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

哺乳动物精子在交配和受精之间必须经历成熟的变化。这些生化和功能的改变，统称为获能，发生在精子穿越雌性生殖道。我们和其他实验室以前的研究已经在几种哺乳动物中鉴定了输卵管特异性糖蛋白（OGP）。尽管该糖蛋白已被报道与输卵管中的卵母细胞相关，但其在受精过程中的生理意义仍然不清楚。该项目的目标是：1）鉴定和分离小鼠OGP的基因，2）表征该基因的结构以供进一步研究，包括生产OGP“敲除”小鼠。用小鼠OGP cDNA作为探针筛选在噬菌体载体pMDaFIX II中由小鼠胚胎干细胞（TT 2）构建的基因组文库（Sendai等，1995，Biol. Reprod. 53：285-294）。基于从该分子的cDNA克隆获得的数据制备几种引物。用长链PCR法或以分离的克隆为模板直接扩增基因组DNA。将PCR产物亚克隆到pBluescript质粒中进行序列分析。OGP基因由12个外显子组成，分布在基因组上约13 kbp处。对应于富含Ser/Thr重复结构的基因位于外显子10。从基因组克隆以及从针对小鼠基因组DNA的反向PCR方法获得的序列数据显示，OGP基因没有典型的TATA盒或GC盒结构。然而，它的启动子区含有三个GGTCA（半回文雌激素反应元件）。这些数据将有助于阐明糖蛋白的生理意义，或使用同源重组进行生殖系统中糖蛋白的功能分析。

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.