Bisphenol A Deranges the Endocannabinoid System of Primary Sertoli Cells with an Impact on Inhibin B Production

Abstract

Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC₅₀) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 μM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB₁) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB₂) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-β, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB₂ receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB₂- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB₂ and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian cells.

Keywords: endocrine disruptor; inhibin B; male reproduction; receptor signalling; spermatogenesis.

Figure 1

Effect of increasing concentrations of BPA on mouse Sertoli cell viability (A), and morphology (B,C), after 48 h of treatment. Magnification, ×200. Data are expressed as means ± SEM of 5 independent experiments. * p < 0.01 vs. vehicle-treated controls (Ctr).

Figure 2

Effect of BPA (0.5 μM for 48 h) on mRNA levels of eCB-binding receptors (CB₁, CB₂, GPR55 and TRPV1) in exposed Sertoli cells, compared to vehicle-treated controls (Ctr). Gene expression is reported as 2^−ΔΔCt values calculated by ΔΔCt method vs. Ctr, posed equal to 1.0. Expression levels of each gene were normalized to β-actin, and data were expressed as means ± SEM of 5 independent experiments. * p < 0.01 vs. Ctr.

Figure 3

Effect of BPA (0.5 μM for 48 h) on mRNA levels of eCB metabolic enzymes (NAPE-PLD, FAAH, DAGL-α, DAGL-β and MAGL) in exposed Sertoli cells, compared to vehicle-treated controls (Ctr). Gene expression was reported as 2^−ΔΔCt values calculated by ΔΔCt method vs. Ctr, posed equal to 1.0. Expression levels of each gene were normalized to β-actin, and data were reported as means ± SEM of 5 independent experiments. ** p < 0.05 vs. Ctr.

Figure 4

Protein expression levels of eCB system in mouse Sertoli cells. (A) Representative Western blot and (B) densitometric analysis of eCB-binding receptors and metabolic enzymes in Sertoli cells exposed to BPA (0.5 μM for 48 h) or to vehicle (Ctr). Data were expressed as mean values (arbitrary units a.u.) ± SEM (n = 3 independent experiments) after normalization with β-actin, used as loading control. * p < 0.05 vs. Ctr.

Figure 5

Production of inhibin B and transferrin by mouse Sertoli cells. Cells were cultured for 48 h in the presence of vehicle (Ctr), or of 0.5 μM BPA alone or with 1 μM of KT109, SR144528 (SR2), IRTX, SR2 + IRTX. Results of 3 independent experiments were expressed as fold increase over controls, posed equal to 1.0. * p < 0.05 vs. Ctr; ^≠ p < 0.05 vs. BPA-treated cells.