In Vitro Exposure to the Endocrine-Disrupting Chemical Climbazole Impairs Human Sperm Motility, Hormonal Signalling, and Mitochondrial Activity

Abstract

This study explores the endocrine-disrupting effects of climbazole (CBZ), an environmental and lifestyle stressor, on male fertility. The impact of CBZ on sperm vitality, motility, and molecular pathways related to hormone receptors and apoptosis was evaluated, in non-capacitated and capacitated conditions. Gene expression of key components, including hormone receptors (ESR1, ESR2, FSHR, AR), apoptosis-related genes (BAX, BCL2), and COX4l1 (involved in mitochondrial function), was analyzed. Protein tyrosine phosphorylation, a marker of capacitation, was also examined using immunofluorescence and Western blot analysis. We demonstrated that CBZ significantly reduced sperm vitality at concentrations above 25 µM and motility at 1 and 10 µM in non-capacitated and capacitated conditions. Changes in tyrosine phosphorylation patterns were also observed. Gene expression analysis revealed an upregulation of ESR1, ESR2, FSHR, and BAX, while AR and COX4l1 expression were downregulated. These findings offer new insights into the potential endocrine-disrupting and cytotoxic effects of CBZ, highlighting its potential role in compromising male reproductive health.

Keywords: climbazole (CBZ); endocrine-disrupting chemicals (EDCs); male fertility; oxidative stress; semen quality.

Figure 1

(A) Percentage of sperm vitality in relation to the time between control, vehicle (DMSO 0.1%) and treatment with CBZ. (B) Percentage of sperm motility in relation to the time between vehicle and treatment with CBZ. Significant differences are indicated with Bonferroni correction * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

(A) Percentage of Acrosome reaction staining in non-capacitation and capacitation conditions. ((B)-upper part) Representative Western blot analysis of P-Tyr in human sperm exposed to Vehicle, 1 and 10 µM of β-tubulin was used as a loading control. ((B)-lower part) Bar graph showing the overall relative intensity of the bands, quantified by computer-assisted densitometric analysis. (B) is representative of 3 independent experiments (C–K). Representative Immunofluorescent images illustrating P-Tyr localization (red) and PSA staining (green) in human sperm, with nuclei counterstained in blue. (C) acrosome, A; (D) Mid piece, MP; (E) Acrosome and Mid piece, A+MP; (F) Equatorial region, EQ; (G) Equatorial region and Principal Piece, EQ+PP; (H) Equatorial region and Mid Piece, EQ+MP; (I) Principal Piece, PP; (J) Equatorial segment and Principal Piece, EQ+PP; (K) Acrosome and Principal Piece, A+PP, Magnification 6300×. (L) Quantification of P-Tyr staining patterns based on subcellular localization in sperm under non-capacitation and capacitation conditions. Data represent the mean proportion of each pattern (±SD). A total of 200 sperm were analyzed per sample. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Normalized gene expression of hormone receptors in sperm treated with CBZ calculated by Delta–Delta Ct (ΔΔCt) method. (A) Estrogen Receptor 1 (ESR1), (B) Estrogen Receptor 2 (ESR2), (C) Follicle Stimulating Hormone Receptor (FSHR) and (D) Androgen Receptor (AR). Significant differences are indicated by Bonferroni-corrected p value: * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 4

Heat maps illustrate the correlation analysis between gene expression of ESR1, ESR2, AR and FSHR in (A) non-capacitated and (B) capacitated sperm. Cells filled in green to red gradient of the heat maps (lower part) represent Spearman’s r; cells filled in yellow to red gradient (upper part) represent p values (empty cells stand for p values greater than 0.05).

Figure 5

Normalized gene expression of (A) BAX and (B) BCL2 in vehicle-treated versus CBZ-treated sperm, calculated using Delta–Delta Ct (ΔΔCt) method. (C) Left panel: Percentage of DNA denaturation. Right panel: Representative image of Acridin orange staining, showing sperm with normally condensed, non-fragmented (green) and decondensed or damaged chromatin (red). (D) TUNEL assay. Sperm DNA fragmentation index (SDF) percentage categorizing sperm with no damage (ND), partial damage (PD) or complete DNA damage (D). Scale bar: 25 µm. Significant differences are indicated by Bonferroni-corrected p value: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6

(A) Normalized gene expression of COX4I1 between vehicle-treated and CBZ-treated samples, calculated by Delta–Delta Ct (ΔΔCt) method. (B) Percentage of Dichlorofluorescein (DCFH-DA) in relation to the time between vehicle and treatment with CBZ. (C) Bar plot showing JC-1 staining with red (high MMP)/green (low MMP) ratio. Significant differences are indicated with Bonferroni-corrected p value: ** p < 0.01; *** p < 0.001; **** p < 0.0001.