Epididymal secretions are critical for mammalian spermatozoa to acquire both forward motility and an ability to recognize and penetrate oocytes. Previous studies identified two glycoproteins, GP-83 and GP-39, which were secreted by the human epididymis and may be related to maturation of sperm function. In this study, GP-83 was purified from human seminal fluid by DEAE-ion exchange, gel filtration chromatography and preparative gel elution. The isoelectric point (pI) of purified GP-83 was 6.57. Monospecific antiserum to GP-83 was induced in male New Zealand rabbits and confirmed on immunoblots. GP-83 was found in fluid, tissue and sperm extracts of corpus and cauda epididymis, but not in the caput. Immunohistochemical localization identified GP-83 in the luminal contents and in the supranuclear region and cell membrane of principal cells of the corpus and cauda epididymis. GP-83 was found on the anterior acrosome in ejaculated spermatozoa, and shifted to the equatorial region after capacitation and the acrosome reaction.
A tricistronic gene mapped between 0.91 and 0.93 map units within the EcoRI D fragment of the human cytomegalovirus unique short region (Us) has been cloned, sequenced, and expressed in vitro. Cloned cDNAs of 2.3, 1.8, and 1.1 kb derived from this region were isolated from a Agtll cDNA library made from virus-infected fibroblasts and used for this study. Two major classes of 3'.coterminal mRNAs, 2.8 and 1.1 kb, were transcribed from this region. Sequence analysis of the cDNAs and the upstream genomic DNA revealed three open reading frames (ORFs), Us18, Us19, and Us20, and a common polyadenylation signal located 15 bases upstream of the poly(A) tail of both the 2.85-and 1.1-kb mRNAs. Protein structure analyses predicted the existence of multiple hydrophobic moieties, suggesting that the Us18, US19, and Us20 polypeptides were transmembrane proteins. The major transcription initiation site, determined by primer extension and S1 nuclease mapping, for the 2.85-kb transcript was located right at the first initiation codon of the Us20 ORF. There was no typical TATA box or CAAT box upstream of the 2.85-kb mRNA cap site except for a TATAAGA sequence that was found about 210 bp downstream from the major cap site. The 1.1-kb transcript was initiated 33 bp upstream of the Us18 translation initiation site, and an atypical TATA box sequence (GATAAGA) was found 22 bp upstream of the transcription start site. Differences in transcription kinetics and sensitivities to metabolic inhibitors suggest that they were regulated by different mechanisms; the 2.85-kb mRNA belongs to the early (13) class of transcripts, while the 1.1-kb mRNA is a late ('y) message. Subgenomic DNA segments derived from the Us18, Us19, and Us20 ORFs were subcloned and overexpressed in Escherichia coli as fusion proteins with glutathione-s-transferase. Western immunoblot analysis with antibodies against the Us18, Us19, and Us20 fusion proteins detected virus-specific polypeptides with molecular sizes of 36, 32, and 43 kDa, respectively. All three antibodies also exhibited a positive immunofluorescence reaction with human cytomegalovirus-infected cells harvested at late stages of infection. Human cytomegalovirus (HCMV) is the fourth herpesvirus to be sequenced and has the greatest genomic complexity among the human viruses discovered to date (3). The viral genome is a double-stranded DNA 230 kb in length. More than 200 open reading frames (ORFs) can be derived from this viral genome (3). Like that of herpes simplex viruses, the HCMV genome is composed of a unique long (UL) and a unique short (Us) segment, each bounded by terminal repeats that permit UL and Us inversions to form four isomeric molecules (9, 13, 14). The HCMV Us region is very different from that of other human herpesviruses. The 38-kb Us region of HCMV contains at least six families of homologous ORFs, and except for the G protein-coupled receptor (GCR) family, none of these show homology to other known human herpesviruses ORFs (25). Each family contains 2 to 13 members or ORFs which occur either...
Mammalian spermatozoa interact with the proteins secreted by the epididymis to develop fertility. Transmembrane proteins that possess a disintegrin and metalloprotease (ADAM) domains are shown to be closely related to spermatogenesis and fertilization. Our previous study demonstrated that GP-83, a glycoprotein secreted by the epididymis, was conjugated to mature sperm. In this study, a 2.1-kilobase (kb) GP-83-expressing insert was isolated from a cDNA library of human epididymis by immunoscreening using GP-83-specific antiserum. The 5' end rapid amplification of cDNA ends (RACE) and 3'-RACE of the 2.1-kb insert elucidated two isoforms of GP-83-encoding cDNA sequences, an alpha-form of 3451 base pairs (bp) and beta-form of 2643 bp. Both forms exhibit the same open reading frame of 2262 bp predicting a peptide of 754 amino acid residues. Deduced amino acid sequence revealed signal sequence, prodomain, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. The GP-83-encoding sequence was recognized as human ADAM7 due to significant homology to other ADAM7s. According to the DNA sequences elucidated in the Human Genome Project, h-ADAM7 was located at chromosome 8p22. Ex vivo expression confirmed that h-ADAM7 cDNA did encode GP-83. Northern blot analysis revealed two transcripts of 4 kb and 3 kb in the epididymis, but not in testis or other major tissues. These results indicate that the GP-83-encoding gene is a human epididymis-associated ADAM7 gene (human ADAM7, h-ADAM7) and may be involved in the sperm-egg interaction.
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