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

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.

HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility

“…For the quantification of cholesterol efflux from ram spermatozoa, we utilized the BODIPY-cholesterol assay, a flow cytometric-based assessment that has been previously validated by our group [ 50 ]. Ram spermatozoa were labeled with BODIPY-cholesterol and prepared for in vitro capacitation as previously described [ 24 ].…”

“…To identify the membrane intact population for assessment of cholesterol efflux (in order to eliminate cells with potential intracellular labeling), suspensions were counterstained with 6 μM PI for 10 min prior to analysis [ 50 ]. BODIPY-cholesterol and PI fluorescence were excited by a 50 mW 488 nm laser and detected with either a 525/40 nm or 585/42 band-pass filter, respectively.…”

Bisphenol A Diglycidyl Ether (BADGE) and Progesterone Do Not Induce Ca2+ Signals in Boar Sperm Cells

“…Hereafter the sperm cells were incubated for at least 1 h at 37°C. For the experiments with capacitated boar sperm cells, the samples were treated similar to in a previous study on boar sperm ( Bernecic et al, 2019 ) and resuspended in a capacitating medium with the following composition: 72.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO 4 , 0.37 mM KH 2 PO 4 , 2.04 mM CaCl 2 , 0.33 mM Na-pyruvate, 21.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, and 25 mM NaHCO 3 , adjusted to pH 7.3–7.4 with NaOH. Human serum albumin (3 mg/mL) was added to the capacitating medium and the sperm cells were incubated for >3 h at 37°C in a 5% CO 2 atmosphere.…”

“…After 1 h at 37 • C, the upper swim-up fraction was carefully removed and after two washes, the sperm concentration was determined by image cytometry (Egeberg et al, 2013) using an NC-3000 (ChemoMetec AS, Denmark) and samples were adjusted to 10 × 10 6 sperm cells/mL in HTF + with human serum albumin (3 mg/mL). Hereafter the sperm cells were incubated for at least 1 h at 37 • C. For the experiments with capacitated boar sperm cells, the samples were treated similar to in a previous study on boar sperm (Bernecic et al, 2019) and resuspended in a capacitating medium with the following composition: 72.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO 4 , 0.37 mM KH 2 PO 4 , 2.04 mM CaCl 2 , 0.33 mM Na-pyruvate, 21.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, and 25 mM NaHCO 3 , adjusted to pH 7.3-7.4 with NaOH. Human serum albumin (3 mg/mL) was added to the capacitating medium and the sperm cells were incubated for >3 h at 37 • C in a 5% CO 2 atmosphere.…”

Male Infertility: Shining a Light on Lipids and Lipid-Modulating Enzymes in the Male Germline

“…It is well established that the sperm ascent of the female reproductive tract is accompanied by a further wave of dynamic changes in their membrane lipid composition. Chief among these changes are the efflux of cholesterol and resultant increase in membrane fluidity, permeability, and fusibility characteristics, which signal the onset of capacitation [118][119][120]. Cholesterol removal is also permissive of membrane remodeling, including the repositioning of receptors and fusion machinery needed to prime the sperm cell for acrosomal exocytosis and downstream oocyte interactions [121][122][123][124].…”