SPERM-ACTIVATED CHANNELS IN ASCIDIAN OOCYTE MEMBRANES

这项建议的目的是研究海鞘内的离子流。 受精精子激活的卵母细胞膜。这个 研究应将精子激活通道的出现与 受精潜力，关于离子的通道的特征 特异性和动力学，并检验通道是 从卵母细胞膜的细胞质一侧被钙激活。 这些实验使用了海鞘，因为去角质的卵母细胞 由于腹水卵母细胞缺乏皮质颗粒，因此容易被膜片钳住： 单通道记录提供了分子描述 精子激活电流，以及皮质颗粒的缺失 通道进入卵母细胞的可能机制 薄膜。此外，膜片钳技术隔离了一个已定义的区域 卵母细胞膜，我们将利用这一特征来限制 通过将精子放入记录吸管进行配子相互作用。这 实验，与添加精子的斑贴实验相结合 到浴缸，并结合全细胞记录 受精流，将有助于确定渠道的位置 受精的精子激活了多少通道， 以及通道从卵母细胞膜的哪一侧打开。 总体目标是了解精子激活通道如何与 受精过程中发生的其他事件，并确定初级 触发卵母细胞激活。这些实验将有助于从整体上 了解精子是如何激活卵母细胞的，它们将进一步 体外受精的科学与实验控制。

The purpose of this proposal is to study the ionic currents in the Ascidian oocyte membrane that are activated by the fertilizing spermatozoon. The research shall correlate the apearance of sperm-activated channels with the fertilization potential, characterize the channels with regard to ion specificity and kinetics, and test the hypothesis that the channels are activated from the cytoplasmic side of the oocyte membrane by Ca. The experiments make use of Ascidians because the dechorinated oocytes are easily patch-clamped and because Ascidian oocytes lack cortical granules: Single-channel recording provides a molecular description of the sperm-activated currents, and the absence of cortical granules elininates a potential mechanism for the introduction of channels into the oocyte membrane. In addition, the patch-clamp technique isolates a defined area of oocyte membrane, and we shall use this feature to restrict the site of gamete interaction by putting sperm in the recording pipette. This experiment, in combination with patch experiments in which sperm are added to the bath, and in combination with the whole-cell recording of fertilization currents, will help to determine where the channels are located, how many channels are activated by the fertilizing spermatozoon, and from which side of the oocyte membrane the channels are opened. The overall aim is to learn how sperm-activated channels interrelate with other events that occur during fertilization, and to determine the primary trigger of oocyte activation. These experiments will aid in the overall understanding of how sperm activate oocytes, and they will further the science and the experimental control of in vitro fertilization.