Nongenomic Steroid- and Ceramide-Induced Maturation in Amphibian Oocytes Involves Functional Caveolae-Like Microdomains Associated with a Cytoskeletal Environment

BUSCHIAZZO, J.; ALONSO, T. S. ; BISCOGLIO, M.; ANTOLLINI, S. S.; BONINI, I. C.

BIOLOGY OF REPRODUCTION (ONLINE)

HighWire Press

Lugar: Madison, WI; Año: 2011 vol. 85 p. 808 - 822

1529-7268

Stimulation of full-grown amphibian oocytes with progesterone initiates a non-transcriptional signalingpathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed thatcholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturationsuggesting involvement of membrane rafts. In the present study, we further characterized caveolae-likemembranes from Rhinella arenarum oocytes biochemically and functionally. The identification by massspectrometry of a non-muscle myosin heavy-chain associated to caveolar membranes evidenced directinvolvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysicalanalysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletionaffected membrane lipid order. In line with this finding, cholesterol removal also affected the localizationof the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturationthat alters the distribution of the raft markers caveolin-1, SRC and GM1, while progesterone seems not toaffect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-inducedmaturation thus suggesting that ceramide is an inducer more vulnerable to changes in the plasmamembrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesteroneinducedmaturation. Functional studies regarding tyrosine phosphorylation raise the possibility that thehormone receptor is located in the non-raft membrane in the absence of ligand and that it translocates tothe caveola when it binds to progesterone. The presence of raft markers and the finding of signalingmolecules from MAPK cascade functionally associated to oocyte light membranes suggest that thiscaveolae rich-fraction efficiently recreates, in part, maturation signaling.BOR Papers in Press. Published on June 8, 2011 as DOI:10.1095/biolreprod.110.090365Copyright