Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca²⁺]_isignal in human sperm

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.

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm

“…Others have observed a biphasic response consisting of a maximal transient peak signal at ∼50–100 seconds followed by a rapid decay to a minimum at ∼150–250 seconds, followed by a lower level signal that was sustained for up to 800 seconds (Strunker et al, 2011; Brenker et al, 2018b ). The sustained component of the biphasic response is likely due to opening of store-operated channels, which promote calcium influx ( Morris et al, 2015 ). The longer-lived calcium response in our studies likely precluded observation of the low-level, sustained signal and was likely due to the following factors: (1) the use of probenecid to block dye efflux, (2) the use of centrifugation to prepare sperm instead of the “swim-up” method, (3) low pH/low K + buffer containing 0.2 mM Ca 2+ instead of human tubal fluid, and (4) the use of Pluronic F-127 to increase dye uptake by others (Strunker et al, 2011).…”

Male infertility in mice lacking the store-operated Ca2+ channel Orai1

“…Previous studies have suggested that store-operated Ca 2+ channels can contribute to the nature of progesterone-mediated Ca 2+ signals in human sperm [26], and thus regulate their hyperactivation in the female reproductive tract [27;28]. Indeed, the testis expresses a number of calcium channels, including the testis-specific CATSPER, voltage-dependent Ca 2+ channels and Ca 2+ -permeable TRP channels [1].…”

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis

“…Indeed, STIM and Orai proteins were documented in human ( 49 ) and mouse sperm ( 60 ) by Western blot and immunocytochemistry. Using cell-penetrating peptides attached to a protein domain that stimulates STIM–Orai activity, Morris et al ( 61 ) were able to induce store-operated Ca 2+ activity at the neck of human sperm, downstream of CatSper stimulation by progesterone. Thus, these findings strongly suggest that as with SPCA, this store-operated Ca 2+ entry complex is spatially separated from our observations of Ca 2+ increase located over the acrosome/apical-acrosome during ACR.…”