Authors
Anway, M.; Cupp, A.; Uzumcu, M.; and Skinner, M.

Title:
Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility

Source:
Science (308):1466-1469, 2005.

Summary:

Disruption of the integrity of the genome can result in genetic or developmental defects in multiple generations of offspring. An external agent has the potential to induce transgenerational effects if it causes a permanent reprogramming of the germ line as a result of either chromosomal or epigenetic alterations. Epigenetic refers, broadly, to anything that affects the cell/tissue that does not alter the sequence of bases in the DNA - it does not necessarily imply heritable changes in phenotype. One possible mechanism of epigenetic DNA modification is interference with normal patterns of DNA methylation. Although exogenous environmental compounds such as endocrine disruptors have demonstrated the ability to cause effects in the first generation of offspring (F1) from an exposed gestating mother, alterations to subsequent generations have not been well examined. Previous studies have demonstrated that mice exposed in utero to diethylstilbestrol (the F1) can produce offspring (F2) that spontaneously produce tumors of the reproductive tract at high frequency (Walker and Haven, 1997; Newbold et al., 1998; Newbold et al., 2000). This suggests that the production of tumors in the F2 generation is mediated through an epigenetic mechanism, as if the effect was the consequence of a gene mutation one would expect that such a specific outcome would occur only infrequently. Further, fertility would be predicted to be reduced due to an increase in dominant lethal mutations. In the current study by Anway et al., altered phenotypes were noted in up to 4 generations of offspring of pregnant rats exposed to endocrine disrupting compounds during the period of gestation corresponding to gonadal sex determination.

Pregnant rats received daily intraperitoneal injections of the pesticides vinclozolin or methoxychlor during embryonic days 8 to 15. The offspring (F1 generation) male rats were mated with F1 generation female rats from different litters. Breeding continued for four generations. Male rats from each of the four generations (F1, F2, F3, F4) were killed either 60 or 180 days after birth. The testes of the rats were obtained for histological examination, sperm count and motility were also assessed. A separate group of female rats received a placebo injection (dimethylsulfoxide buffer). The offspring of this control group was bred the same way as the experimental group. There was a greater than two-fold increase in spermatogenic cell apoptosis in the vinclozolin treatment animals for the F1 to F4 generations as compared to the controls. The sperm count and sperm motility was also decreased in the F1 to F4 generations of the treatment group. The frequency of the germ cell defect, characterized by increased spermatogenic cell apoptosis, did not decline between the F1 and F4 generations. More than 90% of males from all generations exhibited the phenotype. Similar results were observed in the F1 and F2 animals following transient embryonic methoxychlor exposure.

In order to determine if the observed transgenerational phenotype was transmitted through the male germ line, vinclozolon F2 males were crossed with control females. The male offspring of this cross had decreased sperm parameters and an increase in spermatogenic cell apoptosis. Vinclozolin F2 generation females were then crossed with control males. The abnormal phenotype was not observed in the offspring of this cross. These results indicate that this particular endocrine disruptor induced alteration is transmitted through the male germ line.

The high frequency and homogeneity of the observed phenotype throughout the generations of offspring are characteristic of an epigenetic alteration of the male germ line rather than a DNA sequence mutation. The latter mechanism would likely result in a reduction of the phenotypic frequency with each generation, as a result of segregation of the mutation. Methylation of specific gene sequences is the only known epigenetic mechanism capable of influencing germline transmission. This mechanism of germ line reprogramming is supported by the window of exposure to the endocrine disrupting agent in this experiment, which occurred during the period of remethylation programming of the fetal germ line. In addition, altered DNA methylation patterns were identified in the vinclozolin treated male rats upon methylation-sensitive restriction enzyme digestion analysis. The patterns observed represented both hypermethylation and hypomethylation events.

Vinclozolin is a fungicide commonly used in the wine industry. This endocrine disrupting compound has the capability to block cell receptors that are normally activated by androgens. Germ cells in the gonad undergo remethylation during the period of gonadal sex determination and this process appears to be dependent on association with the somatic cells in the gonads. Gonadal sex determination and testis development are initiated by the differentiation of sertoli cells. Sertoli cells contain steroid receptors and are therefore targets for agents capable of disrupting normal steroidal signals. Therefore, exposure to vinclozolin during the period of sex determination and testis development has the ability to influence early testis cellular functions leading to alteration of the normal DNA methylation of the germ line. The results of this study suggest that endocrine disrupting compounds can induce epigenetic changes, which are inherited in subsequent generations. This report of an environmental toxin causing damage in the F2 generation of offspring may influence the way in which vinclozolin and other anti-androgens are regulated, however, subsequent studies are required to confirm these results and to assess how this observed phenomenon translates into human health risk. It must also be noted that the exposure levels used in this experiment are higher than would be expected for environmental exposure levels.

References

Newbold, RR.; Hanson, RB.; Jefferson, WN.; Bullock, BC.; Haseman, J.; McLachlan, JA. Increased tumors but uncompromised fertility in the female descendants of mice exposed developmentally to diethylstilbestrol. Carcinogenesis. 19: 1655-1663. 1998.

Newbold, RR.; Hanson, RB.; Jefferson, WN.; Bullock, BC.; Haseman, J.; McLachlan, JA. Proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol. Carcinogenesis. 21: 1355-1363. 2000.

Walker, BE.; Haven, MI. Intensity of multigenerational carcinogenesis from diethylstilbestrol in mice. Carcinogenesis. 18: 791-793. 1997.