Coculture of preimplantation embryo with an immortalized human oviductal epithelial cells

我们尝试建立了人输卵管上皮永生化细胞系。反复实验后不可能获得永生细胞。我们也用胚胎植入前进行了实验。我们采用逆转录聚合酶链反应（RT-PCR）检测了成纤维细胞生长因子(FGF) -2和FGF受体1 (FGFR-1) mRNA在小鼠胚胎和子宫内膜上皮细胞中的表达。小鼠胚胎中未检测到fgfr -2 mRNA，但在囊胚期有FGFR-1 mRNA表达。这些结果提示FGFR-1在胚胎着床前发育中的重要性。为了研究FGFR-1表达的机制，我们绘制了小鼠FGFR-1基因5'侧编码外显子和内含子的排列图谱。在外显子1和2之间鉴定出一个大的14kb内含子。观察到所有FGFR-1亚型都是通过替代应用产生的。此外，我们还鉴定了该基因的一个转录元件。基础启动子元件定位在-89 ~ -43的5′侧区。DNase I保护实验和凝胶移位分析表明，一个核蛋白可以结合到-62到-42的核苷酸序列上。综上所述，女性生殖道产生的FGF-2可能对着床前胚胎具有旁分泌作用。FGFR-1基因具有独特的基因组结构。从-62到-42的5'侧区域在FGFR-1基因表达中起关键作用。

We have tried establish an immortalized cell line of human tubal epithelium. It is not possible to obtain immortalized cells after repeated experiments. We also have performed experiments using preimplantation embryos. We determined the expression of fibroblast growth factor (FGF) -2 and the FGF receptor 1 (FGFR-1) mRNA in the mouse embryos and in epithelial cells of the uterine endometrium using reverse transcription-polymerase chain reaction (RT-PCR). No FGF-2 mRNA was detected in mouse embryos but FGFR-1 mRNA was expressed at the blastocyst stage. These results suggest importance of FGFR-1 in preimplantation development of embryo. To examine the mechanism of FGFR-1 expression, the arrangement of exons and introns encoding 5'-side of murine FGFR-1 gene was mapped. A large intron with a size of 14kb was identified between exon 1 and 2. All subtypes of FGFR-1 was obserbed to be generated through alternative aplicing. Furthermore, we characterized a transcription element of this gene. The basal promoter element was mapped to the 5'-flanking region from -89 to -43. The DNase I protection assay and gel shift analysis revealed that a nuclear protein could bind to the nucleotide sequence from -62 to -42. In conclusion, FGF-2 produced by the female reproductive tract may exert paracrine effects on preimplantation embryos. FGFR-1 gene has an unique genomic structure. The 5'-flanking region from -62 to -42 plays a pivotal role in FGFR-1 gene expression.