BACKGROUND Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. RESULTS All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). CONCLUSIONS Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

中文翻译：

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体外公猪精子获能过程的荧光分析。

背景技术获能涉及精子在雌性生殖道或体外必须经历的生理变化，以获得结合，渗透和使卵受精的能力。迄今为止，已经开发了几种方法来表征这种复杂的生物过程。本研究的目的是相互比较几种荧光技术，检查其在获能过程中检测分子过程变化的能力，并确定其预测暴露于增溶的透明带后顶体反应（AR）精子百分比的能力（ ZP）。使用四种荧光技术对获能过程进行了分析：1.金霉素（CTC）染色； 2.抗球蛋白抗体（ACR.2）测定； 3.抗磷酸酪氨酸（pY）抗体测定； 4。异硫氰酸荧光素偶联的鬼笔环肽（FITC-phall）测定。所有这些方法均使用荧光显微镜和流式细胞仪进行了测试。结果所有选择的方法均能够检测公猪精子的体外获能过程，但使用荧光显微镜或流式细胞术实验安排和随后的统计分析（KW-ANOVA）时，其结果有显着差异。同样，预测将经历ZP诱导的AR的精子绝对数量的能力也显着不同（CTC和ACR.2给出了最佳预测）。结论我们的研究比较了四种用于表征获能过程的常用方法，强调了它们的差异，并表明所有方法均能够检测出获能进展，CTC和ACR。