Baufeld Anja, Vanselow Jens
Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN);
Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN).
J Vis Exp. 2018 Sep 6(139):58208. doi: 10.3791/58208.
Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (< 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.
卵巢颗粒细胞（GC）是雌二醇合成的主要来源。在排卵前促黄体生成素（LH）峰的诱导下，卵泡膜细胞，尤其是颗粒细胞层的细胞，其形态、生理和分子特征会发生深刻变化，并形成产生孕酮的黄体，黄体负责维持妊娠。细胞培养模型是研究卵泡 - 黄体转化所涉及的潜在调控机制的重要工具。本文介绍的方案重点在于从小到中等大小卵泡（<6 mm）中分离牛颗粒细胞并进行冷冻保存的程序。通过这种技术，可以获得几乎纯净的颗粒细胞群体。冷冻保存程序极大地便于细胞培养工作的时间管理，而无需直接依赖原代组织（卵巢）供应。本方案描述了一种无血清细胞培养模型，该模型模拟了牛颗粒细胞的雌二醇活性状态。在整个方案中讨论了成功进行类固醇活性细胞培养所必需的重要条件。结果表明，增加细胞接种密度会诱导特定反应，表现为基因表达谱和激素产生的改变。此外，该模型为进一步研究颗粒细胞分化及其他应用提供了基础。
