ADA, ADENOSINE AND EMBRYO SURVIVAL

这项建议的总体目标是阐明生理上的 腺苷脱氨酶在母体-胚胎界面的作用。 Ada是一种普遍存在的嘌呤代谢酶，高表达。 在子宫胎盘组织中。在小鼠中，发现高水平的ADA表达 在反子宫蜕膜(母体)和胎盘基底区 (胚胎期)。最近的研究结果表明，子宫内脱氨 腺苷和2‘-脱氧腺苷是ADA的天然底物， 对移植后早期小鼠胚胎的存活至关重要。 这个过程可以被核苷类似物阻断。 (R)-脱氧考福霉素(DCF)，一种有效的ADA抑制剂，其后果 这是胚胎在3到6小时内大量细胞死亡。这个 目前的拨款申请将决定关键角色是否 由抗子宫蜕膜丰富合成的母体ADA扮演 细胞或由基底层滋养层细胞大量合成的胚胎ADA 细胞。假设胚胎存活是严重依赖的。 在母体ADA降解细胞毒性嘌呤核苷(腺苷， 2‘-脱氧腺苷)，由蜕膜萎缩母细胞产生 交叉口。提出了五个具体目标。前三项要求 为验证这一假说而开发转基因小鼠模型的努力： (1)确定ADA基因调控元件 将高水平的报告基因表达导向抗子宫蜕膜 细胞；(2)诱变功能性人ADA互补DNA(CDNA) 保持催化活性的突变形式的序列 对DCF的抗性；以及(3)将耐药的cdna导入 小鼠ADA基因调控信号调控下的小鼠基因组 明确具体目标1.后两个具体目标将直接 检验移植后早期胚胎存活的中心假设 严重依赖于母体腺嘌呤核苷的脱氨基 在抗子宫肌层：(4)测定药物的急性影响 ADA代谢对内源性核苷、胚胎细胞的抑制作用 死亡和生存当母亲被赋予抗药性时， 胚胎，或两者兼而有之；以及(5)确定哪个内源核苷 (腺苷、脱氧腺苷)触发胚胎细胞死亡。这些研究 提供对细胞和分子事件的新见解 对宫内存活至关重要的一段时间内， 据估计，22%的人类妊娠失败，约占 三分之二的怀孕流产。

The overall objective of this proposal is to elucidate the physiological role of adenosine deaminase (ADA) at the maternal-embryonal interface. ADA is an ubiquitous enzyme of purine metabolism that is highly expressed in utero-placental tissues. In mice, high-level ADA expression is found in the antimesometrial decidua (maternal) and basal zone of the placenta (embryonal). Recent findings suggest that intrauterine deamination of adenosine and 2'-deoxyadenesine, which are the natural substrates of ADA, is essential for the survival of early postimplantation mouse embryos. This process can be blocked by the nucleoside analogue (R)-deoxycoformycin (dCF), a potent inhibitor of ADA, the consequence of which is massive cell death in the embryo within 3 to 6 hours. The present grant application will resolve whether the critical role is played by maternal ADA abundantly synthesized by antimesometrial decidual cells or by embryonal ADA abundantly synthesized by basal trophoblast cells. It is hypothesized that embryo survival is critically-dependent upon maternal ADA degradation of cytotoxic purine nucleosides (adenosine, 2'-deoxyadenosine) which are generated at the deciduatrophoblast junction. Five specific aims are proposed. The first three entail efforts to develop a transgenic mouse model for testing the hypothesis: (1) identify the ADA gene regulatory elements that are capable of directing high-level reporter gene expression to antimesometrial decidual cells; (2) mutagenize functional human ADA complementary DNA (cDNA) sequences to mutant forms that retain catalytic activity yet are resistant to dCF; and (3) introduce the drug-resistant cDNA into the mouse genome under control of the murine ADA gene regulatory signals identified in Specific Aim 1. The last two specific aims will directly test the central hypothesis that early postimplantation embryo survival is critically-dependent upon maternal deamination of adenine nucleosides in the antimesometrium: (4) determine the acute impact of pharmacological inhibition of ADA metabolism on endogenous nucleosides, embryonic cell death and survival when drug-resistance is conferred to the mother, the embryo, or both; and (5) determine which endogenous nucleoside (adenosine, deoxyadenosine) triggers embryonic cell death. These studies offer new insights into the cellular and molecular events which are fundamental to intrauterine survival during the immediate period that an estimated 22% of all human pregnancies fail, accounting for about two-thirds of pregnancy miscarriages.