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.

Figure 1

Preparation and flow cytometric analysis of BODIPY-cholesterol labelled boar spermatozoa. (A) To prepare spermatozoa for the BODIPY-cholesterol assay, cells are first labelled with BODIPY-cholesterol in a non-capacitating media (1), then excess label is removed via density gradient centrifugation and spermatozoa can be incubated in various capacitating conditions (2). Following capacitation (2 h), spermatozoa can be analysed with flow cytometry and to determine cholesterol efflux, the percent loss in BODIPY-cholesterol fluorescence relative to the non-capacitating control (NC) was calculated (i.e. BODIPY-cholesterol fluorescence in TALP ÷ BODIPY-cholesterol fluorescence in NC × 100). (B) The resulting density plot of recorded sperm events with BODIPY-cholesterol and counterstained with propidium iodide (PI) to isolate a viable (PI−) and non-viable population (PI+) for analysis. (C) After selecting the viable population only, a loss in BODIPY-cholesterol can be observed following 2 h incubation in capacitating conditions (as indicated by black arrow), TALP and TALP supplemented with cAMP up-regulators (TALP+) when compared to the non-capacitating control (NC).

Figure 2

The percent loss in BODIPY-cholesterol fluorescence (A) and endogenous cholesterol (B) from boar spermatozoa relative to the non-capacitating control. Boar spermatozoa were either labelled with BODIPY-cholesterol prior to exposure to various conditions for 2 h and the efflux of BODIPY-cholesterol was tracked with flow cytometry or alternatively, sperm lipids were extracted following exposure to various conditions and subsequently analysed for endogenous cholesterol loss with mass spectrometry. (A) BODIPY-cholesterol efflux was highest in spermatozoa exposed to TALP supplemented with or without cAMP up-regulators (white and white hatched bar, respectively) or media without bicarbonate but supplemented with cAMP up-regulators (dark gray hatched bar). (B) In contrast, only TALP and TALP with cAMP up-regulators were able to support a significant loss in endogenous cholesterol. When spermatozoa were exposed to TALP devoid of BSA or bicarbonate and supplemented with cAMP up-regulators, there was a significant difference in cholesterol loss quantified by the two methods. Endogenous cholesterol loss was greater in TALP devoid of BSA with cAMP up-regulators compared with BODIPY-cholesterol ((A and B) green hatched bar) and the opposite was observed for TALP devoid of bicarbonate supplemented with cAMP up-regulators ((A and B) red hatched bar). Results are based on five (endogenous cholesterol) six (BODIPY-cholesterol) independent samples and presented as the mean percent BODIPY-cholesterol or endogenous cholesterol remaining ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.0001 indicate differences from the non-capacitating control within a quantification method.

Figure 3

Representative images of viable (A and B) and non-viable (C) BODIPY-cholesterol labelled boar spermatozoa following 2 h in non-capacitating and capacitating conditions. Note that labelling is higher in the apical sperm head than that in the post-equatorial region (indicated with arrows), which is most obvious in sperm that have not undergone capacitation (A). Upon incubation with capacitating conditions, BODIPY-cholesterol fluorescence reduces across the whole sperm head (B), which visually demonstrates the loss of this cholesterol analogue from the plasma membrane during capacitation. In non-viable spermatozoa, as assessed with propidium iodide (PI), BODIPY-cholesterol fluorescence was more intense across the entire sperm head, particularly in the equatorial region (C; indicated with an arrow), demonstrating potential intracellular labelling. Scale bar = 10 µm.

Figure 4

The percentage loss of cholesterol from boar spermatozoa exposed to capacitating conditions as measured by the BODIPY-cholesterol assay (black bars) and mass spectrometric (MS) analysis (gray bars) for populations segregated based on viability. To isolate sperm populations, spermatozoa were stained with propidium iodide (PI) and either BODIPY-cholesterol labelled spermatozoa were gated for these different populations or cells were sorted based on these populations and then sperm lipids were analysed with mass spectrometry. Only in non-viable cells was there a major discrepancy between cholesterol efflux measured by BODIPY-cholesterol and lipid analysis. Results are based on six independent samples for both measures and presented as the mean percent cholesterol remaining ± SEM. *P < 0.05 indicate differences between cholesterol efflux measured by BODIPY-cholesterol and MS analysis for each sperm population.

Figure 5

The percentage change in filipin fluorescence measured from boar spermatozoa incubated in various capacitating and non-capacitating conditions. Following fixation and labelling with filipin complex, filipin fluorescence was quantified with flow cytometry. There was no detectable difference in the percentage change in fluorescence across any of the conditions when compared to the non-capacitating control (black bar). Results are based on six independent samples and presented as the mean percent in filipin-cholesterol compared to the non-capacitating control ± SEMs.

Figure 6

Predominant patterns of fluorescent filipin-cholesterol labelling and the frequency of each of these patterns observed on fixed boar spermatozoa following incubation in capacitating and non-capacitating conditions for 2 h. (A) Uniform filipin labelling was observed across the entire sperm head in non-capacitating cells (1). In capacitating cells, filipin-cholesterol complexes concentrate in the apical and pre-equatorial region of the sperm head (indicated by bracket; 2). As in (2) but there were filipin-cholesterol complexes residing in the post-equatorial region of the sperm head (post-equatorial region indicated by an arrow; 3). Cells that exhibited a loss of filipin-cholesterol complexes were identified by the lower fluorescence when compared to non-responsive cells or those with cholesterol redistribution (indicated by arrows; 4). Scale bar = 10 µm. (B) Spermatozoa exposed to TALP supplemented with or without cAMP up-regulators (white bar and white hatched bar, respectively) or TALP without bicarbonate but supplemented with cAMP up-regulators (dark gray hatched bar) demonstrated a significant decrease in the percentage of non-responsive cells and increase in patterns indicative of cholesterol redistribution and efflux. Results are based on six independent samples and presented as the mean ± SEM. **P < 0.01 and ***P < 0.0001 indicate differences from the non-capacitating control.