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 immature mice 48 h after priming with 5 IU eCG and culture for 3 h in medium containing 14C-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercaptopurine. Tissue extracts from complexes were analyzed by HPLC using either a C18 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 inosine monophosphate (IMP) and then converted to nucleotides. Metabolism favored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encountered, 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 the synthesis of adenyl nucleotides in complexes and partially shunted metabolism in the direction of guanyl nucleotides. However, neither alanosine nor another inhibitor of adenylosuccinate synthetase, hadacidin, significantly influenced the meiotic arrest maintained by hypoxanthine. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotides but did not appreciably affect metabolism to other nucleotides. 6-Mercaptopurine produced an increase in the hypoxanthine-containing peaks, which was consistent with suppression of purine salvage. These results demonstrate that hypoxanthine is readily salvaged by the murine oocyte-cumulus cell complex and that the inhibitor-induced changes in metabolism 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 during the culture period is apparently not required for hypoxanthine-maintained meiotic arrest in vitro.
Downs
S M
SM
Biology Department, Marquette University, Milwaukee, Wisconsin 53233.
D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism* / Q000494:pharmacology; D007288:Inosine / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D008540:Meiosis / Q000187:drug effects; D015122:Mercaptopurine / Q000494:pharmacology; D051379:Mice; D009173:Mycophenolic Acid / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D011687:Purines / Q000494:pharmacology*; D014527:Uric Acid / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D007042:Hypoxanthines; D011687:Purines; D007291:Inosine Monophosphate; D014527:Uric Acid; C000082:alanosine; D019271:Hypoxanthine; D007288:Inosine; D015122:Mercaptopurine; D014969:Xanthine Oxidase; D011683:Purine-Nucleoside Phosphorylase; D000265:Adenylosuccinate Synthase; D009173:Mycophenolic Acid; D000409:Alanine