Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors.
The culture conditions under which hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of D-glucose. The action of hypoxanthine plus D-glucose was reversed by glutamine and higher lactate. D-mannose substituted for D-glucose in supporting the inhibitory action of hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 microM, and guanosine was inhibitory at higher doses. Assays of hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of hypoxanthine or D-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of D-glucose, hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to D-glucose. D-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of hypoxanthine to block embryo development at the two-cell stage depends on the presence of D-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.
Downs
S M
SM
Biology Department, Marquette University, Milwaukee, Wisconsin 53233.
Dow
M P
MP
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002241:Carbohydrates / Q000494:pharmacology; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors; D007042:Hypoxanthines / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D011247:Pregnancy; D011687:Purines / Q000494:pharmacology
D002241:Carbohydrates; D003470:Culture Media; D007042:Hypoxanthines; D011687:Purines; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase; D005947:Glucose