The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation

Abstract

Study question: What is the prevalence of defects in the Ca(2+)-signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF?

Summary answer: This study identifies, for the first time, the prevalence of Ca(2+) store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca(2+) signalling (Ca(2+) store mobilization) for fertilization at IVF.

What is known already: Sperm motility and hyperactivation (HA) are important for fertility, mice with sperm incapable of HA are sterile. Recently, there has been significant progress in our knowledge of the factors controlling these events, in particular the generation and regulation of calcium signals. Both pH-regulated membrane Ca(2+) channels (CatSper) and Ca(2+) stores (potentially activating store-operated Ca(2+) channels) have been implicated in controlling HA.

Study design, size, and duration: This was a prospective study examining a panel of 68 donors and 181 sub-fertile patients attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and ICSI. Twenty-five of the donors gave a second sample (∼4 weeks later) to confirm consistency/reliability of the recorded responses. Ca(2+) signalling was manipulated using three agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation of CatSper channels, potentially enhancing mobilization of stored Ca(2+) by CICR) and 4-aminopyridine (4-AP) (effect on pH equivalent to NH4Cl and mobilizes stored Ca(2+)). The broad-spectrum phosphodiesterase inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was also used for comparison. For patient samples, an aliquot surplus to requirements for IVF/ICSI treatment was examined, allowing direct comparison of Ca(2+) signalling and motility data with functional competence of the sperm.

Materials, setting, methods: The donors and sub-fertile patients were screened for HA (using CASA) and changes in intracellular Ca(2+) were assessed by loading with Fura-2 and measuring fluorescence using a plate reader (FluoStar).

Main results and the role of chance: The relative efficacy of the stimuli in inducing HA was 4-AP >> IBMX > progesterone. NH4Cl increased [Ca(2+)]i similarly to 4-AP and progesterone but did not induce a significant increase in HA. Failure of samples to generate HA (no significant increase in response to stimulation with 4-AP) was seen in just 2% of research donors but occurred in 10% of IVF patients (P = 0.025). All donor samples generated a significant [Ca(2+)]i increase when stimulated with 4-AP but 3.3% of IVF and 28.6% of ICSI patients failed to respond. Amplitudes of HA and [Ca(2+)]i responses to 4-AP were correlated with fertilization rate at IVF (P= 0.029; P = 0.031, respectively). Progesterone reliably induced [Ca(2+)]i responses (97% of donors, 100% of IVF patients) but was significantly less effective than 4-AP in inducing HA. Twenty seven per cent of ICSI patients failed to generate a [Ca(2+)]i response to progesterone (P= 0.035). Progesterone-induced [Ca(2+)]i responses were correlated with fertilization rate at IVF (P= 0.037) but induction of HA was not. In donor samples examined on more than one occasion consistent responses for 4-AP-induced [Ca(2+)]i (R(2) = 0.97) and HA (R(2) = 0.579) were obtained. In summary, the data indicate that defects in Ca(2+) signalling leading to poor HA do occur and that ability to undergo Ca(2+) -induced HA affects IVF fertilizing capacity. The data also confirm that release of stored Ca(2+) is the crucial component of Ca(2+) signals leading to HA and that Ca(2+) store defects may therefore underlie HA failure.

Limitations, reasons for caution: This is an in vitro study of sperm function. While the repeatability of the [Ca(2+)]i and HA responses in samples from the same donor were confirmed, data for patients were from 1 assessment and thus the robustness of the failed responses in patients' needs to be established. The focus of this study was on using 4AP, which mobilizes stored Ca(2+) and is a potent inducer of HA. The n values for other agonists, especially calcium assessments, are smaller.

Wider implications of the findings: Previous studies have shown a significant relationship between basal levels of HA, calcium responses to progesterone and IVF fertilization rates. Here, we have systematically investigated the ability/failure of human sperm to generate Ca(2+) signals and HA in response to targeted pharmacological challenge and, related defects in these responses to IVF success. [Ca(2+)]i signalling is fundamental for sperm motility and data from this study will lead to assessment of the nature of these defects using techniques such as single-cell imaging and patch clamping.

Study funding/competing interest(s): Resources from a Wellcome Trust Project Grant (#086470, Publicover and Barratt PI) primarily funded the study. The authors have no competing interests.

Figure 1

Comparison of four different agonists on HA) in two populations (donors and IVF patients). Box and whisker plots illustrating the data distribution for HA in the baseline (control) and samples treated with 4-AP, IBMX, progesterone and NH₄Cl from donors and IVF patients. The boxes represent the interquartile range and lines within them are the medians. The number in brackets is the sample size. *Highlights that the agonist-induced HA is significantly different to baseline, (a) highlights a significant difference between responses to 4-AP and all other agonists (b) highlights a significant difference between responses to IBMX compared with progesterone and NH₄Cl and (c) highlights a significant difference between responses to progesterone and NH₄Cl. Significance was considered as P < 0.05 assessed by one-way ANOVA and non-parametric ANOVA on ranks Kruskal–Wallis test. Not all samples were tested with each of the agonists.

Figure 2

Ca²⁺ ratio in response to 4-AP and progesterone in three populations (donors, IVF and ICSI patients). Intracellular Ca²⁺ responses induced by 4-AP (upper panel) and progesterone (lower panel) in donors (4-AP n = 36, progesterone n = 37), IVF (4-AP n = 61, progesterone n = 68) and ICSI patients (4-AP n = 7, progesterone n = 11). Each trace shows mean of n fluorimetric (population) responses ± SE. Agonists were added (indicated by black arrow) at 100 s after acquisition of 20 readings at resting level (R). The data for each sample were normalized to pre-stimulus (R) level to facilitate comparison. *Significant difference between the ratio at the peak (P) and the initial resting level (R), (a) significant difference of ratio at peak between donor and ICSI patients, (b) significant difference of ratio at peak between IVF and ICSI patients. ^$Significant difference between the ratio at the sustained phase (S) between donor and ICSI patients (P < 0.001) and between IVF and ICSI patients (P = 0.007). There was no significant difference in the sustained response with progesterone between the groups. Significance was considered as P < 0.05 assessed non-parametric ANOVA on ranks Kruskal–Wallis test.

Figure 3

Intracellular Ca²⁺ in response to 4-AP from four IVF patients including two with a failed Ca²⁺ response. Inset shows % HA from the same patients labelled with the same colours; white bars show % HA in the baseline (control) and coloured bars show % HA in samples treated with 4-AP. Patients 3 and 4 showed a failed intracellular Ca²⁺ and HA responses to 4-AP (IVF fertilization rates for patients 3 and 4–39% and 6% respectively). 4-AP was added to suspensions at 100 s after acquisition of 20 readings at resting level (R) indicated by black arrow. The HA data (inset) are the mean ± SD. *Values are significantly different to baseline (P < 0.05).

Figure 4

Relationship between increment in HA (% cells) stimulated by 4-AP and IVF fertilization rate. (A) 4-AP-induced increment in HA (% cells) was significantly correlated to fertilization rate (R_s = 0.18; P = 0.031, n = 145). (B) Expression of these data in four defined groups according to fertilization rate: FR 1 ≤25%, FR2 >25–≤50%, FR3 >50–≤75%, FR4 >75%. Box and whisker plots show the 4-AP-induced increment in HA for the samples from patients in each group. The boxes represent the inter-quartile range and lines within them are the medians. The number in brackets is the sample size. Significance was assessed by one-way ANOVA.

Figure 5

Model of the calcium-signalling cascade in the human spermatozoon: key points in the pathway affected in sub-fertile men (adapted from Barratt and Publicover, 2012). White boxes show compounds used to manipulate pH_i (NH₄Cl, 4AP), stored calcium (4AP) and cAMP (IBMX). Yellow boxes show signalling components activated by these compounds (pHi, Ca²⁺, cAMP). Blue and red arrows show Ca²⁺-signalling pathways involving CatSper and the calcium store, respectively. The Ca²⁺-store pathway may involve activation of SOCs (not shown; Lefievre et al., 2012). Dashed blue arrow shows the mobilization of stored Ca²⁺ downstream of CatSper activation by CICR. This occurs in a minority of the cells, is sensitive to modulation (e.g. by capacitation) and is responsible for CatSper-mediated hyperactivation. Dashed grey arrow shows possible modulation of Ca²⁺-store mobilization by progesterone through CatSper-independent mechanisms (Sagare-Patil et al., 2012). X represents unknown target mechanism/pathway whereby following mobilization of stored Ca²⁺ hyperactivation is stimulated. ? represents potential pathway subject to further experimentation. The amplitude of the calcium transient (stimulated by both 4AP and progesterone) and the hyperactivation response (to 4AP) were significantly related to IVF fertilization rates suggesting the occurrence of important abnormalities in the Ca²⁺-signalling pathways mediated by CatSper (blue) and the calcium store (red). The calcium signal (4AP and progesterone) in the ICSI patients was significantly lower than in the donors or the IVF patients (∼25%; Fig. 2), providing further evidence of abnormalities in the CatSper functioning/operating complex and calcium store in male infertility. Examination of individual cases demonstrated ∼10% of men undergoing IVF had defective calcium hyperactivation. Although the data are limited, a number of these men did not show a hyperactivation response to IBMX (probably through cAMP, black arrows, ?). Previous studies have indicated that, in humans, increases in cAMP are do not lead to changes in [Ca²⁺]_i (Brenker et al., 2012), thus these men may suffer from a specific defect in hyperactivation as opposed to a Ca²⁺-signalling deficit (Supplementary data, Table SII).