ADA, ADENOSINE AND EMBRYO SURVIVAL

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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.