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.

A STUDY OF THE DIFFUSIBLE FACTOR RELEASED BY THE JELLY OF THE EGG OF THE TOAD, BUFO ARENARUM.

Some properties of an essential factor for fertilization released from the oocyte strings of the toad, Bufo arenarum were studied. All the experiments were carried out with a partially purified preparation of this diffusible factor, and the general procedure consisted in assaying under different conditions its capacity to recover the fertilizability of oocytes previously deprived of this active substance. Using this procedure, a test was devised for estimating the diffusible factor activity. As regards the mechanism of action of this fertilization factor the data reported here are not consistent with the previous assumption that the factor plays its role in fertilization through a simple pH rise of the inseminating medium. Treatment of the gametes with the diffusible factor before insemination proved to be ineffective to promote fertilization; the factor must be present at the very moment of insemination.