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

Morris J, Jones S, Howl J, Lukanowska M, Lefievre L, Publicover S - Mol. Hum. Reprod. (2015)

Bottom Line: Resting [Ca(2+)]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.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 [Ca(2+)]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca(2+)]i transient.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

No MeSH data available.


Related in: MedlinePlus

Effect of cell-penetrating peptides on resting [Ca2+]i in human sperm. In this, and subsequent figures, data are expressed as % change in fluorescence. (a) Effect of 5 µM scrambled KIKK peptide, and (b) effect of 5 µM KIKK peptide. Arrows indicate time of addition of peptide. Traces show mean (±SEM) Rtot for eight experiments. (c) Single-cell traces showing transient fall in [Ca2+]i upon application of KIKKK. (d) Effect of KIKKK in cells showing spontaneous [Ca2+]i oscillations. In a few cells, oscillations ‘paused’ briefly (blue trace) but in the majority they persisted and kinetics appeared unchanged.
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GAV019F1: Effect of cell-penetrating peptides on resting [Ca2+]i in human sperm. In this, and subsequent figures, data are expressed as % change in fluorescence. (a) Effect of 5 µM scrambled KIKK peptide, and (b) effect of 5 µM KIKK peptide. Arrows indicate time of addition of peptide. Traces show mean (±SEM) Rtot for eight experiments. (c) Single-cell traces showing transient fall in [Ca2+]i upon application of KIKKK. (d) Effect of KIKKK in cells showing spontaneous [Ca2+]i oscillations. In a few cells, oscillations ‘paused’ briefly (blue trace) but in the majority they persisted and kinetics appeared unchanged.

Mentions: We have shown previously that CPPs enter mammalian spermatozoa rapidly, with a half-time between 1 and 3 min, dependent on the CPP employed (Jones et al., 2013). Therefore, rather than comparing separate groups of treated and non-treated cells, the effects of CPPs on resting [Ca2+]i was monitored during application. Exposure of human sperm to 5 µM KIKKK induced a transient decrease in [Ca2+]i that was detectable within <30 s, was maximal at ≈90 s (mean decrease in Rtot of 6.6 ± 3.2%; n = 8 experiments; P < 0.05 compared with pre-stimulation) and recovered to control level within ∼3–4 min (Fig. 1). Application of scrambled KIKKK induced a similar effect (P < 0.025 versus pre-stimulation; Fig. 1). The amplitude of this effect varied between cells (Fig. 1c), but both KIKKK and scrambled KIKKK induced a significant decrease in fluorescence (see Methods) in ≈65% of cells. Twenty percent of cells (19.2 ± 2.7%; n = 18 experiments) were spontaneously active, generating [Ca2+]i oscillations under control conditions. In ∼40% of these cells, application of CPPs briefly arrested and reset this activity (KIKKK = 42.8 ± 7.8%, n = 9 experiments; scrambled KIKKK = 39.8 ± 9.0%, n = 9 experiments; t > 0.8), but oscillation amplitude and kinetics were maintained, indicating that basal Ca2+ influx and Ca2+ clearance mechanisms were not affected by KIKKK or scrambled KIKKK peptide treatment (Fig. 1d).Figure 1


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

Morris J, Jones S, Howl J, Lukanowska M, Lefievre L, Publicover S - Mol. Hum. Reprod. (2015)

Effect of cell-penetrating peptides on resting [Ca2+]i in human sperm. In this, and subsequent figures, data are expressed as % change in fluorescence. (a) Effect of 5 µM scrambled KIKK peptide, and (b) effect of 5 µM KIKK peptide. Arrows indicate time of addition of peptide. Traces show mean (±SEM) Rtot for eight experiments. (c) Single-cell traces showing transient fall in [Ca2+]i upon application of KIKKK. (d) Effect of KIKKK in cells showing spontaneous [Ca2+]i oscillations. In a few cells, oscillations ‘paused’ briefly (blue trace) but in the majority they persisted and kinetics appeared unchanged.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487447&req=5

GAV019F1: Effect of cell-penetrating peptides on resting [Ca2+]i in human sperm. In this, and subsequent figures, data are expressed as % change in fluorescence. (a) Effect of 5 µM scrambled KIKK peptide, and (b) effect of 5 µM KIKK peptide. Arrows indicate time of addition of peptide. Traces show mean (±SEM) Rtot for eight experiments. (c) Single-cell traces showing transient fall in [Ca2+]i upon application of KIKKK. (d) Effect of KIKKK in cells showing spontaneous [Ca2+]i oscillations. In a few cells, oscillations ‘paused’ briefly (blue trace) but in the majority they persisted and kinetics appeared unchanged.
Mentions: We have shown previously that CPPs enter mammalian spermatozoa rapidly, with a half-time between 1 and 3 min, dependent on the CPP employed (Jones et al., 2013). Therefore, rather than comparing separate groups of treated and non-treated cells, the effects of CPPs on resting [Ca2+]i was monitored during application. Exposure of human sperm to 5 µM KIKKK induced a transient decrease in [Ca2+]i that was detectable within <30 s, was maximal at ≈90 s (mean decrease in Rtot of 6.6 ± 3.2%; n = 8 experiments; P < 0.05 compared with pre-stimulation) and recovered to control level within ∼3–4 min (Fig. 1). Application of scrambled KIKKK induced a similar effect (P < 0.025 versus pre-stimulation; Fig. 1). The amplitude of this effect varied between cells (Fig. 1c), but both KIKKK and scrambled KIKKK induced a significant decrease in fluorescence (see Methods) in ≈65% of cells. Twenty percent of cells (19.2 ± 2.7%; n = 18 experiments) were spontaneously active, generating [Ca2+]i oscillations under control conditions. In ∼40% of these cells, application of CPPs briefly arrested and reset this activity (KIKKK = 42.8 ± 7.8%, n = 9 experiments; scrambled KIKKK = 39.8 ± 9.0%, n = 9 experiments; t > 0.8), but oscillation amplitude and kinetics were maintained, indicating that basal Ca2+ influx and Ca2+ clearance mechanisms were not affected by KIKKK or scrambled KIKKK peptide treatment (Fig. 1d).Figure 1

Bottom Line: Resting [Ca(2+)]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.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 [Ca(2+)]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca(2+)]i transient.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

No MeSH data available.


Related in: MedlinePlus