Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) impairs Postnatal Testis Development in Mice: The Microtubule Cytoskeleton as a Sensitive Target

Abstract

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE), a widely used flame retardant, has been frequently detected in various environmental compartments, but its health hazard remains largely unknown. Here, we investigated the adverse effects of TBBPA-BDBPE (50 and 1000 μg/kg/day) on postnatal testis development in CD-1 mice and the underlying mechanism. Following the first week of maternal exposure, neonatal mice in the high-dose group exhibited reduced seminiferous tubule area, fewer Sertoli cells and germ cells, and damaged microtubules in Sertoli cells; even microtubule damage was also observed in the low-dose group. When exposure extended to adulthood, male offspring in the high-dose group presented more remarkable alterations in reproductive parameters, including reduced sperm count; in the low-dose group, microtubule damage was also observable, along with blood-testis barrier impairment. Further molecular docking analysis and tubulin polymerization assay indicated that TBBPA-BDBPE could interact with tubulin and disrupt its polymerization. Moreover, we observed attenuated microtubules in mouse Sertoli cells in vitro (TM4) following TBBPA-BDBPE treatment, suggesting that TBBPA-BDBPE impaired testis development possibly by interfering with tubulin dynamics. This study not only highlights the male reproductive hazard of TBBPA-BDBPE but also greatly improved the understanding of the molecular mechanism for male reproductive toxicity of chemicals.

Figure 1

Effects of the first-week lactational exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) on the seminiferous tubule and germ cells in mice on postnatal day 7 (n = 5 litters, three males/litter). (A) Seminiferous tubules whose basement membrane was marked by LAMININ1 immunofluorescence (IF) and their area. (B) Germ cells marked by DAZL IF and their number per tubule. (C) Relative expression of germ cell marker genes (Dazl and Stra8) and Leydig cell marker genes (Cyp17a1 and Stra8). For statistical analysis, at least 100 seminiferous tubules on three cross sections per testis and five litters per group were used. The asterisk (*) indicates a significant difference from the control (p < 0.05).

Figure 2

Effects of the first-week lactational exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) on Sertoli cells and the microtubule cytoskeleton in mice on postnatal day 7 (n = 5 litters, three males/litter). (A) Sertoli cells marked by SOX9 IF and their number per tubule. (B) The microtubule cytoskeleton, labeled by TUBB3 IF, and the microtubule area% of the seminiferous tubule area. (C) Relative expression of Sertoli cell marker gene (Sox9) and microtubule-organizing genes (Map2, Dcx, and Map9). For statistical analysis, at least 100 seminiferous tubules on three cross sections or 20 cross sections (20× field) per testis and five litters per group were used. The asterisk (*) indicates a significant difference from the control (p < 0.05).

Figure 3

Alterations in adult testis and relevant parameters in mice on PND 56 with postnatal exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) (n = 5 litters, two males/litter). (A) Body weight, liver weight, testis weight, and serum testosterone level. (B) Sperm morphology and sperm count in cauda epididymis. (C) Testicular histological images and ectopic immature germ cells marked by SYCP3 IF (red arrow). (C’) Seminiferous tubule area and the percentage of stage VII–VIII seminiferous tubules. (D) Histology of cauda epididymis. Red arrows in parts C and D indicate immature germ cells. For statistical analysis, at least 100 seminiferous tubules on three cross sections per testis and five litters per group were used. The asterisk (*) indicates a significant difference from the control (p < 0.05).

Figure 4

Alterations in spermatogenesis of mice on PND 56 with postnatal exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) (n = 5 litters, two males/litter). (A) Spermatocytes marked by SYCP3 immunofluorescence (IF) and their number per tubule. (B) Sperm marked by ACRV1 IF and their percentage per area. (C) Relative expression of markers for spermatogonia (Plzf), spermatocyte (Ssxb1), and sperm (Prm1 and Prm2). For statistical analysis, at least 100 seminiferous tubules on three cross sections (A) or 20 cross sections (20× field) per testis (B), and five litters per group were used. The asterisk (*) indicates a significant difference from the control (p < 0.05).

Figure 5

Alterations in the microtubule cytoskeleton and the blood–testis barrier (BTB) in adult mice with postnatal exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) (n = 5 litters, two males/litter). (A) The microtubule cytoskeleton, labeled by TUBB3 IF, and its mean gray value per area. (B) The distribution of EZ-Link Sulfo-NHS-LC-Biotin in seminiferous tubules. The histogram illustrates the ratio of the distance traveled by the biotin vs the radius of the seminiferous tubule. For oval-shaped tubules, the radius was calculated as the mean value of the longest and shortest radius of the tubule. For statistical analysis, at least 20 cross sections (20× field) per testis (A) or 100 seminiferous tubules on three cross sections (B), and five litters per group were used. (C) Relative expression of junction marker genes (Tjp1 and Gja1). The asterisk (*) indicates a significant difference from the control (p < 0.05).

Figure 6

Binding affinity of tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) to tubulin (PDB ID: 1SA0) and change of fluorescence intensity during the tubulin polymerizing assay. (A) The left figure shows the 3D structure of the docking status between TBBPA-BDBPE and tubulin (α and β). The right figure shows hydrophobic interactions and halogen bonds. (B) Higher intensity showed stronger tubulin polymerizing activity. The final concentrations of TBBPA-BDBPE were 3, 6, 12, and 24 μmol/L. Paclitaxel (final concentration of 3 μmol/L) and colchicine (final concentration of 3 μmol/L) were used as the positive control and negative control, respectively.

Figure 7

Alterations in cell viability and microtubule cytoskeleton of Sertoli cells (TM4) after 24 h of exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) in vitro (n = 5). (A) The microtubule cytoskeletons were labeled by living cell microtubule fluorescence staining and the nuclear was labeled by DAPI. (B) Statistical results for cell viability and microtubule cytoskeleton amount in TM4 cells.