Andrisani Alessandra, Donà Gabriella, Ambrosini Guido, Bonanni Guglielmo, Bragadin Marcantonio, Cosmi Erich, Clari Giulio, Armanini Decio, Bordin Luciana
Department of Women's and Children's Health .
Syst Biol Reprod Med. 2014 Aug;60(4):239-44. doi: 10.3109/19396368.2014.904952. Epub 2014 Mar 27.
A wide variety of sperm preparation protocols are currently available for assisted conception. They include density gradient separation and washing methods. Both aim at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization. The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Semen samples from 48 healthy donors (normal values of sperm count, motility, morphology, and volume) were analyzed. After separation (density gradient 40/80%), sperm were incubated in various buffers then analysed for reactive oxygen species (ROS) production, viability, tyrosine phosphorylation (Tyr-P), cholera toxin B subunit (CTB) labeling, and the acrosome reaction (AR). The buffers affected ROS generation in various ways resulting either in rapid cell degeneration (when the amount of ROS was too high for cell survival) or the inability of the cells to maintain correct functioning (when ROS were too few). Only when the correct ROS generation curve was maintained, suitable membrane reorganization, evidenced by CTB labeling was achieved, leading to the highest percentages of both Tyr-P- and acrosome-reacted-cells. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.
目前有各种各样的精子制备方案可用于辅助受孕。这些方案包括密度梯度分离法和洗涤法。两者的目的都是分离出尽可能多的有活力精子并使其获能，以便随后使卵母细胞受精。本研究的目的是检测四种商用精子洗涤缓冲液对精子活力和获能的影响。分析了48名健康供者（精子计数、活力、形态和体积的正常值）的精液样本。在分离（密度梯度40/80%）后，将精子在各种缓冲液中孵育，然后分析其活性氧（ROS）产生、活力、酪氨酸磷酸化（Tyr-P）、霍乱毒素B亚基（CTB）标记和顶体反应（AR）。这些缓冲液以各种方式影响ROS的产生，要么导致细胞迅速退化（当ROS量过高而细胞无法存活时），要么导致细胞无法维持正常功能（当ROS过少时）。只有当维持正确的ROS产生曲线时，才能实现由CTB标记证明的合适的膜重组，从而导致Tyr-P细胞和顶体反应细胞的百分比最高。区分精子样本的每种特定病理状态将有助于选择首选的缓冲液处理方法，因为商用缓冲液可显著改变ROS产生和膜重组。
