Functional analysis of oviduct-specific glycoproteins in the reproductive system using homologous recombination

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Mammalian spermatozoa must undergo maturational changes between the events of mating and fertilization. These biochemical and functional alterations, collectively termed capacitation, take place as spermatozoa traverse the famale reproductive tract. Previous studies from our and other laboratories have identified an oviduct-specific glycoprotein (OGP) in several mammalian species. Although the glycoprotein has been reported to associate with oocytes in the oviduct, its physiological significance in the fertilization process remains obscure. The objectives of the project were to : 1) identify and isolate the gene for mouse OGP,and 2) characterize the structure of the gene for further studies, including the production of an OGP "knock out" mouse. The genomic library constructed from the mouse embryonic stem cells (TT2) in thephage vector lambdaFIXII was screened with the mouse OGP cDNA as a probe (Sendai et al, 1995, Biol. Reprod. 53 : 285-294). Several primers were prepared based on the data obtained from the cDNA cloning of the molecule. The genomic DNA wa amplified directly by long PCR method or by using the isolated clone as a template. The PCR products were subcloned into pBluescript plasmid for sequence analysis. The OGP gene consisted of 12 exons distributed approximately 13 kbp on the genome. The gene corresponding to Ser/Thr-rich repeating structures was located on exon 10. The sequence data obtained from the genomic cloning as well as from the inverted PCR method directed against mouse genomic DNA,revealed that the OGP gene had no typical TATA box or GC box structures. However, it contained three GGTCA (half-palindromic estrogen response element) in its promotor region. These data will be useful in elucidating the physiological significance of the glycoprotein, or functional analysis of the glycoprotein in the reproductive system using homologous recombination.