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Participation of IP₃R, RyR and L-type Ca²⁺ channel in the nuclear maturation of Rhinella arenarum oocytes

Toranzo, G. Sánchez, Bühler, M.C. Gramajo, Bühler, M.I.
Zygote 2014 v.22 no.2 pp. 110-123
Rhinella arenarum, agonists, amphibians, caffeine, calcium, calcium channels, calcium signaling, chelating agents, chromatin, endoplasmic reticulum, extracellular space, germinal vesicle, heparin, inositols, meiosis, mitogen-activated protein kinase, mitosis, progesterone, thimerosal
During meiosis resumption, oocytes undergo a series of nuclear and cytosolic changes that prepare them for fertilization and that are referred to as oocyte maturation. These events are characterized by germinal vesicle breakdown (GVBD), chromatin condensation and spindle formation and, among cytosolic changes, organelle redistribution and maturation of Ca²⁺-release mechanisms. The progression of the meiotic cell cycle is regulated by M phase/maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Changes in the levels of intracellular free Ca²⁺ ion have also been implicated strongly in the triggering of the initiation of the M phase. Ca²⁺ signals can be generated by Ca²⁺ release from intracellular Ca²⁺ stores (endoplasmic reticulum; ER) or by Ca²⁺ influx from the extracellular space. In this sense, the L-type Ca²⁺ channel plays an important role in the incorporation of Ca²⁺ from the extracellular space. Two types of intracellular Ca²⁺ receptor/channels are known to mediate the intracellular Ca²⁺ release from the ER lumen. The most abundant, the inositol 1,4,5-trisphosphate receptor (IP₃R), and the other Ca²⁺ channel, the ryanodine receptor (RyR), have also been reported to mediate Ca²⁺ release in several oocytes. In amphibians, MPF and MAPK play a central role during oocyte maturation, controlling several events. However, no definitive relationships have been identified between Ca²⁺ and MPF or MAPK. We investigated the participation of Ca²⁺ in the spontaneous and progesterone-induced nuclear maturation in Rhinella arenarum oocytes and the effect of different pharmacological agents known to produce modifications in the Ca²⁺ channels. We demonstrated that loading competent and incompetent oocytes with the intracellular calcium chelator BAPTA/AM produced suppression of spontaneous and progesterone-induced GVBD. In our results, the capacity of progesterone to trigger meiosis reinitiation in Rhinella in the presence of L-type Ca²⁺ channel blockers (nifedipine and lanthane) indicated that spontaneous and progesterone-induced maturation would be independent of extracellular calcium influx, but would be sensitive to intracellular Ca²⁺ deprivation. As demonstrated by the effect of thimerosal and heparin in Rhinella arenarum, the intracellular increase in Ca²⁺ during maturation is also mediated mainly by IP₃R. In addition, our results using caffeine, an agonist of the RyR, could suggest that Ca²⁺ release from ryanodine-sensitive stores is not essential for oocyte maturation in Rhinella. The decrease in MPF activity with NaVO₃ negatively affected the percentage of thimerosal-induced GVBD. This finding suggests that Ca²⁺ release through the IP₃R could be involved in the signalling pathway that induces MPF activation. However, the inhibition of MAP/ERK kinase (MEK) by PD98128 or P90 by geldanamycin produced a significant decrease in the percentages of GVBD induced by thimerosal. This finding suggests that Ca²⁺ release per se cannot bypass the inhibition of the MAPK activity.