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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD030302-01
Application #
3331651
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1993-04-01
Project End
1997-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Knudsen, T B (2000) HPLC-based mRNA differential display. Methods Mol Biol 136:345-9
Blackburn, M R; Wubah, J A; Chunn, J L et al. (1999) Transitory expression of the A2b adenosine receptor during implantation chamber development. Dev Dyn 216:127-36
Ibrahim, M M; Razmara, M; Nguyen, D et al. (1998) Altered expression of mitochondrial 16S ribosomal RNA in p53-deficient mouse embryos revealed by differential display. Biochim Biophys Acta 1403:254-64
Wubah, J A; Ibrahim, M M; Gao, X et al. (1996) Teratogen-induced eye defects mediated by p53-dependent apoptosis. Curr Biol 6:60-9
Blackburn, M R; Kellems, R E (1996) Regulation and function of adenosine deaminase in mice. Prog Nucleic Acid Res Mol Biol 55:195-226
Blackburn, M R; Datta, S K; Wakamiya, M et al. (1996) Metabolic and immunologic consequences of limited adenosine deaminase expression in mice. J Biol Chem 271:15203-10
Ibrahim, M M; Weber, I T; Knudsen, T B (1995) Mutagenesis of human adenosine deaminase to active forms that partially resist inhibition by pentostatin. Biochem Biophys Res Commun 209:407-16
Puffinbarger, N K; Hansen, K R; Resta, R et al. (1995) Production and characterization of multiple antigenic peptide antibodies to the adenosine A2b receptor. Mol Pharmacol 47:1126-32
Blackburn, M R; Wakamiya, M; Caskey, C T et al. (1995) Tissue-specific rescue suggests that placental adenosine deaminase is important for fetal development in mice. J Biol Chem 270:23891-4
Gao, X; Blackburn, M R; Knudsen, T B (1994) Activation of apoptosis in early mouse embryos by 2'-deoxyadenosine exposure. Teratology 49:1-12