Cyclic AMP reversal of hypoxanthine-arrested preimplantation mouse embryos is EDTA-dependent.
Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the hypoxanthine-induced block. While IBMX alone is unable to reverse the hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.
Biology Department, Marquette University, Milwaukee, Wisconsin 53201-1881, USA.
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Dienhart
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Downs
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D004357:Drug Synergism; D004492:Edetic Acid / Q000494:pharmacology*; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D005947:Glucose / Q000737:chemistry; D019271:Hypoxanthine / Q000494:pharmacology*; D008297:Male; D008540:Meiosis / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology
D003470:Culture Media; D010726:Phosphodiesterase Inhibitors; D019271:Hypoxanthine; C067297:8-aminohexylamino cAMP; D004492:Edetic Acid; D000242:Cyclic AMP; D005947:Glucose; D015056:1-Methyl-3-isobutylxanthine