INVOLVEMENT OF PURINE NUCLEOTIDE SYNTHETIC PATHWAYS IN GONADOTROPIN-INDUCED MEIOTIC MATURATION IN MOUSE CUMULUS CELL-ENCLOSED OOCYTES

This study was carried out to test the hypothesis that purine nucleoti de-generating pathways are required for ligand-stimulated oocyte matur ation in meiotically arrested cumulus cell-enclosed oocytes. Oocytes f rom hormonally primed, immature mice were cultured overnight in Eagle' s minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (t o maintain meiotic arrest, plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these co nditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, o r adenosine were included during the first 3 hr. Further more, preincu bation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The ph osphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, could not mimi c this protective effect of hypoxanthine. Azaserine and aminopterin, i nhibitors of purine de novo synthesis, blocked hormone-triggered matur ation in dbcAMP-arrested oocytes, but had little effect on hypoxanthin e-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that c an be taken up by cells and phosphorylated to form AICAR, which can en ter the purine de novo pathway at a point distal to the sites of azase rine inhibition. FSH was stimulatory to purine de novo synthesis, whil e azaserine, aminopterin, hypoxanthine, and AICA riboside all suppress ed de novo synthesis in the presence or absence of FSH, with dbcAMP ha ving no effect. HPLC analysis of C-14-hypoxanthine metabolism in oocyt e-cumulus cell complexes revealed that changes in the pattern of purin e metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathway s are vital participants in the mechanism(s) regulating hormone-induce d meiotic maturation, and that either the de novo or salvage pathway c an fulfill this nucleotide requirement. (C) 1997 Wiley-Liss, Inc.