非洲爪蟾卵母细胞中的亮氨酸转运：不同去滤泡程序的功能和形态学分析

Marciani P, Castagna M, Bonasoro F, Carnevali M D, Sacchi V F

Istituto di Fisiologia Generale e di Chimica Biologica, Facoltà di Farmacia, Milano, Italy.

Comp Biochem Physiol A Mol Integr Physiol. 1998 Apr;119(4):1009-17. doi: 10.1016/s1095-6433(98)00018-x.

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.

去除卵泡细胞的特定方法会影响处于Ⅴ期的非洲爪蟾卵母细胞对L-亮氨酸的摄取。在100 mM NaCl存在的情况下，通过胶原酶处理进行酶解去卵膜时，L-亮氨酸摄取量降低了67.5%±5.7%，而机械去卵膜后摄取量降低了30.5%±6.4%。在有卵泡的卵母细胞中，0.1 mM L-亮氨酸的Na⁺依赖性摄取量为18.6±4.6 pmol·卵母细胞⁻¹·40 min⁻¹，在经胶原酶去卵膜的卵母细胞中为5.6±1.9 pmol·卵母细胞⁻¹·40 min⁻¹（平均值±标准误）。如果在转运期后发生去卵膜，去除卵泡层不会影响L-亮氨酸的摄取；因此，放射性标记的L-亮氨酸不会被摄取到因去卵膜过程而被去除的隔室中。在有卵泡和去卵膜的卵母细胞中观察到的不同L-亮氨酸摄取率并非由于L-亮氨酸与膜的非特异性结合。L-亮氨酸动力学表明，缺乏卵泡层的卵母细胞中L-亮氨酸的Vmax和Km值低于被完整卵泡层包裹的对照卵母细胞。根据胶原酶处理去卵膜后第二天获得的数据计算，Na⁺依赖性L-亮氨酸转运的Vmax和Km值分别为：16±1.5 pmol·卵母细胞⁻¹·40 min⁻¹和57±21 μmol（平均值±标准差）。在有卵泡的卵母细胞中，0.1 mM L-亮氨酸的Na⁺激活曲线是双曲线型的，而在去卵膜的卵母细胞中是S型的。与转运实验同时进行的形态学分析表明，去卵膜后，形成卵黄膜的纤维倾向于排列成更规则的正交阵列，胶原酶处理后卵母细胞微绒毛的数量减少。机械去卵膜对卵母细胞微绒毛没有明显影响，然而该过程并没有完全去除所有卵泡细胞。胶原酶处理对卵母细胞造成的损伤是可逆的，大多数卵母细胞在随后的体外培养中其功能和结构特征得到改善。鉴于L-亮氨酸Vmax值增加以及显微镜观察显示微绒毛装置恢复，恢复过程似乎涉及蛋白质合成。