Research Article: Testicular Dysgenesis Syndrome and Long-Lasting Epigenetic Silencing of Mouse Sperm Genes Involved in the Reproductive System after Prenatal Exposure to DEHP

Date Published: January 13, 2017

Publisher: Public Library of Science

Author(s): Ludwig Stenz, Jessica Escoffier, Rita Rahban, Serge Nef, Ariane Paoloni-Giacobino, Jean-Marc A Lobaccaro.


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.

Partial Text

The adverse impact of bis(2-ethylhexyl) phthalate (DEHP) on the animal reproductive system has been documented since the 1980s. In mice, DEHP was found to induce phenotypic alterations partially analogous to the human testicular dysgenesis syndrome (TDS) [1]. The increasing incidence of TDS was reported to be due to adverse environmental influences, but the exact mechanisms causing the disorder have still to be elucidated [2, 3]. At present, the disorder is thought to originate during fetal growth and to be associated with an alteration in testosterone production. 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].

We showed that prenatal exposure to DEHP in C57BL/6J mice resulted in statistically significant lower litter sizes at the highest dose tested, compatible with decreased embryo survival. F1 males were bred up to mature adulthood at P100 and five F1 D300 were compared to five CTL subjects. Results demonstrated a reduced AGD, lighter testes, severe decreased sperm quality, atrophy of the germinal tissue thickness, and a lower amount of seminiferous tubules with lumens full of elongated spermatids (Fig 1). These observations converge to an acquired hypogonadism at adulthood and a phthalate-induced phenotype resembling human TDS. The reduction of fertility in males prenatally exposed to DEHP remains to be further analyzed by detailed recordings of litter sizes, plug productions and pregnancy efficiency in the second filial generation (F2). F2 mice should be obtained by crossing F1 D300 and F1 CTL males with new females, thus producing comparable F2 litters. In addition, testosterone supplementation experiments are strongly needed. In the absence of both results, the impact on fertility and interferences with testosterone remains speculative in the present study.




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