PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice

Abstract

It was reported that 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide and a possible endocrine disruptor, can disturb spermatogenesis, but the precise mechanism is not understood. Since 2,4-D is a weak peroxisome proliferator in hepatocytes and peroxisome proliferator-activated receptor α (PPARα) is also expressed in Leydig cells, this study aimed to investigate the link between PPARα and 2,4-D-mediated testicular dysfunction. 2,4-D (130 mg/kg/day) was administered to wild-type and Ppara-null mice for 2 weeks, and the alterations in testis and testosterone/cholesterol metabolism in Leydig cells were examined. Treatment with 2,4-D markedly decreased testicular testosterone in wild-type mice, leading to degeneration of spermatocytes and Sertoli cells. The 2,4-D decreased cholesterol levels in Leydig cells of wild-type mice through down-regulating the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 and reductase, involved in de novo cholesterogenesis. However, the mRNAs encoding the important proteins involved in testosterone synthesis were unchanged by 2,4-D except for CYP17A1, indicating that exhausted cholesterol levels in the cells is a main reason for reduced testicular testosterone. Additionally, pregnancy rate and the number of pups between 2,4-D-treated wild-type male mice and untreated female mice were significantly lower compared with those between untreated couples. These phenomena were not observed in 2,4-D-treated Ppara-null males. Collectively, these results suggest a critical role for PPARα in 2,4-D-induced testicular toxicity due to disruption of cholesterol/testosterone homeostasis in Leydig cells. This study yields novel insights into the possible mechanism of testicular dysfunction and male infertility caused by 2,4-D.

Keywords: 2,4-dichlorophenoxyacetic acid; Cholesterol; Leydig cell; PPARα; Testicular toxicity; Testosterone.

Fig. 1

2,4-D treatment decreased serum/testicular testosterone levels in a PPARα-dependent manner. 2,4-D methyl ester was dissolved in corn oil (4 mL/kg/day) just prior to administration to male Sv/129 wild-type (WT) or Ppara-null (KO) mice (16–20 weeks of age, 25–30 g of body weight) by daily gavage at 130 mg/kg/day. For the control groups, the same volume of corn oil was given as a vehicle (Veh). After treatment for 14 days, the mice were killed and serum/testicular testosterone (a, b) and serum luteinizing hormone (LH) levels (c) measured. Values were expressed as mean ± SEM (n = 5–8). Statistical analysis was performed using ANOVA test with Bonferroni’s correction. **P < 0.01; NS not significant between the 2,4-D-treated and Veh-treated mice in the same genotype

Fig. 2

2,4-D treatment caused atrophy of seminiferous tubules and injury of seminiferous epithelium in a PPARα-dependent manner. The paraffin-embedded sections were stained with periodic acid–Schiff and hematoxylin and subjected to light microscopy. Representative photomicrographs obtained from vehicle (Veh)- or 2,4-D-treated Sv/129 wild-type (WT) or Ppara-null (KO) mice were shown. Arrows indicate degenerated germ cells. Bars represent 100 μm in left photos and 50 μm in right ones

Fig. 3

Electron microscopic evaluation of Sertoli cells and Leydig cells. The testes were fixed by whole-body perfusion using 4 % paraformaldehyde and ultrathin sections were double-stained with uranyl acetate and lead citrate after cytochemical staining for catalase using alkaline DAB. Representative photomicrographs obtained from vehicle (Veh)- or 2,4-D-treated Sv/129 wild-type (WT) or Ppara-null (KO) mice were demonstrated. Left column Sertoli cells. Arrows indicate vacuoles which were observed only in 2,4-D-treated WT mice. Bars represent 2 μm. Right column Leydig cells. An inset in the top panel is a magnified photomicrograph showing organelles and lipid droplets in a Leydig cell from the control WT mice. Black and white arrowheads in the inset indicate mitochondria and peroxisomes, respectively. L lipid droplet, N nucleus of Leydig cell. Bars represent 2 μm. in regular photos and 0.5 μm in the inset

Fig. 4

2,4-D treatment depleted cholesterol contents in Leydig cells in a PPARα-dependent manner. a, b Total cholesterol (TC) levels in isolated Leydig cells (a) and serum (b) were determined using the samples obtained from vehicle (Veh)- or 2,4-D-treated Sv/129 wild-type (WT) or Ppara-null (KO) mice. Values were expressed as mean ± SEM (n = 5). Statistical analysis was performed using ANOVA test with Bonferroni’s correction. *P < 0.05; ***P < 0.001; NS not significant between the 2,4-D-treated and Veh-treated mice in the same genotype. c The abundance of cholesterol ester in Leydig cells was assayed using frozen testis sections obtained from vehicle (Veh)- or 2,4-D-treated Sv/129 wild-type (WT) or Ppara-null (KO) mice and cytochemical staining according to the method of Emeis et al. Arrows indicate cholesterol-rich particles

Fig. 5

Quantification of mRNA levels of genes associated with cholesterol metabolism in Leydig cells. Leydig cells were isolated and purified from vehicle (Veh)- or 2,4-D-treated Sv/129 wild-type (WT) or Ppara-null (KO) mice and were subjected to qPCR analysis. a The mRNA levels of genes encoding scavenger receptor BI (Scarb1) and low-density-lipoprotein receptor (Ldlr). b The mRNA levels of genes encoding HMG-CoA synthase 1 (Hmgcs1) and reductase (Hmgcr), involved in de novo cholesterol synthesis. c The mRNA levels of genes encoding sterol-responsive element-binding protein 2 (Srebf2). The mRNA levels of these mRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA and expressed as mean ± SEM (n = 3). Statistical analysis was performed using ANOVA test with Bonferroni’s correction. *P < 0.05; **P < 0.01; NS not significant between the 2,4-D-treated and Veh-treated mice in the same genotype

Fig. 6

Quantification of mRNA levels of genes associated with testosterone metabolism in Leydig cells. The cDNA samples used in Fig. 5 were adopted. The mRNA levels of genes encoding steroidogenic acute regulatory protein (Star), peripheral-type benzodiazepine receptor (Acbd3), cytochrome P450 (CYP) 11A1 (Cyp11a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), CYP17A1 (Cyp17a1), and hydroxysteroid (17-beta) dehydrogenase 3 (Hsd17b3) were measured, normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA, and expressed as mean ± SEM (n = 3). Statistical analysis was performed using ANOVA test with Bonferroni’s correction. *P < 0.05; NS not significant between the 2,4-D-treated and vehicle (Veh)-treated mice in the same genotype. WT wild-type mice, KO Ppara-null mice

Fig. 7

2,4-D treatment reduced pregnancy rate and new born pup number in a PPARα-dependent manner. Male Sv/129 wild-type (WT) or Ppara-null (KO) mice (6–8 weeks of age) were mated with untreated female wild-type mice at the similar age and body weight that has never experienced pregnancy (1 male vs. 3 females in each cage). During mating, male mice were treated with vehicle (Veh) or 2,4-D at four different doses (13, 50, 130, or 260 mg/kg/day) every day (n = 10 males/treatment group/genotype). The incidence of first pregnancy rate (a), miscarriage rate (b), and the total number of pups delivered (c) were assessed for 12 weeks

Fig. 8

Proposed mechanism of PPARα-mediated 2,4-D-induced testicular toxicity. Testosterone synthesized in Leydig cells plays an important role for the maintenance of normal spermatogenesis. Source of testosterone is cholesterol contained in circulating lipoproteins and newly synthesized from acetyl-CoA. 2,4-D mainly suppresses mRNAs of Hmgcs1/Hmgcr, rate-limiting enzymes of de novo cholesterogenesis, via PPARα signaling and reduces testicular cholesterol levels. PPARα-mediated disruption of cholesterol/testosterone homeostasis in Leydig cells causes Sertoli cell/spermatocyte damage and testicular dysfunction