Nongenomic Steroid- and Ceramide-Induced Maturation in Amphibian Oocytes Involves Functional Caveolae-Like Microdomains Associated with a Cytoskeletal Environment
Nongenomic Steroid- and Ceramide-Induced Maturation in Amphibian Oocytes Involves Functional Caveolae-Like Microdomains Associated with a Cytoskeletal Environment
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DOI：
10.1095/biolreprod.110.090365
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发表时间：
2011-10-01
影响因子：
3.6
通讯作者：
Bonini, Ida C.
中科院分区：
文献类型：
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作者：
Buschiazzo, Jorgelina;Alonso, Telma S.;Bonini, Ida C.
用孕酮刺激成熟的两栖类卵母细胞启动一个非转录信号通路，该通路在Cdc 2/细胞周期蛋白B的激活和重新进入减数分裂中会聚。我们观察到甲基-β-环糊精（MbetaCD）介导的胆固醇消耗抑制减数分裂成熟，表明膜筏的参与。在本研究中，我们进一步的特点小窝样膜沙蚕卵母细胞的生化和功能。通过质谱鉴定与小窝膜相关的非肌肉肌球蛋白重链，显示了直接参与卵母细胞筏结构的潜在细胞骨架环境的证据。使用荧光探针Laurdan的生物物理分析显示，MbetaCD介导的胆固醇消耗影响膜脂质秩序。与这一发现一致，胆固醇去除也影响了筏标记物脂质GM 1的定位。结果表明，神经酰胺是一种有效的成熟诱导剂，改变筏标记小窝蛋白-1，SRC和GM 1的分布，而孕酮似乎不影响膜微区的完整性。胆固醇耗竭对神经酰胺诱导的成熟有更大的影响，从而表明神经酰胺是一种更容易受到质膜变化影响的诱导剂。MbetaCD处理延迟了孕酮诱导的成熟中的酪氨酸磷酸化和MAPK活化。关于酪氨酸磷酸化的功能研究提出了这样的可能性，即激素受体在没有配体的情况下位于非筏膜中，并且当它与孕酮结合时，它易位到小窝。筏标记物的存在和MAPK级联信号分子的功能相关的卵母细胞光膜的发现表明，这种小窝丰富的馏分有效地重建，部分，成熟信号。
Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.