Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation

Abstract

To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K(+), by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K(+) channels.

Keywords: ENac; SLO3; capacitation; flow cytometry; membrane potential.

© 2015 by the Society for the Study of Reproduction, Inc.

Figures

FIG. 1

Flow cytometry analysis reveals that capacitated sperm are composed of two subpopulations depicting different Ems. A–C) Whole-population analysis. Em was measured in mouse sperm in Whitten medium by using 1 μM DiSC3(5) and 1 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse mitochondrial potential. Representative fluorescence traces were used to measure resting Em, which show noncapacitated (A) or capacitated (B) sperm, and the calibration obtained by adding 1 μM valinomycin followed by sequential additions of K+. C) Summary of the sperm Em under different conditions (mean ± SEM; n = 3; *P ≤ 0.05). D–I) Flow cytometry analysis. After swimming in Whitten Hepes-buffered medium for 10 min, cauda epididymal sperm were capacitated, or not, in Whitten Hepes-buffered medium containing BSA with the addition of 15 mM HCO3− (CAP), or not (NON), for 1 h. Then, the sperm were loaded with 15 μM of DiSBAC2(3) fluorescent voltage sensor in Whitten Hepes-buffered medium containing NaHCO3, or not, at 37°C. After 30-min incubation, PI was added and the sperm population analyzed by flow cytometry. D and E) Two-dimensional dot plot SSC versus FSC analysis of NON capacitated sperm in the absence (D) or in the presence (E) of 0.1 % triton. These analyses allowed differentiating events corresponding, or not, to sperm cells as explained in Results. F and G) Sperm events were selected for DiSBAC2(3) versus PI two-dimensional fluorescence dot plot analysis of sperm incubated under conditions that do not support capacitation (F: NON) or do support capacitation (G: CAP). Two-dimensional dot plots shown in F and G were used to distinguish between sperm with low (live) and high (dead) PI staining. H and I) Live sperm populations of sperm incubated under conditions that do not support (NON) or do support (CAP) capacitation were used independently for histogram analysis depicting percentage of sperm versus DiSBAC2(3) fluorescence.

FIG. 2

Sperm belonging to the hyperpolarized capacitated subpopulation has not undergone the AR. A) Flow cytometry analyses of sperm containing GFP in their acrosomes. Cauda epididymal sperm containing GFP in their acrosomes were obtained from Acr-GFP CF1 mice in noncapacitating medium. These sperm were incubated in media that support capacitation and were loaded with DiSBAC2(3) for 60 min. Before flow cytometry, PI was added to the sperm suspension. Two-dimensional DiSBAC2(3) versus PI fluorescence dot plots of Acr-GFP sperm were used to analyze CAP (A) live and dead sperm. Live populations were then analyzed for their individual hyperpolarization states using DiSBAC2(3) fluorescence histograms. The spontaneous AR in the capacitated hyperpolarized live sperm population was further analyzed by GFP fluorescence histograms in which only one subpopulation of cells containing high GFP with intact acrosomes can be observed (lower left panel). In contrast, in the capacitated depolarized live sperm population (lower right panel), two populations of cells containing high GFP with intact acrosomes and or low GFP without acrosomes can be distinguished. B) Dot plot analysis of GFP-containing versus DisBac2(3) fluorescence. Results from A were analyzed using the GFP content versus DisBac2(3) fluorescence. C) Percentage of hyperpolarized versus depolarized cells in acrosome intact (AI) or AR sperm. The experiment shown in A and B was repeated seven times and the percentage of hyperpolarized versus depolarized sperm calculated in each repeat for AI or AR sperm. Data are expressed as mean percentage ± SEM; n = 7; ***P ≤ 0.001.

FIG. 3

The capacitation-associated hyperpolarization is dependent on the extracellular HCO3− concentration. Cauda epididymal sperm were capacitated in Whitten Hepes-buffered medium containing BSA and different HCO3− concentrations during 1 h and loaded with DiSBAC2(3), as described in the Materials and Methods, for 30 min. A) DiSBac2(3) does not affect capacitation-associated phosphorylation processes. Sperm incubated in conditions that support (CAP) or do not support (NON) capacitation for 1 h and loaded with DiSBAC2(3), or not, for 30 min were analyzed by Western blot using anti-phospho PKA substrates (α-pPKAs, left panel) or anti-phosphotyrosine antibodies (α-PY, right panel). B–G) Flow cytometry analysis of Em of sperm incubated with increasing HCO3− concentrations. Two-dimensional fluorescence dots plots of DiSBAC2(3) versus PI of sperm incubated with 0 (B), 7 (C), 15 (D), 30 (E), and 45 (F) mM of HCO3−. G) The experiment was repeated three times and the percentage of hyperpolarized live sperm plotted versus the extracellular HCO3− concentration. Data represent average percentage values ± SEM; n = 3; ***P ≤ 0.001.

FIG. 4

cAMP agonists induced hyperpolarization in conditions that do not support capacitation. Cauda epididymal sperm were recovered in media without HCO3−, which does not support capacitation. Aliquots of the sperm suspension were incubated in conditions described below for an additional hour and then loaded with DiSBAC2(3), as described in the Materials and Methods, for 30 min. A–C) PI versus DiSBAC2(3) two-dimensional dot plots of sperm incubated in the absence of HCO3− (A, NON), in media in the presence of 24 mM HCO3− (B, CAP), or in the absence of HCO3− and in the presence of 1 mM db-cAMP and 100 μM IBMX (C, db-cAMP). The respective live populations were then analyzed for their hyperpolarization states using DiSBAC2(3) fluorescence histograms. Vertical arrows show the correspondence of the dot plots (A–C) with the histograms (D–F). Data in G represent mean percentage of hyperpolarized live sperm ± SEM; n = 3; ***P ≤ 0.001; NS, nonsignificant) from three independent experiments.

FIG. 5

PKA inhibition by H-89 blocks hyperpolarization of sperm incubated in capacitation-supporting media. Cauda epididymal sperm were incubated in media that support capacitation in the absence (A, CAP) or in the presence of increasing concentrations of H-89 (1, 5, 10, 20, and 50 μM; B–F, respectively) for 1 h and then loaded with DiSBAC2(3), as described in the Materials and Methods, for 30 min. Sperm populations were analyzed using DiSBAC2(3) versus PI two-dimensional dot plots of sperm in the aforementioned conditions. Three independent experiments were conducted and data (G) are presented as the mean ± SEM percentage of hyperpolarized live sperm (***P ≤ 0.001).

FIG. 6

Amiloride induced the sperm membrane hyperpolarization. Cauda epididymal sperm were incubated in the presence (B, CAP) or in the absence of HCO3− without (A, NON) or with the addition of increasing concentrations of amiloride (0, 5, 20, and 40 μM; C–E, respectively). After 1-h incubation in each condition, the sperm were loaded with DisBACa(3), as described in the Materials and Methods, for 30 min and then analyzed by flow cytometry. Data are presented as DiSBAC2(3) versus PI two-dimensional dot plots. Three independent experiments were conducted, and the mean ± SEM of hyperpolarized live sperm versus amiloride concentration is presented in F (***P ≤ 0.001). NS, nonsignificant.

FIG. 7

Clofilium, a SLO3 channel antagonist, blocked membrane hyperpolarization of sperm incubated in conditions that support capacitation. Cauda epididymal sperm were incubated in media that support capacitation in the absence (A, CAP) or in the presence of increasing concentrations of Clofilium (0.1, 0.5, 1, 5, 10, 25, and 50 μM; B–F, respectively). After 1 h, sperm were loaded with DiSBAC2(3) for 30 min, as described in the Materials and Methods. Data are presented as DiSBAC2(3) versus PI two-dimensional dot plots. Data from four independent experiments are presented in G as mean ± SEM percentage of hyperpolarized live sperm (**P ≤ 0.01 and ***P ≤ 0.001).

FIG. 8

Sperm from Slo3−/− mice do not undergo capacitation-associated hyperpolarization. Cauda epididymal sperm from control (A) and Slo3−/− (B) mice were incubated for 1 h in conditions that support capacitation and loaded with DiSBAC2(3), as described in the Materials and Methods, for 30 min. The capacitation-associated hyperpolarization was analyzed using DiSBAC2(3) versus PI two-dimensional fluorescence dots plots. Live sperm populations of sperm incubated under conditions that support capacitation from control and Slo3−/− mice were used for histogram analysis depicting percentage of sperm versus DiSBAC2(3) fluorescence (C and D, respectively). Merged data are presented in E. Three independent experiments were conducted, and the mean ± SEM of hyperpolarized live sperm are presented (F) (***P ≤ 0.001). G and H) Western blots using anti-p-PKAS or anti-PY antibodies of sperm extracts from control mice (G) and Slo3−/− mice (H) incubated in conditions that support (CAP) or do not support (NON) capacitation in the presence or absence of 50 μM of clofilium, as indicated in the figure text.