Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability
Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability
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DOI：
10.1530/reprod/118.1.145
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发表时间：
2000-01-01
期刊：
影响因子：
--
通讯作者：
Roldan, ERS
中科院分区：
文献类型：
--
作者：
Pintado, B;de la Fuente, J;Roldan, ERS
这项研究旨在评估核酸染色(如碘化丙啶和Hoechst 33258)和胞浆染色曙红是否识别出相同比例的非活细胞。将保存长达72小时的猪精子和保存在吸管中并在染色前解冻的公牛精子的亚样单独或联合暴露于碘化丙胺或Hoechst 33258。用伊红-黑松素染色子样品，然后用Giemsa染色。单独使用碘化丙啶鉴定的非活细胞比例与与其他荧光探针联合使用时观察到的比例相同。Hoechst 33258也观察到了类似的结果。然而，对暴露于这两种菌株的子样本的直接显微镜检查发现，用碘化丙啶染色的精子中有一部分不含有Hoechst 33258。在使用的不同实验条件下，在公猪和公牛的精子中发现了一致的这一点。定量结果显示，碘化丙啶阳性细胞比例明显高于Hoechst 33258阳性细胞。此外，碘化丙啶染色的细胞比例高于伊红染色的细胞，但Hoechst 33258和伊红染色的细胞数没有差异。尽管观察到更多的细胞含有碘化丙啶，但碘化丙啶染色的细胞比例与Hoechst 33258或曙红染色的比例呈正相关。综上所述，这些结果表明，荧光探针识别无活性精子细胞的能力存在差异，在使用染色方案分析精子活力时，或在功能研究中将活力用作区分因素时，如与顶体胞吐有关的研究，应考虑这一点。
This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.