Abstract
The endocrine disruptor bis(2-ethylhexyl) phthalate (DEHP) has been shown to exert adverse effects on the male animal reproductive system. However, its mode of action is unclear and a systematic analysis of its molecular targets is needed. In the present study, we investigated the effects of prenatal exposure to 300 mg/kg/day DEHP during a critical period for gonads differentiation to testes on male mice offspring reproductive parameters, including the genome-wide RNA expression and associated promoter methylation status in the sperm of the first filial generation. It was observed that adult male offspring displayed symptoms similar to the human testicular dysgenesis syndrome. A combination of sperm transcriptome and methylome data analysis allowed to detect a long-lasting DEHP-induced and robust promoter methylation-associated silencing of almost the entire cluster of the seminal vesicle secretory proteins and antigen genes, which are known to play a fundamental role in sperm physiology. It also resulted in the detection of a DEHP-induced promoter demethylation associated with an up-regulation of three genes apparently not relevant for sperm physiology and partially related to the immune system. As previously reported, DEHP induced an increase in mir-615 microRNA expression and a genome-wide decrease in microRNA promoter methylation. A functional analysis revealed DEHP-induced enrichments in down-regulated gene transcripts coding for peroxisome proliferator-activated receptors and tumor necrosis factor signaling pathways, and in up-regulated gene transcripts coding for calcium binding and numerous myosin proteins. All these enriched pathways and networks have been described to be associated in some way with the reproductive system. This study identifies a large new array of genes dysregulated by DEHP that may play a role in the complex system controlling the development of the male reproductive system.
Highlights
The adverse impact of bis(2-ethylhexyl) phthalate (DEHP) on the animal reproductive system has been documented since the 1980s
The impact of fetal disruptor bis(2-ethylhexyl) phthalate (DEHP) exposure on male reproductive parameters was investigated in F1 males born from mice receiving either 20 μl corn oil vehicle (CTL) or 300 mg/kg/day DEHP diluted in corn oil (D300) per os each day from E9 to E19
anogenital distance (AGD) and testes weight were significantly reduced in the D300 group compared to CTL mice, (1.9 cm ± 0.16 in CTL vs. 1.5 cm ± 0.06 cm in D300 [p = 1,2Ã10−3] and 214 mg ± 14 in CTL vs. 193 mg ± 15 in D300 [p = 0.049], respectively) (Fig 1C & 1D)
Summary
The adverse impact of bis(2-ethylhexyl) phthalate (DEHP) on the animal reproductive system has been documented since the 1980s. Due to the impossibility to study the mechanisms that give rise to TDS in the human fetus, animals exposed in utero to phthalate are used as a mimicking model system [3]. Evidence suggests that DEHP alters testosterone production and/or distribution, both essential for testes development. In a study using human testes cells in culture, DEHP 10−5 or 10−4 M or mono(2-ethylhexyl) phthalate (MEHP), the major DEHP metabolite, were found to inhibit testosterone production [4]. Fit docking simulations predicted that DEHP would bind with a higher affinity to the SHBG steroid pocket than DHT [6]. When occurring in a critical testis development period, competition between DEHP and DHT to bind to SHBG might induce a decrease in testosterone production, which may explain DEHP-induced TDS
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