Direct action of endocrine disrupting chemicals on human sperm

Abstract

Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca(2+) increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca(2+) levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.

Keywords: Ca2+ signalling; CatSper; endocrine disrupting chemical; human sperm.

Figure 1. Structurally diverse EDCs directly activate CatSper in human sperm

A Composition of the compound library comprising 96 EDCs. B Bisphenol A (BPA) does not evoke Ca²⁺ signals in human sperm. ΔF/F₀ (%) indicates the percentage change in fluorescence (ΔF) with respect to the mean basal fluorescence (F₀) before the application of buffer, progesterone (2 μM) or chemicals. C 4-Methylbenzylidene camphor (4-MBC)-evoked Ca²⁺ signals in human sperm; progesterone = 2 μM. D Mean (n = 4–6) Ca²⁺ signal amplitudes evoked by EDCs at 0.1, 1, and 10 μM (blue), compared to signal amplitudes evoked by progesterone (2 μM, red) and buffer (black). Shaded area: buffer ± 3 SD; EDCs that evoked amplitudes > mean buffer + 3 SD were defined as ‘active’. E 4-MBC-induced Ca²⁺ signals (10 μM) in the absence (buffer) and presence of the CatSper inhibitor MDL12330A (100 μM). F Triclosan (TCS)-induced Ca²⁺ signals (10 μM) in the absence (buffer) and presence of the CatSper inhibitor MDL12330A (100 μM). G Relative inhibition of EDC (3-30 μM)- and progesterone (2 μM)-induced Ca²⁺ signals by MDL12330A (100 μM) [TCS, 4-octylphenol (4-OP), benzophenone-3 (BP-3), 4,4′-DDT, n-nonylparaben (n-NP), 4-MBC: n = 3; progesterone, 3-benzylidene camphor (3-BC), α-zearalenol, di-n-butyl phthalate (DnBP), homosalate (HMS), padimate O (OD-PABA): n = 4]. H 4-MBC (10 μM) reversibly enhanced monovalent whole-cell CatSper currents (NaDVF + 4-MBC) in human sperm, recorded in Na⁺-based divalent-free solution (NaDVF), in the absence of intracellular divalent ions. Voltage was stepped from 0 ± 80 mV in increments of 10 mV. HS: currents recorded in the presence of extracellular Ca²⁺ and Mg²⁺. I Current-voltage relation of currents shown in (H). J Increase in monovalent CatSper currents at −60 mV evoked by DnBP (100 μM; n = 4), 4-MBC (10 μM; n = 9), TCS (10 μM; n = 4), and progesterone (2 μM; n = 6). Data information: All values are given as mean ± SD.

Figure 2. EDCs compete with physiological ligands for CatSper activation

A Representative dose-response relationship of progesterone in the absence (buffer) and presence of 4-MBC (10 μM) or α-zearalenol (10 μM). B Mean EC₅₀ values of progesterone in the absence (buffer) and presence (upper panel) of 3-CMO-progesterone (100 nM) or prostaglandin E1 (PGE1; 100 nM), and in the presence (lower panel) of 4-MBC (10 μM), α-zearalenol (10 μM), or TCS (10 μM). The number of experiments is indicated in the bars. C Representative dose-response relationship of PGE1 in the absence (buffer) and presence of 4-MBC (10 μM) or α-zearalenol (10 μM). D Mean EC₅₀ values of PGE1 in the absence (buffer) and presence (upper panel) of PGF1α (300 nM) or progesterone (100 nM) and in the presence (lower panel) of 4-MBC (10 μM), α-zearalenol (10 μM), or TCS (10 μM). The number of experiments is indicated in the bars. Data information: All values are given as mean ± SD.

Figure 3. EDCs evoke motility responses and acrosomal exocytosis in human sperm

A Images: Flagella waveform of head-tethered human sperm at rest (left, buffer) and following perfusion with buffer (middle) or 4-MBC (6.8 μM, right). Successive, aligned, and superimposed images yielding a quasi stop-motion image, illustrating one flagellar beating cycle (Scale bar: 15 μm). Graph: Time course of beat frequency (black) and flagellar asymmetry (blue) before and after perfusion with buffer and 4-MBC. An increase in the asymmetry index indicates a more asymmetric flagella waveform. (1) and (2) indicate perfusion of sperm with buffer and 4-MBC, respectively. B Mean fold change in asymmetry index and beat frequency after perfusion with buffer (n = 15), 4-MBC [6.8 μM (n = 8) and 30 μM (frequency: n = 4, asymmetry: n = 3)], and progesterone (1 μM; n = 4). C Percentage of acrosome-reacted sperm bathed in progesterone, 3-benzylidene camphor (3-BC), 4-MBC, and TCS (all 10 μM) relative to the vehicle (DMSO) control (all conditions: n = 5). Data information: All values are given as mean ± SD.

Figure 4. EDCs evoke Ca²⁺ responses at physiologically relevant concentrations

A α-Zearalenol-evoked Ca²⁺ signals. B Normalized dose-response relationships of 4-MBC, α-zearalenol, di-n-butyl phthalate (DnBP), progesterone, and prostaglandin E1 (PGE1). Dashed lines: EC₅₀ and EC₀₂. C EC₅₀ values for the EDCs (3-BC, OD-PABA, n-NP, HMS, 4-OP: n = 3; 4-MBC: n = 4; α-zearalenol, TCS, DnBP: n = 5; BP-3: n = 6). We failed to determine the EC₅₀ for 4,4′-DDT. Gray arrow heads: EC₀₂; red arrow heads: maximal reported concentrations in human blood (Table 1). D Ca²⁺ signals evoked by pico- to nanomolar 4,4′-DDT concentrations. Data information: All values are given as mean ± SD.

Figure 5. EDCs cooperate to evoke Ca²⁺ responses

A Ca²⁺ signals evoked by DnBP, 4-OP, BP-3, TCS, HMS, n-NP, 4-MBC, OD-PABA, α-zearalenol, and 3-BC alone (concentration = EC₀₂, black) and by a mixture of the chemicals, containing each chemical at its EC₀₂ concentration (red). B Mean Ca²⁺ signal amplitudes evoked by individual EDCs and by the mixture used in (A) (n = 3). All values are given as mean ± SD. C Ca²⁺ signals evoked by different progesterone concentrations in comparison with the Ca²⁺ response evoked by the EDC mixture used in (A).