Epigenetic and Phenotypic Transgenerational Changes Induced by Vinclozolin in the Mouse Germ Line

Carlos M. Guerrero-Bosagna; Trevor Covert; Muksitul Haque; Sean Leonard; Eric Nilsson; Michael K. Skinner

doi:10.1093/biolreprod/85.s1.119

Endocrine disrupting chemicals are known for their ability to alter development and reproduction in mammals. One of the periods most sensitive to endocrine disruptor s is embryonic gonadal sex determination, when the germ line is undergoing epigenetic programming and DNA re-methylation. These environmental compounds can induce physiological changes in mammalian mothers, leading to epigenetic alterations in the developing fetus and the emergence of disease conditions such as cancers, reproductive defects or obesity. Interestingly, in addition to one-generation effects, the incidence of epigenetic changes or diseases resulting from exposure to endocrine disruptors can be transgenerationally transmitted. The mechanism behind this transmission would be the alteration of epigenetic DNA features in the male germ line. Previous work in rats have shown that the exposure of pregnant F0 mothers to intra-peritoneal injections of vinclozolin (fungicide), while the F1 undergoes early development, is capable of inducing adult onset disease states in the F1 generation. These phenotypes can be transmitted through the male germ line and maintained across the following generations. Vinclozolin early embryonic exposure also induces a number of transgenerational epigenetic changes in DNA methylation of promoter regions, detected in the sperm epigenome of F3-generation male rats. The present study replicates these results previously obtained with the rat model now using the mouse model and also expands the analysis to interrogate changes in females. The effects of transient exposure to the endocrine disruptors vinclozolin and flutamide during germ line development were analyzed in two strains of mice, one inbred (129) and one outbred (CD-1), in F1, F2 and F3 generations. Transgenerational increases in apoptosis with vinclozolin in the two mouse strains were observed with the vinclozolin but not the flutamide treatment. Increases in the incidence of diseased animals were also observed across generations, when analyzing testes, prostate and kidney. Also, a transgenerational disease in females was observed, which corresponds to an increase in the number of ovarian cysts following vinclozolin exposure observed from the F1 to F3 generations in a dose dependent manner. In addition to phenotypic effects, a number of epigenetic changes were also detected in the F3 generation when analyzing the epigenome in promoter regions of the sperm DNA. These changes were detected using methylated DNA immunoprecipitation follow by chip comparative arrays (MeDip-chip) and then confirmed regionally with quantification of the changed regions through qPCR of this immunoprecipitated DNA (qChIP). These findings show that the transgenerational transmission of these disease states is accompanied by transgenerational changes in the sperm epigenome. Further studies are required to identify if specific epigenetic changes may associate with particular disease states. Both phenotypic and epigenetic results presented here can have profound implications for the study of endocrine disrupting chemicals on toxicological or epidemiological research.

Epigenetic and Phenotypic Transgenerational Changes Induced by Vinclozolin in the Mouse Germ Line

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