Department of Cell Biology and Histology, School of Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Department of Physiology, Faculty of Veterinary Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Curr Protoc Toxicol. 2020 Dec;86(1):e100. doi: 10.1002/cptx.100.
We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm-binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS-PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm-binding assay Alternate Protocol 2: Sperm preparation by the swim-up method.
我们最近描述了一种新的模型，用于研究哺乳动物物种中的配子相互作用。该模型通过使用磁性琼脂糖珠来重现卵母细胞的球形表面，这些珠子被一层参与配子相互作用的重组蛋白（如 ZP2 或 IZUMO1 受体 JUNO）和一层卵丘细胞所覆盖，从而在某种程度上模拟了天然的卵丘 - 卵母细胞复合体。一旦生成，这个 3D 模型可以用于精子结合测定，以获得有关配子相互作用分子基础的有价值信息，因为可以使用不同的重组蛋白来覆盖珠表面，从而生成各种模型，用于多种物种。此外，由于该模型能够诱捕精子，因此可以研究结合精子的生理状态，这使其成为选择具有高受精能力的精子、揭示家畜养殖中心的亚生育动物或进行毒理学研究的有力工具。在这里，我们描述了如何使用猪精子作为示例，并使用来自透明带的 ZP2 蛋白作为感兴趣的重组蛋白，生成和使用该模型进行精子结合测定。© 2020 威利父子公司。基本方案 1：体外 3D 模型的生成备选方案 1：将卵丘细胞结合到模型上基本方案 2：通过 SDS-PAGE 电泳和 Western blot 进行模型质量控制支持方案 1：免疫化学以确认表面上正确的蛋白分布珠支持方案 2：重组缀合蛋白的洗脱基本方案 3：精子结合测定备选方案 2：通过游泳法制备精子。
