Chemical activation inRhinella arenarumoocytes: effect of dehydroleucodine (DhL) and its hydrogenated derivative (2H-DhL)

Abstract

Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.

Participation of PLA2 and PLC in DhL-induced activation ofRhinella arenarumoocytes

“…DhL could activate PLC with the consequent production of IP 3 , which would induce Ca 2+ release from the endoplasmic reticulum. These possibilities agree with the observation that DhL requires no extracellular Ca 2+ to exert its effect; however, intracellular Ca 2+ release through IP 3 Rs and RyRs is critical (Medina et al, 2014).…”

“…Dehydroleucodine (DhL), a sesquiterpenic lactone of the guaianolide group, is the active principle of a wild plant, Artemisa douglasiana Besser. Treatments of mature oocytes of R. arenarum with DhL were able to induce oocyte activation in a dose-dependent manner, the process being independent of extracellular Ca 2+ but dependent on intracellular free Ca 2+ (Medina et al , 2014). This DhL-induced activation is genuine, as oocytes are capable of inducing pronuclei formation.…”

“…This DhL-induced activation is genuine, as oocytes are capable of inducing pronuclei formation. Experiments with antagonists of RyRs (ruthenium red) and IP 3 Rs (heparin) revealed that both Ca 2+ -mobilizing systems were activated by lactones and that the blockage of either of the systems alone was not sufficient to prevent total activation induced with DhL (Medina et al , 2014).…”

Development and Validation of a Novel HPLC Method for the Analysis and Quantification of Dehydroleucodine in Plant Extracts

“…2670/2672/2673; Fax: 00-54-261-4494117; E-mail: apenissi@fcm.uncu.edu.ar tinal cytoprotective effects [7][8][9][10][11], mast cell stabilizer action [12,13], aromatase inhibitory activity [14], antioxidant properties [15,16], adipocyte differentiation effects [17] and antimicrobial activities against Pseudomonas aeruginosa [18]. DhL also induces the activation of matured oocytes of Rhinella arenarum in vitro [19,20] and the expression of TP73 and TP53 in human astrocytoma cells [21].…”