human synaptophysin. The base sequence indicates a molecule consisting of 259 amino acids, with four hydrophilic and four hydrophobic regions. In order to further characterize the h-Sp-1 molecule, we synthesized the probable region of amino acids with high antigenicity based on the amino acid sequence (amino acid nos. 174-198) and immunized rabbits to prepare an antiserum. In our experimental model of fertilization between human sperm and zona pellucida-free hamster ova, partial inhibition of fertilization was observed. We were able to synthesize a large quantity of recombinant protein by inserting the h-Sp-1 gene into a baculovirus vector and infecting spodoptera frugiperda culture cells (sf9 insect cells). The synthesized protein had a molecular weight of 30 kDa. We then immunized Balb/c mice with this protein to prepare a monoclonal antibody (G3G9), which was used to localize the h-Sp-1 molecule in sperm and tissues (e.g. testis). The h-Sp-1 molecule was present in the cell membrane from the head to tail of human sperm. Staining of the testis and epididymis also showed h-Sp-1 to be present in spermatogonia, spermatocyte, sperm and epididymal duct epithelium. These findings suggest that the h-Sp-1 molecule is expressed in sperm and testes and plays a role in fertilization.
Kanazawa
Ri-Ichiro
R
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan.
Komori
Shinji
S
Sakata
Kazuko
K
Tanaka
Hiroyuki
H
Sawai
Hideaki
H
Tsuji
Yoshiyuki
Y
Koyama
Koji
K
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005306:Fertilization / Q000187:drug effects; D019204:GTP-Binding Proteins; D005822:Genetic Vectors; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D013094:Spermatozoa / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D013194:Staining and Labeling; D016708:Synaptophysin / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D007106:Immune Sera; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016708:Synaptophysin; D004247:DNA; D019204:GTP-Binding Proteins; C483942:SPAG1 protein, human