Oocyte maturation in the toad Rhinella arenarum (Amphibia, Anura): Evidence of cAMP involvement in steroid production and action.

In this work, we describe the participation of the adenylate cyclase/3'-5'-cyclic adenonsine monophosphate (cAMP) pathway in the seasonal follicular secretion of progesterone (P4 ) and testosterone (T), and its relationship with the maturation of Rhinella arenarum oocytes. Under gonadotropin stimulation, P4 secretion was the dominant steroid produced during the reproductive period, resulting in 100% germinal vesicle breakdown (GVBD) in oocytes in vitro; in contrast, T and estradiol (E2 ) secretion increased (∼16 nM/20 follicles and ∼80 pM/20 follicles, respectively) during the non-reproductive period, but only yielded 50% GVBD. Treatment of the follicles with dibutyryl-cAMP or forskolin induced a significant increase in T secretion during both periods, but P4 secretion did not significantly change and GVBD did not occur. These results suggest that high cAMP levels in the oocyte maintain meiotic arrest and prevent the induction effect of follicular steroids. An increase in cAMP levels in denuded oocytes, however, negatively regulated T-induced maturation since treatment with increasing db-cAMP or forskolin inhibited their maturation. Therefore, we hypothesize that an elevation in T during the non-reproductive period favors its aromatization to E2 , leading to follicle growth. During the reproductive period, P4 production might promote oocyte maturation when environmental conditions are favorable for reproduction. Together, the results indicate that steroidogenesis is seasonal and depends on gonadotropic activity in R. arenarum.

The role of calcium in the nuclear maturation of Bufo arenarum oocytes.

In Bufo arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca(2+) influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca(2+)-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca(2+)-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.