Nongenomic action of progesterone mediated by caveolae-like microdomains. Ceramide as maturation inducer

BUSCHIAZZO J.; BONINI I. C.; ALONSO T. S.

Buenos Aires, Argentina

Congreso; 4th International Meeting of the Latin American Society of Developmental Biology (LASDB); 2008

Sociedad Latinoamericana de Biolgía del Desarrollo (LASDB)

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The initial action of progesterone at its plasma membrane receptor triggers enzyme activations that not only modify membrane fluidity but also release a cascade of lipid messengers, such as ceramide and multiple diacylglycerol species. Previous findings have reported that treatment with either sphingomyelinase or soluble forms of ceramide induces meiosis resumption in Xenopus and Rana oocytes. In this work, ceramide is compared with progesterone for its ability to induce germinal vesicle breakdown (GVBD) indicative of maturation in Bufo oocytes. In addition, methyl-β-cyclodextrin (MβCD) was used for cellular cholesterol modulation in order to assess membrane raft involvement in progesterone-induced oocyte maturation. Oocytes arrested in G2 of meiosis I were incubated for 24 h with C2-ceramide or the corresponding dihydroceramide solubilized in dimethylsufoxide (DMSO) and added to ND 96 solution at different concentrations (15, 25, 50, 100, 150 µg/ml). Dihydroceramide and DMSO alone were used as negative controls. Progesterone-induced maturation was achieved by incubation of full-grown oocytes in ND 96 with progesterone 5 µg/ml final concentration under the same conditions. To remove cellular cholesterol, oocytes were pretreated with 5-50 mM MβCD at room temperature for 60 minutes and subjected to in vitro maturation with progesterone or ceramide. Cholesterol repletion was performed using cholesterol/MβCD complexes to assess the reversibility of the removal. Cholesterol-enriched low-density membranes containing an immunodetectable caveolin-like protein were isolated from Bufo arenarum oocytes using a detergent-free protocol. Treatment of oocytes with exogenous C2-ceramide induced GVBD at 100 µg/ml concentration with a maximal response at 150 µg/ml in which the effect was similar to progesterone-induced levels without evidence of cytotoxicity. Oocytes did not undergo GVBD at low ceramide concentrations. The inactive form of soluble ceramide (C2-dihydroceramide) and DMSO at the concentrations assayed did not induced GVBD in any case. Oocytes incubated with progesterone under the same conditions showed more than 95% of GVBD. If membrane order is essential for the maturation signaling, then the possible disruption of membrane microdomains by MβCD should inhibit the steroid-induced response. Cholesterol content of oocytes treated with 25 mM and 50 mM MβCD decreased when compared to control oocytes. In both concentrations, cholesterol was incorporated in Bufo oocytes in a reversible manner reaching the level of the control oocytes. When MβCD-treated oocytes were incubated with progesterone to induce meiotic maturation, GVBD underwent a significant decrease compared to control oocytes. We found that MβCD at 25 mM reduced the GVBD response by 23% and with 50 mM MβCD the inhibition was higher (41%) indicating that inhibition of maturation was dose-dependent. Cholesterol repletion showed a recovery of the ability to mature of MβCD-treated oocytes. At 25 mM MβCD the reversibility was near the control suggesting that at this concentration the drug does not result toxic for oocytes. Oocytes treated with 50 mM MβCD not only failed to recover the control oocyte ability to mature but also became fragile and sometimes wrinkled. Under our experimental conditions, MβCD-treated oocytes that were not incubated with progesterone did not show GVBD indicating that the drug alone does not induce the maturation response. Compatible with a disorganization of membrane rafts produced by MβCD, drug treatment affected the localization of the rafts markers caveolin and c-Src among membrane fractions. In the case of ceramide-induced maturation, MβCD  pretreatment partially inhibited GVBD. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes and indicate that ceramide is an effective inducer of maturation. It is not known whether the steroid/ceramide pathways activate different steps or if there are different pools of ceramide or different sites of ceramide generation associated with specific physiological responses.