BODIPY-cholesterol can be reliably used to monitor cholesterol efflux from capacitating mammalian spermatozoa

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Abstract

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.

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Bernecic, N. C., Zhang, M., Gadella, B. M., Brouwers, J. F. H. M., Jansen, J. W. A., Arkesteijn, G. J. A., … Leahy, T. (2019). BODIPY-cholesterol can be reliably used to monitor cholesterol efflux from capacitating mammalian spermatozoa. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-45831-7

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