CYCLIC-AMP REVERSAL OF HYPOXANTHINE-ARRESTED PREIMPLANTATION MOUSE EMBRYOS IS EDTA-DEPENDENT

Hypoxanthine can block preimplantation mouse embryo development in vit ro 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 hypoxan thine-induced arrest is determined by the culture conditions and strai n of mouse. Whitten's and KSOM/AA are two embryo culture media that su pport 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 t est the hypothesis that reversal of the hypoxanthine block by cAMP-ele vating 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/A A; furthermore, removal of EDTA from KSOM/AA rendered this medium inca pable of supporting high levels of development to blastocyst (9%), whe reas high numbers of blastocysts (80%) formed in Whitten's medium, whi ch does not contain the chelator. Consequently, Whitten's medium was u sed to test our hypothesis. It has previously been demonstrated that t he phosphodiesterase inhibitor, IBMX, can reverse the developmental ar rest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt s olution, but in the present study the addition of IBMX to Whitten's me dium resulted in a block to development and failed to reverse the hypo xanthine arrest. These disparate effects can be explained by the prese nce or absence of EDTA. Supplementing Whitten's medium with EDTA rever ses the IBMX effect, but not the hypoxanthine-induced block. While IBM X alone is unable to reverse the hypoxanthine block in Whitten's mediu m, development is greatly enhanced by the simultaneous addition of EDT A and IBMX. Similar results were obtained with the cAMP analogue, 8-AH A-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is criticall y dependent on the presence of EDTA. We contrast this with the situati on in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.