Bisphenol S Reduces Pig Spermatozoa Motility through Different Intracellular Pathways and Mechanisms than Its Analog Bisphenol A

Abstract

Bisphenol A (BPA: 2,3-bis (4-hydroxyphenyl) propane) is an environmental chemical widely used in the manufacturing of epoxy polymers and many thermoplastic consumer products. Serious concerns about its safety led to the development of analogs, such as BPS (4-hydroxyphenyl sulfone). Very limited studies about BPS's impact on reproduction, specifically in spermatozoa, exist in comparison with BPA. Therefore, this work aims to study the in vitro impact of BPS in pig spermatozoa in comparison with BPA, focusing on sperm motility, intracellular signaling pathways and functional sperm parameters. We have used porcine spermatozoa as an optimal and validated in vitro cell model to investigate sperm toxicity. Pig spermatozoa were exposed to 1 and 100 μM BPS or BPA for 3 and 20 h. Both bisphenol S and A (100 μM) significantly reduce pig sperm motility in a time-dependent manner, although BPS exerts a lower and slower effect than BPA. Moreover, BPS (100 μM, 20 h) causes a significant increase in the mitochondrial reactive species, whereas it does not affect sperm viability, mitochondrial membrane potential, cell reactive oxygen species, GSK3α/β phosphorylation or phosphorylation of PKA substrates. However, BPA (100 μM, 20 h) leads to a decrease in sperm viability, mitochondrial membrane potential, GSK3β phosphorylation and PKA phosphorylation, also causing an increase in cell reactive oxygen species and mitochondrial reactive species. These intracellular effects and signaling pathways inhibited might contribute to explaining the BPA-triggered reduction in pig sperm motility. However, the intracellular pathways and mechanisms triggered by BPS are different, and the BPS-caused reduction in motility can be only partially attributed to an increase in mitochondrial oxidant species.

Keywords: BPS; bisphenols; motility; phosphorylation; spermatozoa; toxicology.

Figure 1

Effects of BPS and BPA on pig spermatozoa motility. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). The percentage of motile spermatozoa was evaluated at 3 h (A) (F = 22.171; p < 0.001) and 20 h (B) (F = 32.106; p < 0.001), and the results are depicted in box-and-whisker plots. The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Circles represent outliers. Each experiment was performed 6 times (n = 6). Boxes with different letters are statistically different from each other, p < 0.05.

Figure 2

Impact of BPS and BPA on progressive motility. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). The percentage of progressive spermatozoa was evaluated at 3 h (A) (F = 30.376; p < 0.001) and 20 h (B) (F = 20.819; p < 0.001) (n = 6) and depicted in box-and-whisker plots. The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 3

Impact of BPS and BPA on rapid and progressive spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C for 3 h with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Each experiment was performed 6 times (n = 6), and the percentages of rapid and progressive (A) (F = 22.436; p < 0.001) and rapid spermatozoa (B) (F = 17.573; p < 0.001) are depicted in box-and-whisker plots. The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 4

Effects of BPS and BPA on the viability of pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). This experiment was performed 6 times (n = 6), and the percentages of SYBR14-positive and PI-negative spermatozoa are depicted in box-and-whisker plots for 3 h treatment (A) (F = 1.753; p = 0.170) and 20 h (B) (F = 32.372; p < 0.001). The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 5

Effects of BPS and BPA on reactive oxygen species (ROS) content of pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). This experiment was performed 5 times (n = 5), and the means of relative fluorescence intensity (RFI) of CellROX-positive spermatozoa are expressed in box-and-whisker plots for 3 h treatment (A) (F = 0.873; p = 0.150) and 20 h (B) (F = 7.224; p < 0.001). The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 6

Effects of BPS and BPA on dihydrorhodamine 123 (DHR) oxidation of pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Each experiment was performed 7 times (n = 7). The mean relative fluorescence intensity (RFI) of DHR was evaluated at 3 h (A) (F = 0.156; p = 0.950) and 20 h (B) (F = 6.209; p < 0.001), and the results are depicted in box-and-whisker plots, where whiskers extend to the largest and smallest data points and the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Circles represent outliers. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 7

Effects of BPS and BPA on spermatozoa mitochondrial membrane potential (ΔΨm). Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Each experiment was performed 6 times (n = 6), and the percentage of spermatozoa exhibiting relatively higher ΔΨm was evaluated at 3 h (A) (F = 0.158; p = 0.950) and 20 h (B) (F = 53.844; p < 0.001). The results are depicted in box-and-whisker plots. The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 8

Effects of BPS and BPA on pig sperm plasma membrane lipid organization. Pig spermatozoa were incubated in TBM at 38.5 °C with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). This experiment was performed 8 times (n = 8). The mean relative fluorescence intensity (RFI) of M540-positive/Yo-Pro-1-negative spermatozoa is depicted in box-and-whisker plots for 3 h (A) (F = 0.598; p = 0.660) and 20 h (B) (F = 1.506; p = 0.220). The whiskers extend to the largest and smallest data points; the box extends from the upper quartile to the lower quartile and is crossed by a line at the median of the data. Circles represent outliers. Boxes with different letters are statistically different from each other, p < 0.05.

Figure 9

Effects of BPS and BPA on the phosphorylation of intracellular signaling pathways mediated by PKA in pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C for 3 and 20 h with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Upper panel: Sperm proteins (10 μg) were analyzed by Western blotting using anti-phospho-PKA substrates as primary antibodies. Each experiment was performed 6 times, and representative films are shown. Loading controls using anti-α-tubulin antibody (lower films) were performed for each experiment in the same membrane. Arrows indicate cross-reactive bands (Bands I–IV) of sperm phosphorylated proteins that are substrates of PKA. Lower panel: Densitometry analysis of Bands I–IV is shown, bars in colors represent the different treatment in each case and values are expressed as mean ± SEM of arbitrary units. Band I (F = 1.831; p = 0.150), Band II (F = 7.188; p < 0.001), Band III (F = 5.292; p < 0.05), Band IV (F = 3.257; p < 0.05) for 3 h treatment; Band I (F = 4.533; p < 0.05), Band II (F = 15.792; p < 0.001), Band III (F = 9.749; p < 0.001), Band IV (F = 3.427; p < 0.05) for 20 h treatment. Statistical differences are shown with * (p < 0.05).

Figure 10

Effects of BPS and BPA on the intracellular signaling pathways mediated by GSK3α/β in pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C for 3 and 20 h with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Upper panel: Sperm proteins (10 μg) were analyzed by Western blotting using anti-phospho GSK3α/β as primary antibody. Arrows indicate the reactive sperm bands corresponding to phosphorylated forms of GSK3α (upper arrow) and GSK3β (lower arrow). Each experiment was performed 6 times, and representative films are shown. Loading controls using α-tubulin antibodies (lower films) were performed for each experiment. Lower panel: Densitometry analysis of P-GSK3α and P-GSK3β bands is shown, bars in colors represent the different treatment in each case and values are expressed as the mean ± SEM of arbitrary units. P-GSK3α (F = 1.555; p = 0.210) and P-GSK3β (F = 0.435; p = 0.780) at 3 h treatment and P-GSK3α (F = 0.513; p = 0.720) and P-GSK3β (F = 4.713; p < 0.05) at 20 h treatment. Statistical differences are shown with * (p < 0.05).

Figure 11

Effects of BPS and BPA on the tyrosine phosphorylation of intracellular signaling pathways in pig spermatozoa. Pig spermatozoa were incubated in TBM at 38.5 °C for 3 and 20 h with 5% CO₂ in the absence (control) or presence of BPA (1 and 100 μM) or BPS (1 and 100 μM). Upper panel: Sperm proteins (10 μg) were analyzed by Western blotting using anti-phospho-tyrosine as primary antibody. Arrows indicate cross-reactive bands (Bands I and II) of sperm tyrosine-phosphorylated proteins. Each experiment was performed 6 times, and representative films are shown. Loading controls using GSK3β antibodies (lower films) were performed for each experiment. Lower panel: Densitometry analysis of Bands I (referred as Group I) and II (referred as Group II) and II is shown, and values are expressed as the mean ± SEM of arbitrary units. Band I (F = 2.394; p = 0.070) and Band II (F = 0.992; p = 0.430) at 3 h treatment and Band I (F = 2.951; p < 0.05) and Band II (F = 2.314; p = 0.080) at 20 h treatment.