Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm

Abstract

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca 2+ ] i and the occurrence of AE. Em hyperpolarization turned [Ca 2+ ] i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca 2+ ] i increase. The addition of progesterone (P4) or K + to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca 2+ ] i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca 2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction

“…In vitro, the incubation of human sperm in capacitating conditions is associated with changes in the plasma membrane [ 87 ], protein phosphorylation [ 88 ], membrane potential (Vm) hyperpolarization [ 89 ] and elevation of the [Ca 2+ ]i and pHi. All are necessary for cells to undergo the AR [ 47 , 90 ]. The AR is more likely to be suppressed in cells which exhibit Vm and pH-dependent [Ca 2+ ]i oscillations [ 90 , 91 ], which occur spontaneously in ~35% of the capacitated cells [ 47 ], and ~20% develop oscillations in response to the P4 [ 30 ].…”

“…All are necessary for cells to undergo the AR [ 47 , 90 ]. The AR is more likely to be suppressed in cells which exhibit Vm and pH-dependent [Ca 2+ ]i oscillations [ 90 , 91 ], which occur spontaneously in ~35% of the capacitated cells [ 47 ], and ~20% develop oscillations in response to the P4 [ 30 ]. The Vm and pHi changes associated with capacitation are heterogeneous [ 89 , 92 , 93 , 94 , 95 ].…”

“…The normalisation of the background-corrected fluorescence data was, as described previously [ 90 ], using ΔF = ((F − Fbl)/Fbl) × 100%, where ΔF is the percentage change in intensity; F is the fluorescence intensity at time t; and Fbl is the mean baseline fluorescence prior to the compound addition. The fluorescence data were normalized to the maximum ΔF response to the ionophore and the data expressed as ΔF (% ionophore).…”

High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding

“…In addition, FBC in live sperm itself emerges as a limitless and therefore valuable tool. Considering only the proper Frontiers in Cell and Developmental Biology frontiersin.org combination of fluorophores, this system could be applied in the future to determine novel blockers of acrosome reaction (by using for example the mouse model with EGFP in their acrosomes (Hasuwa et al, 2010) or FM4-64 when the desired extension to humans is performed (Balestrini et al, 2021)), intracellular [Ca 2+ ] i increase (Fluo-4, Fluo-3 or Fura), intracellular pH alkalinization (BCECF), as well as other biochemical and/or functional events associated with sperm capacitation.…”

A Minimal Model Shows that a Positive Feedback Loop Between sNHE and SLO3 can Control Mouse Sperm Capacitation

“…Our model could be adapted and applied to human sperm by adjusting the activation curves of SLO3 and taking into account the voltage-gated hydrogen channel 1, HV1, which has been shown to be the dominant proton conductance in human sperm (Lishko et al, 2010). However, as this transporter is not activated by hyperpolarization as it is the case for sNHE, another feedback loop could take place, between hSLO3 and Catsper, as it has been shown that the variations in hyperpolarization and intracellular calcium concentration of human spermatozoa are interconnected during capacitation (Balestrini et al, 2021). The proposed minimal model has allowed us to identify the core molecular mechanism which is likely to control murine sperm capacitation.…”