Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY-cholesterol assay to quantify cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY-cholesterol assay was validated with endogenous cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY-cholesterol assay was able to detect and quantify cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of cholesterol efflux. Finally, the BODIPY-cholesterol assay proved to be the superior method to quantify cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing cholesterol redistribution. Taken together, the BODIPY-cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of cholesterol efflux from spermatozoa during in vitro capacitation.

获能是精子在受精之前经历的最后一个成熟步骤。胆固醇从精子膜到细胞外环境的外排是获能过程中的关键步骤，但是目前量化该过程的方法并不理想。在这项研究中，我们验证了使用BODIPY-胆固醇测定来量化体外精子中的胆固醇流出捕获能力，以公猪为模型物种。新型流式细胞术BODIPY-胆固醇测定已通过内源性胆固醇损失进行了验证，该损失可通过质谱法进行测定，并与菲林标记法进行比较。暴露于一系列条件后，BODIPY-胆固醇测定法能够检测和量化类似于质谱法测得的胆固醇外流。对存活力进行复染的能力是该测定法的独特功能，使我们能够强调仅为了可靠地测量胆固醇外流而分离存活细胞的重要性。最后，尽管菲律宾血脂仍可用于评估胆固醇的再分配，但相对于菲律宾血脂标记，BODIPY-胆固醇测定法被证明是量化胆固醇外流的更好方法。总而言之，BODIPY-胆固醇测定是一种简单的方法，体外获能。