Rat Oocyte Maturation: Effects of Protein Synthesis Inhibitors

Abstract

Puromycin has been reported to block the meiosis of cultured mouse oocytes before extrusion of the first polar body, but to allow resumption of meiosis as revealed by germinal vesicle breakdown (GVB) (Schultz and Wassarman, 1977a). The conclusion from this was that GVB occurs independently of protein synthesis. In the present study rat oocytes were treated with puromycin during two successive culture periods. During the first period resumption of meiosis was prevented either by the concomitant presence of dibutyryl-cyclic AMP (dbcAMP) or by incubation of oocytes within their intact preovulatory follicles. Isolated oocytes, with or without cumulus cells, were then transferred to plain medium or medium with puromycin alone for a second culture period. Cumulus-surrounded oocytes matured spontaneously in culture. DbcAMP prevented GVB, if added within 1 h from the start of culture, whereas puromycin did not prevent GVB but blocked polar body formation, thus confirming earlier observations. When oocytes were first cultured for 0.5-4 h in the presence of both puromycin and dbcAMP and then cultured for 4 h with puromycin alone, GVB was prevented in a “time dependent” way: the longer the first culture period the lower the rate of GVB. The inhibition of GVB was, however, fully reversed when no drugs were present during the second culture. The block of GVB produced by puromycin was sustained even when the second culture was continued for 18 h. Cycloheximide, but not puromycin aminonucleoside (PAN), produced the same effects as puromycin. Similar results were obtained when denuded oocytes were used, suggesting that the effects of puromycin were directly on the oocyte and not mediated via the cumulus cells. When follicle-enclosed oocytes were incubated for 3 h with puromycin and the oocytes then isolated and cultured with puromycin, GVB was also inhibited. The rise in oxygen consumption seen in maturing oocytes could be prevented in oocytes maturing in medium containing puromycin. Also this effect of puromycin was reversible, indicating that puromycin did not inflict permanent damage on the oocyte. The interpretation of these data is that resumption of meiosis is dependent on some proteins with high turnover rates existing in the oocyte before the start of meiosis. Polar body formation might also be explained in a similar way.