Involvement of G protein and purines in Rhinella arenarum oocyte maturation.

We investigated the participation of G(αi) protein and of intracellular cAMP levels on spontaneous and progesterone-mediated maturation in Rhinella arenarum fully grown follicles and denuded oocytes. Although progesterone is the established maturation inducer in amphibians, Rhinella arenarum oocytes obtained during the reproductive period (competent oocytes) resume meiosis with no need for an exogenous hormonal stimulus if deprived of their enveloping follicular cells, a phenomenon called spontaneous maturation. In amphibian oocytes, numerous signalling mechanisms have been involved in the rapid, non-genomic, membrane effects of progesterone, but most of these are not fully understood. The data presented here demonstrate that activation of the G(αi) protein by Mas-7 induced maturation in non-competent oocytes and also an increase in GVBD (germinal vesicle breakdown) in competent oocytes. Similar results were obtained with intact follicles independent of the season. The activation of adenylyl cyclase (AC) by forskolin seems to inhibit both spontaneous and progesterone-induced GVBD. In addition, the high intracellular levels of cAMP caused by activation of AC by forskolin treatment or addition of db-cAMP inhibited maturation that had been induced by Mas-7 and in a dose-dependent manner. Treatment with H-89, a protein kinase A (PKA) inhibitor, was able to trigger GVBD in a dose-dependent manner in non-competent oocytes and increased the percentages of GVBD in oocytes competent to mature spontaneously. The results obtained with whole follicles and denuded oocytes were similar, which suggested that effects on AC and PKA were not mediated by follicle cells. The fact that Mas-7 was able to induce maturation in non-competent oocytes in a similar manner to progesterone and to increase spontaneous maturation suggests that G(αi) activation could be an important step in meiosis resumption. Thus, the decrease in cAMP as a result of the regulation of the G proteins on AC and the inactivation of PKA by H-89 could contribute to the activation of MPF (maturation promoting factor) and induce maturation of the oocytes of Rhinella arenarum.

Spontaneous maturation in Bufo arenarum oocytes: follicle wall involvement, respiratory activity, and seasonal influences.

The various somatic cell layers (surface epithelium, theca, and follicle cells) that envelope the amphibian oocyte were removed one by one. The various follicular preparations thus obtained were tested for their ability to undergo germinal vesicle breakdown (GVBD) in the presence or absence of progesterone during different seasonal periods. Intact follicles, follicles without epithelium, or defolliculated oocytes (without epithelium and theca layers) did not undergo GVBD when cultured in Ringer solution, unless progesterone was added to the culture medium. In contrast, denuded oocytes (lacking all follicular layers) cultured in a medium with no hormones underwent GVBD in percentages similar to those of denuded oocytes incubated with progesterone. This ability of denuded oocytes to undergo spontaneous maturation was found to be related to the respiratory activity of the oocyte, which is in turn a seasonal variable in Bufo arenarum. The different respiratory activity of the full-grown oocyte according to the seasonal period was expressed by the respiratory control quotient (around 1.0 during winter and 4.0 during summer). In fact, only those oocytes with a respiratory control quotient over 2.0 were able to undergo spontaneous nuclear maturation. The maturation obtained without exogenous hormonal stimulus was genuine as indicated by the ability of these oocytes to induce pronuclear formation after sperm injection.