Impact of Chronic Multi-Generational Exposure to an Environmentally Relevant Atrazine Concentration on Testicular Development and Function in Mice

Abstract

A common herbicide, atrazine, is associated with poor health. Atrazine acts as an endocrine disruptor at supra-environmental levels. Little research, however, has been conducted regarding chronic exposure to environmental atrazine concentrations across generations. This study utilized comprehensive endpoint measures to investigate the effects of chronic exposure to a conservative atrazine concentration (0.02 ng/mL), measured in Australian waterways, on male mice fertility across two generations. Mice were exposed through the maternal line, from the pre-conception period and through the F1 and F2 generations until three or six months of age. Atrazine did not impact sperm function, testicular morphology nor germ cell parameters but did alter the expression of steroidogenic genes in the F1, down-regulating the expression of Cyp17a1 (Cytochrome P450 family 17, subfamily A member 1; p = 0.0008) and Ddx4 (DEAD-box helicase 4; p = 0.007), and up-regulating the expression of Star (Steroidogenic acute regulatory protein; p = 0.017). In the F2, atrazine induced up-regulation in the expression of Star (p = 0.016). The current study demonstrates that chronic exposure to an environmentally relevant atrazine concentration perturbs testicular steroid-associated gene expression that varies across generations. Future studies through the paternal and combined parental lineages should be undertaken to further elucidate the multigenerational effects of atrazine on male fertility.

Keywords: atrazine; endocrine disruptor; multi-generational; testis.

Figure 1

Representative images of H&E-stained testis section from F1 mice exposed to 0.02 ng/mL atrazine or the control for 6 months. (a) control (×100), (b) control (×200), (c) ATZ (×100), (d) ATZ (× 200). No visual difference in gross morphology was observed between treatment groups. Scale bar at 100× is 200 µm, and at 200× is 100 µm. Arrow indicates the tubule lumen and the dashed line indicates the basement membrane of the tubule.

Figure 2

F1 and F2 testis gene expression assessed by qRT-PCR. (a) Star, Steroidogenic acute regulatory protein, (b) Cyp17a1, Cytochrome P450 family 17, subfamily A member 1, (c) Hsd17β11, Hydroxysteroid 17-beta dehydrogenase 11, (d) Hsd17β3, Hydroxysteroid 17-beta dehydrogenase 3, (e) Cyp19a1, Cytochrome P450 family 19, subfamily A member 1, (f) Srd5α1, Steroid 5 alpha-reductase 1 and (g) Nr5a1, Nuclear receptor subfamily 5 group A member 1 (SF-1). Genes were normalized to the geometric mean of B-actin, TBP, and Rlp19. Results were represented as a fold change in gene expression relative to the control, using the Pfaffl method [46]. Data are means ± SEM. Significance between the control (n = 8–10) and ATZ (n = 8–10) is indicated by an asterisk (* p < 0.05 or *** p < 0.001). There were >4 L per treatment, per generation.

Figure 3

F1 and F2 testis gene expression assessed by qRT-PCR. (a) Ddx4, DEAD-box helicase 4–germ, cells, (b) Hsd3b1, Hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta-isomerase 1–Leydig cells, and (c) Gata6, GATA binding protein 6–Sertoli cells. Genes were normalized to the geometric mean of B-actin, TBP, and Rlp19. Results were represented as a fold change in gene expression relative to the control, using the Pfaffl method [46]. Data are mean ± SEM. Significance between the control (n = 8–10) and ATZ (n = 8–10) is indicated by an asterisk (** p < 0.01). There were >4 litters per treatment, per generation.

Figure 4

Representative immunofluorescence of testis sections at 200X of the F1 control (a,c) and ATZ exposed (b,d) 6-month cohort. (a,b) immunolabelling of SOX9 (Sertoli cells, red) and of proliferating cells with an antibody raised to phospho-histone H3 (P-HH3, green), with a DAPI counterstain (showing the nuclei, blue). White arrows indicate proliferating cells and white arrow heads indicate Sertoli cells (c,d) immunolabelling of GATA4 (Sertoli and Leydig cell, green) and of DDX4 (germ cells, red), with a DAPI counterstain (showing the nuclei, blue). White arrows indicate Leydig cells, white arrowhead indicates Sertoli cells and white asterisks indicate germ cells. Scale bar = 100 µm. ATZ = atrazine.

Figure 5

Analysis of atrazine exposure on the number of cell sub-types within the testis at 6 months of age in the F1 cohort. (a) DDX4 area was represented as a percentage area per tubule. (b) Sertoli cells were normalized to a set area and are represented as number of cells per unit area. (c) The number of proliferating cells were normalized to the total testis section area and represented as cells per total area. No significant differences were found between the control (n = 6) and ATZ (n = 6–8) treatment for all parameters. Data are mean ± SEM. There were > 3 L per treatment.