Bisphenol A Diglycidyl Ether (BADGE) and Bisphenol Analogs, but Not Bisphenol A (BPA), Activate the CatSper Ca2+ Channel in Human Sperm

Abstract

Aim: Evidence suggests that bisphenol A diglycidyl ether (BADGE), bisphenol A (BPA), and BPA analogs can interfere with human male fertility. However, the effect directly on human sperm function is not known. The CatSper Ca²⁺ channel in human sperm controls important sperm functions and is necessary for normal male fertility. Environmental chemicals have been shown to activate CatSper and thereby affect Ca²⁺ signaling in human sperm. BPA has previously been investigated for effects on Ca²⁺ signaling human sperm, whereas the effects of other BPA analogs are currently unknown. The aim of this study is thus to characterize the effect of BADGE, BPA, and the eight analogs BPG, BPAF, BPC, BPB, BPBP, BPE, BPF, BPS on Ca²⁺ signaling, and CatSper in human sperm. Methods: Direct effects of the bisphenols on Ca²⁺ signaling in human sperm cells were evaluated using a Ca²⁺ fluorimetric assay measuring changes in intracellular Ca²⁺. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function was assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm-mucus penetration assay. Results: At 10 μM the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP induced Ca²⁺ signals in human sperm cells, whereas BPE, BPF, BPS, and BPA had no effect. The efficacy of the chemicals at 10 μM is BPG > BPAF > BPC > BADGE > BPB > BPBP. Dose-response relations of BPG, BPAF, BPC, BADGE, BPB, and BPBP yielded EC50-values in the nM-μM range. The induced Ca²⁺ signals were almost completely abolished using the CatSper inhibitor RU1968, indicating an effect of the bisphenols on CatSper. All bisphenols, except BPBP, were found to dose-dependently inhibit progesterone-induced Ca²⁺ signals, with BPG and BPAF displaying inhibition even in low μM doses. BPG and BPAF were shown to affect human sperm function in a progesterone-like manner. Conclusion: Our results show that the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP can affect Ca²⁺ signaling in human sperm cells through activation of CatSper. This could potentially disrupt human sperm function by interfering with normal CatSper-signaling and thus be a contributing factor in human infertility, either alone or in mixtures with other chemicals.

Keywords: CatSper; bisphenol; endocrine disruption; fertility; male reproduction.

Figure 1

Normalized dose response curves (mean ± SD) for “positive hit” bisphenols. (A) BPG, (B) BPAF, (C) BPC, (D) BADGE, (E) BPB, and (F) BPBP, n = 3–7.

Figure 2

Ca²⁺ signals induced by (A) BPG, (B) BPAF, (C) BPC, (D) BADGE, (E) BPB, (F) BPBP, (G) the endogenous CatSper ligand progesterone, and (H) HTF⁺ buffer in the absence and presence of CatSper inhibitor RU1968, 30 μM. (I) Mean inhibition (in %) of the induced Ca²⁺signal ±SD in the presence of RU1968, 30 μM (n = 3).

Figure 3

Changes (mean ± SD) in pH_(i) induced by the bisphenols (5–50 μM), HTF⁺ buffer and positive control NH₄Cl at 30 mM (n = 3).

Figure 4

Inhibition of Ca²⁺ signals induced by 100 nM progesterone after 5 min of pre-incubation with the negative control HTF and different concentrations the bisphenols: (A) BPG, (C) BPAF, (E) BPC, (G) BADGE, (I) BPB, and (K) BPBP. Normalized dose response relations (n = 3−5) of Ca²⁺ signals induced by 100 nM progesterone after 5 min of pre-incubation with the different concentrations the bisphenols: (B) BPG, (D) BPAF, (F) BPC, (H) BADGE, (J) BPB, and (L) BPBP.

Figure 5

Human sperm cells at 2 cm into a viscous medium (mean ± SEM) after treatment with negative control (HTF⁺ with 0.1% DMSO “HTF”), positive controls (5 μM progesterone “Prog” and prostaglandin E1 “PGE1”), 10 μM BPG, and 10 μM BPAF (n ≥ 5). Statistics from multiple comparison between negative control and treatments: ****adjusted P ≤ 0.0001; **adjusted P = 0.0029; *adjusted P ≤ 0.0295.

Figure 6

Percentage live acrosome reacted sperm cells (mean ± SEM) after 30 min treatment of capacitated human sperm cells with negative control (HTF⁺ with 0.2% DMSO “HTF”), positive control (10 μM progesterone “Prog”), 10 μM BPG, and 10 μM BPAF (n ≥ 8). Statistics from multiple comparison between negative control and treatments: ****adjusted P ≤ 0.0001; *adjusted P ≤ 0.0249.