Disturbed relaxin signaling pathway and testicular dysfunction in mouse offspring upon maternal exposure to simazine

Abstract

Simazine is a triazine herbicide that is being widely applied worldwide and commonly detected in surface and groundwater. Despite its popular use in controlling weeds and algae, very limited information is available regarding its toxicity. In the present study, pregnant mice were orally exposed to low doses (0, 5, 50, or 500 µg/kg body weight per day) of simazine during gestation and lactation, during which no overt maternal toxic response was detected, and their offspring was assessed. Simazine-exposed male offspring showed decreased body, testicular, and epididymis weight, increased testicular apoptosis, and decreased sperm concentrations. Differentially-expressed genes in the testes of male offspring exposed to simazine were identified by DNA microarray, revealing 775 upregulated and 791 downregulated genes; among these, the relaxin-family peptide receptor 1 (Rxfp1), which is the receptor for relaxin hormone, was significantly downregulated. In addition, the expression of target genes in the relaxin pathway, including nitric oxide synthase 2 (Nos2) and Nos3, was significantly decreased in simazine-exposed F1 testes. Moreover, simazine inhibited NO release, and knockdown of Rxfp1 blocked the inhibitory action of simazine on NO production in testicular Leydig cells. Therefore, the present study provides a better understanding of the toxicities associated with the widely used herbicide simazine at environmentally relevant doses by demonstrating that maternal exposure interferes with the pleotropic relaxin-NO signaling pathway, impairing normal development and reproductive activity of male offspring.

Figure 1. Schematic of the in vivo animal experiment.

Dams (F0) were mated, and pregnant mice were exposed to different daily doses of simazine from gestational day (GD) 12 to postnatal day (PND) 20. An assessment of body and organ weights, anogenital distance (AGD), computer-assisted sperm analysis (CASA), immunohistochemistry, western blot, microarray and real-time PCR of their male offspring (F1) was performed at the indicated time points.

Figure 2. Increased testicular and ovarian apoptosis of mouse offspring exposed to simazine.

(A) TUNEL-positive control section pretreated with DNase I (a), TUNEL-negative control section without terminal deoxynucleotidyl transferase treatment (b). The TUNEL assay was conducted on the cross-sections of 8-week-old mice testes (c and d) prepared from groups exposed to corn oil (c) or simazine (50 µg/kg) (d). (B) Real-time PCR analysis of Bcl-2 family members in the testes of control and simazine-exposed offspring was shown. The data were normalized to the expression level of β-actin and are presented as relative fold changes. The results are the mean ± SEM of six independent testicular analyses performed in triplicate for each group.

Figure 3. Summary and confirmation of microarray analysis.

(A) Positively or negatively regulated DEGs in the testes of simazine (500 µg/kg)-exposed adult F1 mice compared with the control, classified by their roles in biological processes. (B) The expression levels of several of the differentially upregulated genes in the testes of F1 offspring exposed to simazine identified in the microarray analysis were confirmed by qRT-PCR. The results are reported as the mean ± SEM of six independent testicular analyses performed in triplicate for each group. The data were normalized to the expression level of β-actin and are presented as relative fold changes.

Figure 4. Decreased expression of Rxfp1 in the testis of F1 male mice exposed to simazine.

(A) Testicular sections of young adult F1 male control (a) or 500 µg/kg simazine-exposed (b) mice were immunostained for Rxfp1. (B) Six independent testicular lysates of the control and the 500 µg/kg groups were prepared, and western blot analyses were performed using anti-Rxfp1 antibodies (left panel). Equal loading of each sample was confirmed by immunoblotting of the same membrane with anti-β-actin antibody. The results of a quantitative analysis of the Rxfp1 expression shown in the left panel using Multi Gauge V3.0 software are presented in the right panel. The asterisk indicates a significant difference compared with the control (p<0.05).

Figure 5. Reduced expression of target genes in the relaxin pathway in the testis of F1 mice exposed to simazine.

(A) The expression of crucial target genes (Nos2, Nos3, Vegf, Mmp9) in the relaxin pathway was determined by qRT-PCR on the testis of young adult F1 control or 500 µg/kg simazine-exposed mice. The data (mean ± SEM) are from six testicular analyses performed in triplicate for each group and are shown as relative fold changes. Asterisks indicate significant differences compared with the control (*p<0.05; **p<0.005). (B) The expression of Nos2 protein was further determined by western blot analysis (left panel), and its expression was quantified after normalization to β-actin (n = 6) (right panel). Significantly different values between groups are indicated by an asterisk (p<0.05).

Figure 6. Simazine-induced decreased Rxfp1 and Nos2 expression and reduced NO production in testicular cells in vitro.

Rat Leydig cells (LC540) were exposed to simazine (0, 0.01, 0.1, or 1 µM) for 36 h, and the mRNA levels of Rxfp1 (A) and Nos2 (B) were analyzed by qRT-PCR. The relative expression levels of Rxfp1 and Nos2 normalized to β-actin are shown. (C) LC540 cells were exposed to various concentrations of simazine for 36 h, and the levels of NO produced were determined by measuring nitrite concentration. (D) LC540 knockdown cells were prepared by transfection with a scrambled sequence or siRNAs for Rxfp1, and reduced Rxfp1 expression was demonstrated by western blotting. Subsequently, knocked-down cells were treated with simazine for 36 h, and the levels of NO produced were determined. For all experiments (A-D), three independent experiments were performed in triplicate, and different letters denote significant values (p<0.05).

Figure 7. A schematic representation of the relaxin pathway when disturbed by simazine.

Simazine inhibits testicular Rxfp1 expression, which consequently may limit the intracellular signaling of relaxin in the testis. The compromised relaxin-Rxfp1 signaling is further expected to downregulate the expression of relaxin target genes such as Nos2, Nos3, Vegf, and Mmp9, leading to the inhibition of NO release.