Meiotic arrest in incompetent rat oocytes is not regulated by cAMP

Abstract

Fully grown, but not growing, mammalian oocytes spontaneously resume meiosis in vitro. Resumption of meiosis, also known as oocyte maturation, is associated with a drop in intraoocyte concentrations of cAMP followed by activation of the maturation promoting factor (MPF). Microtubule-associated-protein (MAP) kinase has been suggested as a substrate for the active p34cdc2 kinase, the catalytic subunit of MPF. Our study was designed to explore the mechanism of regulation of meiotic arrest in growing rat oocytes. Confirming previous observations we showed that in our rat colony oocytes do not acquire the competence to spontaneously resume meiosis earlier than 22 days postpartum. We further demonstrated that follicle-enclosed oocytes from 20-day-old female rats fail to resume meiosis in response to luteinizing hormone, follicle-stimulating hormone, a gonadotropin-releasing hormone analog, or forskolin, all of which are known to induce maturation in competent oocytes. Immunoblot analysis using highly specific anti p34cdc2 antibodies revealed that incompetent oocytes express the catalytic subunit of MPF at amounts that are not different from that found in competent oocytes. In addition, highly specific anti MAP kinase antibodies detected the presence of similar quantities of two isoforms (42 and 44 kDa) of MAP kinase in competent and incompetent oocytes. Measurements of cAMP revealed that as compared to competent oocytes, incompetent oocytes contain somewhat lower levels of this nucleotide (1.42 +/- 0.3 and 1.17 +/- 0.07 fmole/oocyte, respectively). However, considering the difference in protein content, the calculated concentrations seem to be similar. Furthermore, similar to competent oocytes, intracellular concentrations of cAMP in incompetent oocytes dropped significantly (from 1.17 +/- 0.07 to 0.77 +/- 0.12 fmole/oocyte) 2 hr after isolation from the follicle. We hereby suggest that (a) in mammals, similar to amphibians, the term meiotic incompetence can be extended to include inability to resume meiosis in response to hormonal stimulation; (b) it is not the lack of p34cdc2 or downstream regulatory elements, such as MAP kinase, that prevents growing oocytes from resuming meiosis; and (c) unlike fully grown oocytes, resumption of meiosis in growing oocytes is not subjected to negative regulation by cAMP.