Synergistic activation of CatSper Ca²⁺ channels in human sperm by oviductal ligands and endocrine disrupting chemicals

Abstract

STUDY QUESTION

Does the chemosensory activation of CatSper Ca²⁺ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists?

SUMMARY ANSWER

We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically.

WHAT IS KNOWN ALREADY

In human sperm, the sperm-specific CatSper channel controls the intracellular Ca²⁺ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization.

STUDY DESIGN, SIZE, DURATION

For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions.

PARTICIPANTS/MATERIALS, SETTING, METHODS

We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca²⁺ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method.

MAIN RESULTS AND THE ROLE OF CHANCE

Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca²⁺ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel.

LIMITATIONS, REASONS FOR CAUTION

This is an in vitro study. Future studies have to assess the physiological relevance in vivo.

WIDER IMPLICATIONS OF THE FINDINGS

These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the “No Observed Adverse Effect Level (NOAEL)” or “No Observed Effect Concentration (NOEC)” are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the ‘Innovative Medical Research’ of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen’s Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests.

Introduction

Male fertility disorders are common and their cause is often unknown (Skakkebaek et al., 2016), but exposure to endocrine disrupting chemicals (EDCs) is suspected to be involved (Gore et al., 2015). EDCs are usually examined in studies aiming to elucidate the action of single substances on organisms, organs, tissues, cells or particular proteins, whereas the quantitative analysis of combined EDC actions has received less attention (Kortenkamp, 2014). In daily life, however, humans are exposed to a plethora of EDCs originating from a broad range of sources. Therefore, human body fluids are contaminated with complex mixtures of EDCs (Kortenkamp, 2014) that act via multiple binding sites and mechanisms. This might create not only additive and sub-additive but also synergistic actions among the chemicals. A prime example for combined EDC actions is human sperm: at concentrations present in human body fluids, structurally diverse EDCs activate the sperm-specific Ca²⁺ channel CatSper (Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016) that is essential for sperm function and male fertility (Williams et al., 2015). Thereby, the chemicals interfere with various sperm functions (Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016). In fact, EDCs mimic the action of the oviductal CatSper ligands progesterone and prostaglandins (Schiffer et al., 2014; Rehfeld et al., 2016) that act via a steroid- and prostaglandin-binding site, respectively, to activate the channel (Lishko et al., 2011; Strünker et al., 2011). It has been proposed that progesterone binds to the receptor alpha/beta hydrolase domain-containing protein 2 (ABHD2) (Miller et al., 2016): at rest, CatSper is inhibited by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the flagellar membrane. Upon progesterone binding, ABHD2 degrades 2-AG and, thereby, relieves CatSper from inhibition (Miller et al., 2016). CatSper activation by prostaglandins, however, does not involve ABHD2 (Miller et al., 2016) but an as yet unknown receptor. Progesterone- and prostaglandin-induced Ca²⁺ influx has been implicated in sperm capacitation (Sumigama et al., 2015), chemotaxis (Eisenbach and Giojalas, 2006; Publicover et al., 2008), hyperactivation (Alasmari et al., 2013a, 2013b; Rennhack et al., 2018) and acrosomal exocytosis (Tamburrino et al., 2014; Rennhack et al., 2018). Individual EDCs compete either with progesterone or prostaglandins to activate CatSper and, thereby, desensitize sperm for the respective physiological ligand (Schiffer et al., 2014; Rehfeld et al., 2016). Notably, when applied in complex low-dose mixtures, EDCs seem to co-operate to elevate Ca²⁺ levels in human sperm (Schiffer et al., 2014; Rehfeld et al., 2016). Thus, human sperm can serve as a model to elucidate the action of EDC mixtures in mechanistic terms. To this end, we investigated the combined action of progesterone and prostaglandins as well as that of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol in sperm. BCSA and α-Zearalenol act via the steroid- and prostaglandin-binding site, respectively (Schiffer et al., 2014; Rehfeld et al., 2016).

Materials and Methods

Reagents

BCSA was obtained from Life Chemicals (Munich, Germany). Progesterone-3-(O-carboxymethyl)oxime (CMO-progesterone), α-zearalenol, progesterone, ionomycin, prostaglandin E1 (PGE1), prostaglandin F1-α (PGF1-α) and NH₄Cl were obtained from Sigma-Aldrich (MO, USA). Stock solutions of ligands/chemicals were prepared in dimethylsulfoxide. NH₄Cl was dissolved in buffer. Human serum albumin was obtained from Irvine Scientific (CA, USA). Ca²⁺ indicators were obtained from Invitrogen (CA, USA).

Semen samples

Human semen samples were obtained from healthy donors with their prior written consent. Semen samples were produced by masturbation and ejaculated into plastic containers. After ejaculation, the samples were allowed to liquefy for 15–30 min at 37°C.

Ethical approval

Approval was obtained from the ethical committees of the medical association Westfalen-Lippe and the medical faculty of the University of Münster: 4INie.

Purification of motile sperm cells via swim-up

Motile spermatozoa were isolated from the ejaculates by swim-up separation for 1 h at 37°C in human tubular fluid (HTF) medium containing (all mM): 97.8 NaCl, 4.69 KCl, 0.2 MgSO₄, 0.37 KH₂PO₄, 2.04 CaCl₂, 0.33 Na-pyruvate, 21.4 Na-lactate, 2.78 glucose, 21 HEPES, and 4 NaHCO₃, adjusted to pH 7.3–7.4 with NaOH, as described before (Strünker et al., 2011). The sperm were washed two times (700g, 20 min, room temperature) and the sperm concentration was adjusted to 1 × 10⁷/ml in HTF fortified with human serum albumin (3 mg/ml) (HTF⁺).

Measurement of changes in [Ca²⁺]_i

Changes in [Ca²⁺]_i were measured in 384 multi-well plates in a fluorescence plate reader (FLUOstar Omega, BMG Labtech, Germany) at 30°C, as described previously (Schiffer et al., 2014). Sperm were loaded with the fluorescent Ca²⁺ indicator Fluo-4FF-AM, Fluo-4-AM or Mag-Fluo-4-AM (10 μM) for 45 min at 37°C. After incubation, excess dye was removed by centrifugation (700g, 10 min, room temperature). Sperm were resuspended in HTF at a concentration of 5 × 10⁶/ml. Each well was filled with 50 μl of the sperm suspension. Fluorescence was excited at 485 nm and emission was recorded at 520 nm with bottom optics. Fluorescence was recorded before and after injection of 25 μl of serially diluted ligands and ligand mixtures, of buffer with vehicle (negative control), and of a reference (progesterone 10 μM, PGE1 5 μM, and NH₄Cl 30 mM, in the experiments with Fluo-4FF and mag-Fluo-4; ionomycin 5 μM for experiments with Fluo-4) to determine the response to a [Ca²⁺]_i increase that saturates the indicator. The ligands, mixtures thereof, and controls were injected simultaneously using a multichannel pipette. Each condition was measured in duplicate and the two fluorescence traces were averaged. To determine the dose–response relations, the signal amplitudes were normalized to the reference and fitted (nonlinear, least squares) using a modified Hill equation: $y=Vmax∗xnkn+xn$⁠, with y being the effect of the ligand at concentration x, Vmax the maximal response amplitude, k the K_1/2 value and n the Hill coefficient of the fit.

Electrophysiology

We recorded from sperm in the whole-cell configuration, as described before (Strünker et al., 2011). Seals between pipette and sperm were formed either at the cytoplasmic droplet or in the neck region in standard extracellular solution containing (all mM): 135 NaCl, 5 KCl, 1 MgSO4, 2 CaCl₂, 5 glucose, 1 Na-pyruvate, 10 lactic acid, 20 HEPES, adjusted to pH 7.4 with NaOH. Monovalent currents were recorded in a sodium-based divalent-free solution (NaDVF) containing (all mM): 140 NaCl, 40 HEPES, and 1 EGTA, adjusted to pH 7.4 with NaOH; the pipette (10–15 MΩ) solution contained (all mM): 130 Cs-aspartate, 50 HEPES, 5 EGTA, 5 CsCl, adjusted to pH 7.3 with CsOH. Data were not corrected for liquid junction potentials.

Analysis of synergism

We employed curve-shift analysis (Zhao et al., 2010), curvilinear isobolographic analysis (Grabovsky and Tallarida, 2004), and the combination-index method (Chou and Talalay, 1984) to assess the nature of the combined action of the ligands.

Using the modified Hill equation: $y=VmaxB∗(B+beq)nBkBnB+(b+beq)nB$⁠, we extrapolated the expected additive response of the mixture as a function of b + b_eq, with (b) being the given concentration of ligand B in the mixture. This procedure yields the predicted additive dose–response relation for the mixtures.

Curvilinear isobolographic analysis (Grabovsky and Tallarida, 2004): the curvilinear isoboles indicate all possible mixtures of two ligands expected to evoke a given response level if the ligands act purely in an additive manner, plotted in a diagram that depicts the respective concentrations of the more effective ligand B (b) and the less effective ligand A (a) on the y- and x-axis, respectively. The isobole for a given relative response level (y) was calculated as a function of (a) with the following formula: $b(a)=by−beq(a)$ with b_y being the concentration of the more efficacious ligand B evoking that response level on its own, and b_eq(a) representing the concentration of ligand B equivalent to (a), calculated according to Equation (1). This analysis was performed for relative response levels up to the maximal level evoked by the more efficacious ligand B. For a given response level, the experimentally determined ligand mixture evoking that particular response level is plotted in the diagram. A superimposition of the experimentally determined mixtures on the respective isobole indicates additivity, whereas mixtures plotting below or above the isobole indicate synergism and sub-additivity, respectively.

The combination index (Chou and Talalay, 1984) was calculated using the effective concentrations (EC) evoking a particular relative response level, i.e. the concentration evoking 10% of the maximal response (EC10), 20% (EC20), 30% (EC30), etc., extrapolated from the dose–response relations for the individual ligands and the mixtures thereof with the following formula: $CI=(ECxmixAECxA)+(ECxmixBECxB)$⁠, with CI being the combination index, ECx_mixA and ECx_mixB the concentrations of ligand A and B in a given mixture evoking x% of the maximal relative response level, and ECx_A and ECx_B being the concentrations of ligand A and B that evoke that response level on their own. This analysis was used for relative response levels up to the maximal response level reached by both ligands. A CI = 1 indicates an additive action, a CI > 1 indicates sub-additivity, whereas a CI < 1 indicates synergism.

Data analysis

All data are presented as mean ± SD. Data analysis and fitting of dose–response relations were performed using Origin 2015 (OriginLab, Northampton, MA, USA).

Results

Synergistic activation of human CatSper by progesterone and PGE1

We studied Ca²⁺ signals in human sperm evoked by progesterone, PGE1 and mixtures thereof; the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored via the fluorescent Ca²⁺ indicator Fluo-4FF (K_D = 9.7 μM) that registers Ca²⁺ up to a concentration of about 100 μM. Progesterone and PGE1 evoked a rapid and transient Ca²⁺ increase (Fig. 1A, B); as a reference, to gauge the maximal response level, we recorded a Ca²⁺ response that saturated the indicator. Analysis of the normalized dose–response relations yielded constants of half-maximal activation (K_1/2) of 101 ± 113 nM (n = 24) for progesterone and 28 ± 13 nM for PGE1 (n = 18) (Fig. 1D). The amplitudes of the progesterone- and PGE1-induced Ca²⁺ signals saturated at relative response levels of 0.76 ± 0.1 (n = 24) and 0.49 ± 0.1 (n = 18), respectively (Fig. 1D). Thus, PGE1 is more potent but less efficacious than progesterone to evoke Ca²⁺ responses in human sperm. Next, we studied the Ca²⁺ signals evoked by 2:1 mixtures of progesterone and PGE1. Similar to the individual ligands, the mixtures evoked a rapid and dose-dependent Ca²⁺ transient (Fig. 1C). The nature of the combined action of the ligands was evaluated by curve-shift analysis (Zhao et al., 2010). The analysis of combined drug actions relies on the dose equivalence principle, also referred to as Loewe Additivity (Loewe and Muischnek, 1926). Quite general, from the individual dose–response relationships of progesterone and PGE1, one can determine for any concentration of PGE1 the equivalent concentration of progesterone evoking the same response level. Therefore, if progesterone and PGE1 acted purely additively, the response level evoked by a particular progesterone/PGE1 mixture could be predicted by transforming the PGE1 concentration in the mixture into the equivalent progesterone concentration. Accordingly, we modeled the dose–response relation for the 2:1 progesterone/PGE1 mixtures that is expected for a purely additive action (Fig. 1E, black) and compared it to the experimentally determined dose–response relation (Fig. 1E, red). The experimental relation saturated at a considerably higher response level (0.94 ± 0.08 versus 0.78 ± 0.05; n = 5) and was left-shifted towards lower ligand concentrations. For a given response level, we calculated the ratio [Prog + PGE1]_exp/[Prog + PGE1]_pred between the progesterone (Prog) and PGE1 concentration in a given mixture extrapolated from the experimentally determined dose–response relation and the progesterone and PGE1 concentration predicted by the modeled additive dose–response relation to reach that particular response level. For an additive action, this ratio is 1, whereas a ratio >1 and <1 indicates sub-additivity and synergism, respectively. At response levels ≥0.1, the ratio was less than 1 and it decreased with increasing response levels, i.e. higher ligand concentrations in the mixture, from 0.59 ± 0.29 to 0.02 ± 0.04 (n = 5) at response levels of 0.1 and 0.7, respectively (Fig. 1F). Altogether, these results indicate that progesterone and PGE1 activate CatSper synergistically and that the synergism is enhanced at high compared to low ligand concentrations. Of note, progesterone and PGE1 also acted synergistically in 1:1 or 1:2 mixtures (Supplementary Fig. S1). Furthermore, we studied the Ca²⁺ responses evoked by mixtures of ligands that compete for the same binding site to activate CatSper – competitive ligands cannot act synergistically (Chou and Talalay, 1984). Indeed, for mixtures of progesterone and progesterone-3-(O-carboxymethyl)oxime (CMO-progesterone) (Fig. 1G, H, Supplementary Fig. S2) or of PGE1 and PGF1-α (Fig. 1I, J; Supplementary Fig. S2), the experimentally determined dose–response relations superimposed on that predicted for a purely additive action. Consequently, at all response levels, the ratio [Prog + CMO-Prog]_exp/[Prog + CMO-Prog]_pred and [PGE1 + PGF1-α]_exp/[PGE1 + PGF1-α]_pred was ≥1 (Fig. 1F). Moreover, in addition to the curve-shift analysis, we employed isobolographic analysis (Grabovsky and Tallarida, 2004) (Supplementary Fig. S3 A–C, E) and the combination-index method (Chou and Talalay, 1984) (Supplementary Fig. S3F) to study the action of the mixtures. These methods confirmed that progesterone and PGE1, but not derivatives of progesterone or prostaglandins, act synergistically to increase [Ca²⁺]_i in human sperm. Of note, to unveil this synergistic ligand action, a medium- to low-affinity Ca²⁺ indicator is required, e.g. Fluo-4FF or mag-Fluo-4 (KD = 22 μM) (Supplementary Fig. S4): the common high-affinity indicator Fluo-4 (KD = 335 nM) already becomes saturated with Ca²⁺ during the responses evoked by progesterone and PGE1 alone (Brenker et al., 2014) (Supplementary Fig. S4). This masks not only the enhanced efficacy of progesterone compared to PGE1, but also the synergistic action of the ligands (Supplementary Fig. S4).

To scrutinize the synergistic action of progesterone and PGE1 by an independent technique, we recorded CatSper currents in human sperm by whole-cell patch clamping (Fig. 2A–C). The amplitude of monovalent CatSper currents at −80 mV was −9.9 ± 2.8 pA (Fig. 2C, control); superfusion of sperm with a saturating (1 μM) concentration of progesterone or PGE1 increased the current amplitudes to −85 ± 60 and −38 ± 24 pA (Fig. 2A–C), respectively, reflecting the more efficacious activation of CatSper by progesterone compared to PGE1. Moreover, superfusion of sperm with a progesterone/PGE1 mixture (1 μM/1 μM) increased the amplitude to −357 ± 40 pA (Fig. 2A–C), which exceeds the amplitude expected for a purely additive action (~−124 pA) by about threefold. Altogether, these data clearly show that the oviductal ligands progesterone and PGE1 synergistically activate CatSper in human sperm.

Synergistic activation of human CatSper by EDCs

Finally, we studied the combined action of EDCs. Human CatSper is activated by structurally diverse EDCs that mimic the action of the oviductal ligands (Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016). Prime examples of EDCs that activate CatSper are the UV filter BCSA and the growth promoter α-Zearalenol that act via the steroid- and prostaglandin-binding site, respectively (Schiffer et al., 2014; Rehfeld et al., 2016). BCSA and α-Zearalenol alone evoked a transient and dose-dependent Ca²⁺ increase (Fig. 3A, B) that was suppressed by the CatSper inhibitor RU1968 (Rennhack et al., 2018) (Supplementary Fig. S5), reinforcing that the chemicals act via CatSper. Analysis of the normalized dose–response relation yielded K_1/2 values of 19.9 ± 7.5 μM for BCSA and 2.2 ± 0.8 μM for α-Zearalenol (n = 6) (Fig. 3D). Moreover, the Ca²⁺ amplitudes saturated at response levels of 0.75 ± 0.08 and 0.33 ± 0.13, respectively (n = 6) (Fig. 3D). Thus, α-Zearalenol is more potent but less efficacious than BCSA to activate CatSper. Next, we tested the action of 2:1 mixtures of BCSA and α-Zearalenol. Similar to the individual ligands, the mixtures evoked a rapid and dose-dependent Ca²⁺ transient (Fig. 3C). Compared to the predicted additive dose–response relation, the experimentally determined dose–response relation was left-shifted to lower ligand concentrations, but settled at a similar maximal response level (Fig. 3E); at response levels ≥0.1, the ratio [BCSA + α-Zearalenol]_exp/[BCSA + α-Zearalenol]_pred was less than 1 and decreased with increasing response levels (Fig. 3F). At a level of 0.1 and 0.7, the ratio was 0.83 ± 0.22 and 0.17 ± 0.06 (n = 6) (Fig. 3F), respectively. These results demonstrate that BCSA and α-Zearalenol act synergistically in human sperm and that the synergism is enhanced at high versus low concentrations of the chemicals; isobolographic analysis (Supplementary Fig. S3D, E) and the combination-index method (Supplementary Fig. S3F) yielded similar results. Yet, the synergism between the EDCs is not as pronounced as that between progesterone and PGE1: over the entire range of response levels, the experimental/predicted concentration ratio was less for the mixtures of physiological ligands than for that of the EDCs (Fig. 3H). Finally, we examined the action of mixtures of progesterone and α-Zearalenol. Compared to the predicted additive dose–response relation, the experimentally determined dose–response relation was left-shifted to lower ligand concentrations, indicating that also progesterone and α-Zearalenol act synergistically in human sperm (Fig. 3F, G).

Discussion

Mutations in CATSPER genes (Avidan et al., 2003; Avenarius et al., 2009; Zhang et al., 2007; Hildebrand et al., 2010; Smith et al., 2013; Jaiswal et al., 2014) and CatSper dysfunction (Williams et al., 2015) are associated with male infertility in humans, indicating that CatSper represents a central signaling node in human sperm. In fact, CatSper serves as a polymodal sensor, integrating diverse chemical and physical cues (Brenker et al., 2012; Miki and Clapham, 2013) that assist sperm for fertilization. Progesterone and prostaglandins have been proposed to guide sperm to the egg (Eisenbach and Giojalas, 2006; Publicover et al., 2008) and to facilitate the penetration of its vestments (Schaefer et al., 1998; Tamburrino et al., 2014). However, the role of these hormones during fertilization has not been definitely established (Baldi et al., 2009), not least due to the challenge of experimentally emulating the complex chemical, topographical, and hydrodynamic landscapes of the female genital tract (Suarez and Pacey, 2006; Suarez, 2008; Kirkman-Brown and Smith, 2011; Miki and Clapham, 2013). The results presented here underscore that fertilization is orchestrated by a complex interplay of various chemical cues. Progesterone and prostaglandins are released by cells lining the oviduct (Ogra et al., 1974; Vastik-Fernandez et al., 1975; Libersky and Boatman, 1995) and the cumulus cells surrounding the oocyte (Schuetz and Dubin, 1981). The concentrations of these hormones in the oviduct are in the nanomolar range (Ogra et al., 1974; Libersky and Boatman, 1995; Munuce et al., 2006; Lamy et al., 2016), whereas micromolar progesterone concentrations prevail within the cumulus oophorous (Osman et al., 1989); the concentration of prostaglandins within the cumulus oophorous is unknown. Thus, throughout their journey across the oviduct, until fusion with the oocyte, sperm are exposed to steroids and prostaglandins at the same time. Yet, previous studies have examined the action of these ligands independent of each other. Here, we show that, in fact, steroids and prostaglandins activate CatSper in a strongly synergistic fashion and, thereby, elevate [Ca²⁺]_i to levels that are not reached by each ligand alone. Future studies have to take this synergism into account: it needs to be addressed how the combined action of steroids and prostaglandins affects human sperm functions, such as the swimming behavior and acrosomal exocytosis. This might provide further insight into the ligand control of sperm behavior during fertilization. CatSper is also controlled by the membrane potential (V_m) and the intracellular pH (pH_i). However, the interplay between V_m, pH_i, and ligands to control CatSper during fertilization is unknown.

Previous studies indicated that EDCs in reproductive fluids might disturb the precisely coordinated sequence of events underlying fertilization (Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016). Activation of CatSper by these chemicals could evoke motility responses and the acrosome reaction at the wrong time and place (Tavares et al., 2013; Schiffer et al., 2014). Furthermore, the desensitization of sperm to progesterone and prostaglandins (Schiffer et al., 2014; Rehfeld et al., 2016) might lead sperm astray on their way to the egg and could hamper the penetration of its vestments. Here, we demonstrate that the EDCs BCSA and α-Zearalenol synergistically activate Ca²⁺ influx via CatSper in human sperm. This finding suggests that even low-dose EDC mixtures in reproductive fluids might affect human sperm in vivo, reinforcing concerns regarding the negative impact of EDCs on male reproductive health. The concentrations of EDCs, including BCSA and α-Zearalenol, in reproductive fluids are largely unknown. Therefore, to strengthen and extend our conclusions, more data concerning the molecular identities and concentrations of EDCs in seminal and oviductal fluids are required. In particular, studies using animal models, for example non-human primates, are required to scrutinize whether EDCs indeed disturb the fertilization process. Nevertheless, our data challenge the common risk-assessment strategy for EDCs: based on the NOAEL standard, safety thresholds are set for individual EDCs. Our findings demonstrate that this standard procedure is prone to underestimate the risk of adverse health effects, because synergistic actions among EDCs are not taken into account (Kortenkamp, 2014). Supporting this notion, synergistic actions of EDCs were also observed in studies performed in human cell lines in vitro (Kim et al., 2005; Delfosse et al., 2015; Gan et al., 2015) and hepatocytes (Delfosse et al., 2015) as well on purified pregnane X receptors (Delfosse et al., 2015). Altogether, this highlights the need to implement concepts for risk assessments that account for the combined action of chemicals.

Acknowledgements

We thank Jolanta Körber, Sabine Forsthoff, Joachim Esselmann and Ina Lund for technical assistance.

Authors’ roles

All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. T.S. and N.E.S. conceived the project. A.R. and C.B. designed and coordinated the experiments and drafted the manuscript. A.R., C.B., C.S, M.K., U.B.K., N.E.S., and T.S. performed experiments, acquired, analyzed and/or interpreted data. All authors revised the manuscript critically for important intellectual content and approved the manuscript.

Funding

This work was supported by the German Research Foundation (SFB 645 to T.S. and U.B.K., CRU326 to T.S.), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17 to T.S.), the ‘Innovative Medical Research’ of the University of Münster Medical School (BR121507 to C.B.), an EDMaRC research grant from the Kirsten and Freddy Johansen’s Foundation (to N.E.S.), and the Innovation Fund Denmark (InnovationsFonden, Grant number 14-2013-4 to N.E.S.).

Conflict of interest

The authors declare that they have no conflict of interest and no actual or potential competing financial interests.

References

Author notes