HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ANALYSIS OF HYPOXANTHINE METABOLISM IN MOUSE OOCYTE-CUMULUS CELL COMPLEXES - EFFECTS OF PURINE METABOLIC PERTURBANTS

This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from imm ature mice 48 h after priming with 5 IU eCG and culture for 3 h in med ium containing C-14-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercapt opurine. Tissue extracts from complexes were analyzed by HPLC using ei ther a C-18 reversed-phase column (for separation of purine bases and nucleosides) or an ion exchange column (for separation of nucleotides) . Most of the hypoxanthine taken up by complexes was salvaged to inosi ne monophosphate (IMP) and then converted to nucleotides. Metabolism f avored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encounte red, and metabolism to inosine via purine nucleoside phosphorylase was negligible. A similar pattern of hypoxanthine metabolism was observed in extracts of oocytes that had been denuded after the culture period . Addition of alanosine to the culture medium significantly reduced th e synthesis of adenyl nucleotides in complexes and partially shunted m etabolism in the direction of guanyl nucleotides. However, neither ala nosine nor another inhibitor of adenylosuccinate synthetase, hadacidin , significantly influenced the meiotic arrest maintained by hypoxanthi ne. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotid es but did not appreciably affect metabolism to other nucleotides. B-M ercaptopurine produced an increase in the hypoxanthine-containing peak s, which was consistent with suppression of purine salvage. These resu lts demonstrate that hypoxanthine is readily salvaged by the murine oo cyte-cumulus cell complex and that the inhibitor-induced changes in me tabolism are consistent with the presumed mechanism of action of each inhibitor. In addition, whereas metabolism favors conversion of IMP to adenyl nucleotides, synthesis of adenyl nucleotides by this route dur ing the culture period is apparently not required for hypoxanthine-mai ntained meiotic arrest in vitro.