Limits...
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.However, in cells pretreated with KIKKK, the transient [Ca(2+)]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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus

Pretreatment with KIKK prolongs the progesterone-induced [Ca2+]i transient in human sperm. (a) Effects of 5 µM KIKKK peptide (red) and 5 µM scrambled KIKKK peptide (grey) on progesterone-induced biphasic [Ca2+]i response in human sperm. Black shows response to progesterone in cells with no pretreatment. Each trace is the mean of Rtot plots (average of all cells in an experiment) from nine experiments (±SEM). Arrows indicate time of addition of progesterone. (b) Enlarged plot of boxed section from (a) with error bars (±SEM). (c) Mean rate of decay of Rtot (from 1.5 to 2 min after application of progesterone; mean ± SEM from nine sets of parallel experiments). **P < 0.01 compared with KIKKK pretreatment (paired t-test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4487447&req=5

GAV019F2: Pretreatment with KIKK prolongs the progesterone-induced [Ca2+]i transient in human sperm. (a) Effects of 5 µM KIKKK peptide (red) and 5 µM scrambled KIKKK peptide (grey) on progesterone-induced biphasic [Ca2+]i response in human sperm. Black shows response to progesterone in cells with no pretreatment. Each trace is the mean of Rtot plots (average of all cells in an experiment) from nine experiments (±SEM). Arrows indicate time of addition of progesterone. (b) Enlarged plot of boxed section from (a) with error bars (±SEM). (c) Mean rate of decay of Rtot (from 1.5 to 2 min after application of progesterone; mean ± SEM from nine sets of parallel experiments). **P < 0.01 compared with KIKKK pretreatment (paired t-test).

Mentions: We then investigated whether KKKK could affect Ca2+ signals induced by the CatSper agonist progesterone. Experiments were carried out as ‘parallel’ sets, including a control to assess the effect of progesterone with no pretreatment and experiments using pretreatment with KIKKK and with scrambled KIKKK peptide. Stimulation of human sperm with 3 μM progesterone induces a biphasic [Ca2+]i response, comprising an early [Ca2+]i transient which decays within 2 min, followed by a slowly developing sustained component (Blackmore et al., 1990; Kirkman-Brown et al., 2000). This biphasic response was clearly visible in Rtot plots (mean normalized responses of all cells in an experiment) of control experiments (Fig. 2a). Exposure to 5 μM KIKKK CPP prior to stimulation with 3 μM progesterone did not affect the mean amplitude or rise time of the [Ca2+]i transient in Rtot traces (P > 0.75 compared with parallel controls and cells pretreated with scrambled peptide; Fig. 2a). However, in KIKKK-pretreated cells, the decay of the transient was clearly slower than in control experiments (untreated and pretreated with scrambled peptide). During the steepest part of the falling phase of the [Ca2+]i transient (from 1.5 to 2 min after progesterone application), the rate of decay was significantly reduced in KIKKK-pretreatment experiments compared with parallel controls (no pretreatment and scrambled peptide pretreatment; Fig. 2c), such that in 8 of 9 experiments the amplitude of the early sustained response (Rtot assessed 2.5–3.3 min after application of progesterone) was significantly greater in cells pretreated with KIKKK (Fig. 2b; P < 0.03; paired t-test). This difference between control and pretreated cells persisted into the plateau phase of the [Ca2+]i signal in some sets of experiments, but this was inconsistent and at 7.5 min after progesterone addition the three conditions did not differ statistically (P > 0.2).Figure 2


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)

Pretreatment with KIKK prolongs the progesterone-induced [Ca2+]i transient in human sperm. (a) Effects of 5 µM KIKKK peptide (red) and 5 µM scrambled KIKKK peptide (grey) on progesterone-induced biphasic [Ca2+]i response in human sperm. Black shows response to progesterone in cells with no pretreatment. Each trace is the mean of Rtot plots (average of all cells in an experiment) from nine experiments (±SEM). Arrows indicate time of addition of progesterone. (b) Enlarged plot of boxed section from (a) with error bars (±SEM). (c) Mean rate of decay of Rtot (from 1.5 to 2 min after application of progesterone; mean ± SEM from nine sets of parallel experiments). **P < 0.01 compared with KIKKK pretreatment (paired t-test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

GAV019F2: Pretreatment with KIKK prolongs the progesterone-induced [Ca2+]i transient in human sperm. (a) Effects of 5 µM KIKKK peptide (red) and 5 µM scrambled KIKKK peptide (grey) on progesterone-induced biphasic [Ca2+]i response in human sperm. Black shows response to progesterone in cells with no pretreatment. Each trace is the mean of Rtot plots (average of all cells in an experiment) from nine experiments (±SEM). Arrows indicate time of addition of progesterone. (b) Enlarged plot of boxed section from (a) with error bars (±SEM). (c) Mean rate of decay of Rtot (from 1.5 to 2 min after application of progesterone; mean ± SEM from nine sets of parallel experiments). **P < 0.01 compared with KIKKK pretreatment (paired t-test).
Mentions: We then investigated whether KKKK could affect Ca2+ signals induced by the CatSper agonist progesterone. Experiments were carried out as ‘parallel’ sets, including a control to assess the effect of progesterone with no pretreatment and experiments using pretreatment with KIKKK and with scrambled KIKKK peptide. Stimulation of human sperm with 3 μM progesterone induces a biphasic [Ca2+]i response, comprising an early [Ca2+]i transient which decays within 2 min, followed by a slowly developing sustained component (Blackmore et al., 1990; Kirkman-Brown et al., 2000). This biphasic response was clearly visible in Rtot plots (mean normalized responses of all cells in an experiment) of control experiments (Fig. 2a). Exposure to 5 μM KIKKK CPP prior to stimulation with 3 μM progesterone did not affect the mean amplitude or rise time of the [Ca2+]i transient in Rtot traces (P > 0.75 compared with parallel controls and cells pretreated with scrambled peptide; Fig. 2a). However, in KIKKK-pretreated cells, the decay of the transient was clearly slower than in control experiments (untreated and pretreated with scrambled peptide). During the steepest part of the falling phase of the [Ca2+]i transient (from 1.5 to 2 min after progesterone application), the rate of decay was significantly reduced in KIKKK-pretreatment experiments compared with parallel controls (no pretreatment and scrambled peptide pretreatment; Fig. 2c), such that in 8 of 9 experiments the amplitude of the early sustained response (Rtot assessed 2.5–3.3 min after application of progesterone) was significantly greater in cells pretreated with KIKKK (Fig. 2b; P < 0.03; paired t-test). This difference between control and pretreated cells persisted into the plateau phase of the [Ca2+]i signal in some sets of experiments, but this was inconsistent and at 7.5 min after progesterone addition the three conditions did not differ statistically (P > 0.2).Figure 2

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.However, in cells pretreated with KIKKK, the transient [Ca(2+)]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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus