Abstract
Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca2+]i signalling of human sperm, including a contribution to the transient [Ca2+]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localized to the flagellum, by progesterone. To further investigate the potential involvement of SOCs in the generation of [Ca2+]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM–Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca2+. Resting [Ca2+]i temporarily decreased upon application of KIKKK peptide (3–4 min), but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence-specific. However, in cells pretreated with KIKKK, the transient [Ca2+]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pretreated with scrambled KIKKK peptide. Examination of single-cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 min in a subpopulation of cells. We hypothesize that SOCs contribute to the progesterone-induced [Ca2+]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca2+]i transient.
Highlights
[Ca2+]i plays a central role in the control of sperm function
CatSper, a sperm-specific cation channel localised to the flagellum, is the primary Ca2+-influx channel in mammalian sperm, is central to the regulation of [Ca2+]i and experiments in CatSper-null mice have shown that the channels play a key role in both regulation of motility and the early phase of zona pellucida-induced acrosome [Ca2+]i signalling (Carlson et al, 2003, Xia and Ren, 2009)
Release of Ca2+ stored in the acrosome is required for completion of the acrosome reaction and the Ca2+ stored at the sperm neck plays a key role in regulation of motility (Alasmari et al, 2013, Costello et al, 2009)
Summary
[Ca2+]i plays a central role in the control of sperm function. Maturation of the ejaculated sperm to acquire competence to fertilise (capacitation), the regulation of motility pattern (behaviour and chemotaxis) and control of the secretion of the acrosomal vesicle (acrosome reaction), which releases hydrolytic enzymes and exposes proteins required for fertilisation at the sperm surface, are all controlled separately through [Ca2+]i signals. In a sub-population of cells this plateau phase includes repeated [Ca2+]i elevations (oscillations) (Aitken and McLaughlin, 2007, Harper et al , 2004, Kirkman-Brown et al , 2004, Sanchez-Cardenas et al , 2014) which are typically irregular/chaotic but are ‘organised’ by ryanodine and by caffeine, consistent with Ca2+-induced-Ca2+-release (CICR) from the store at the sperm neck (Harper et al, 2004) These oscillations modify flagellar beat (Harper et al, 2004) and may suppress the acrosome reaction (Sanchez-Cardenas et al, 2014). To further investigate the potential involvement of SOCs in the generation of [Ca2+]i signals in human sperm, we have used a CPP (KIKKK) that mimics the basic patch on the SOAR region of STIM. KIKKK may act directly to stimulate CCE by binding Orai or may interact with the acidic patch on STIM, interfering with the refolding that would normally render STIM quiescent upon store refilling
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