Epigenetic transgenerational actions of endocrine disruptors and male fertility

Abstract

Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (that is, F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germ line. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germ line and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology.

Fig. 1.

Transgenerational phenotype after vinclozolin treatment of F₀ gestating mothers. (A) Spermatogenic cell apoptosis, (B) epididymal sperm counts, and (C) epididymal sperm motility in PND60 to 180 control and vinclozolin off-spring Sprague-Dawley rats in the F₁, F₂, F₃, and F₄ generations, and vinclozolin F₂ generation male outcross (VOC) to wild-type control females, and vinclozolin F₂ generation female reverse outcross (RVOC) to wild-type control males. Statistically significant differences between control and vinclozolin treatment generations are indicated by (*) for P G 0.001 with a two-way analysis of variance test. The n value for each bar ranged between 10 and 30 animals. Detailed methods are provided in SOM.

Fig. 2.

Testis histology from control (A) and vinclozolin treatment (B) 100-day-old F₃ generation animals, ×200 magnification. The vinclozolin F₃ generation male is a representative infertile male. Arrow in (A) identifies the tails of elongate spermatozoa in the seminiferous tubule lumen; arrowhead labels spermatocytes in the tubule epithelial layer. Arrow in (B) identifies the lack of germ cells in the seminiferous tubule. Methods are provided in SOM.

Fig. 3.

DNA methylation analysis from control and vinclozolin offspring testis. (A) Representative gel images of the PCR-based methylation-sensitive Hpa II restriction enzyme digest analysis with representative band (arrow) affected in PND6 testis from control and vinclozolin treatment animals. Each lane represents a different individual animal (n = 4). (B) Location of selected sequences on specific chromosomes for two representative DNA sequences with altered DNA methylation patterns termed clone 7 and 17. (C) Methylation-sensitive restriction enzyme PCR analysis of the methylation state of clone 17 (i.e., cytokine-inducible SH2 protein) gene in epididymal sperm from F₂ and F₃ generations from control and vinclozolin-treated animals. The bands presented are representative of sperm DNA collected from different animals from different litters and are consistent in four out of eight F₂ animals and two out of five F₃ animals analyzed. Methods are provided in SOM.