Matthew D. Anway

Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility

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.

Transgenerational effects of the endocrine disruptor vinclozolin on the prostate transcriptome and adult onset disease.

The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study.

Transgenerational epigenetic imprints on mate preference

Environmental contamination by endocrine-disrupting chemicals (EDC) can have epigenetic effects (by DNA methylation) on the germ line and promote disease across subsequent generations. In natural populations, both sexes may encounter affected as well as unaffected individuals during the breeding season, and any diminution in attractiveness could compromise reproductive success. Here we examine mate preference in male and female rats whose progenitors had been treated with the antiandrogenic fungicide vinclozolin. This effect is sex-specific, and we demonstrate that females three generations removed from the exposure discriminate and prefer males who do not have a history of exposure, whereas similarly epigenetically imprinted males do not exhibit such a preference. The observations suggest that the consequences of EDCs are not just transgenerational but can be “transpopulational”, because in many mammalian species, males are the dispersing sex. This result indicates that epigenetic transgenerational inheritance of EDC action represents an unappreciated force in sexual selection. Our observations provide direct experimental evidence for a role of epigenetics as a determinant factor in evolution.

Transgenerational Epigenetic Programming of the Brain Transcriptome and Anxiety Behavior

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Further analysis of this transgenerational phenotype on the brain demonstrated reproducible changes in the brain transcriptome three generations (F3) removed from the exposure. The transgenerational alterations in the male and female brain transcriptomes were distinct. In the males, the expression of 92 genes in the hippocampus and 276 genes in the amygdala were transgenerationally altered. In the females, the expression of 1,301 genes in the hippocampus and 172 genes in the amygdala were transgenerationally altered. Analysis of specific gene sets demonstrated that several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation. An investigation of behavior demonstrated that the vinclozolin F3 generation males had a decrease in anxiety-like behavior, while the females had an increase in anxiety-like behavior. These observations demonstrate that an embryonic exposure to an environmental compound appears to promote a reprogramming of brain development that correlates with transgenerational sex-specific alterations in the brain transcriptomes and behavior. Observations are discussed in regards to environmental and transgenerational influences on the etiology of brain disease.

Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

Epigenetic programming of the germ line: effects of endocrine disruptors on the development of transgenerational disease

Epigenetic programming of the germ line occurs during embryonic development in a sex-specific manner. The male germ line becomes imprinted following sex determination. Environmental influences can alter this epigenetic programming and affect not only the developing offspring, but also potentially subsequent generations. Exposure to an endocrine disruptor (i.e. vinclozolin) during embryonic gonadal sex determination can alter the male germ-line epigenetics (e.g. DNA methylation). The epigenetic mechanism involves the alteration of DNA methylation in the germ line that appears to transmit transgenerational adult onset disease, including spermatogenic defects, prostate disease, kidney disease and cancer.

Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease

Endocrine disruptor exposure during gonadal sex determination was previously found to induce male rat adult onset transgenerational disease (F1-F4 generation), and this was associated with an alteration in the epigenetic (i.e., DNA methylation) programming of the male germ line. The current study was designed to characterize the transgenerational disease phenotypes of the female adult offspring. Pregnant rats (F0 generation) were treated transiently with vinclozolin (i.e., fungicide with anti-androgenic activity) on embryonic (E) days E8-E14 of gestation. F1 control and vinclozolin generation offspring from different litters were mated to produce F2 offspring, and similarly F2 generation animals produced F3 generation offspring. Observations demonstrated that 9 out of 105 pregnant rats (8.6%) from the vinclozolin F1-F3 generations exhibited uterine hemorrhage and/or anemia late in pregnancy. None (0 out of 82) of the control F1-F3 generation females had similar pregnancy problems. Complete blood cell counts and serum chemistry profiles demonstrated that selected vinclozolin generation animals, but not controls, exhibited marked regenerative anemia in late pregnancy. Examination of kidney histology revealed moderate or severe glomerular abnormalities in 67% of the vinclozolin F2 and F3 generation adult females compared with 18% of the controls. Adult female vinclozolin generation animals also developed various types of tumors in 6.5% of the animals (11 out of 170), while 2% of control-line animals (3 out of 151) developed mammary tumors. Observations demonstrate that vinclozolin exposure during gonadal sex determination promotes a transgenerational increase in pregnancy abnormalities and female adult onset disease states.

Comparative anti-androgenic actions of vinclozolin and flutamide on transgenerational adult onset disease and spermatogenesis

Exposure of gestating female rats to the anti-androgenic endocrine disruptor vinclozolin has been shown to induce transgenerational adult onset disease phenotypes. The current study, was designed to compare the actions of vinclozolin to the known anti-androgenic compound flutamide. The gestating female rats were exposed to intraperitoneal injections during embryonic day 8-14 (E8-E14) to 100mg/kg/day vinclozolin or flutamide at either 5mg or 20mg/kg/day. As previously observed, vinclozolin induced a transgenerational testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm number. In contrast, the flutamide exposures resulted in a testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm numbers in the F1 generation only, and not the F2 and F3 generation adult males. Interestingly, some of the low dose (5mg/kg) flutamide F2 generation offspring developed spinal agenesis and supernummery development (polymelia) of limbs. Although the actions of vinclozolin and flutamide appear similar in the F1 generation males, the transgenerational effects of vinclozolin do not appear to be acting through the same anti-androgenic mechanism as flutamide.

Seminiferous Cord Formation and Germ‐Cell Programming: Epigenetic Transgenerational Actions of Endocrine Disruptors

Abstract: The molecular and cellular control of embryonic testis development was investigated through an analysis of the embryonic testis transcriptome to identify potential regulatory factors for male sex determination and testis morphogenesis. One critical factor identified is neurotropin 3 (NT3). At the onset of male sex determination, Sertoli cells initiate differentiation and express NT3 to act as a chemotactic factor for mesonephros cells to migrate and associate with Sertoli‐germ cell aggregates to promote cord formation. Promoter analysis suggests that NT3 may be an initial downstream gene to SRY and helps promote testis morphogenesis. Endocrine disruptors were used to potentially interfere with embryonic testis development and further investigate this biological process. The estrogenic pesticide methoxychlor and antiandrogenic fungicide vinclozolin were used. Previous studies have shown that methoxychlor and vinclozolin both interfere with embryonic testis cord formation and cause increased spermatogenic cell apoptosis in the adult testis. Interestingly, transient in vivo exposure to endocrine disruptors at the time of male sex determination caused a transgenerational phenotype (F1‐F4) of spermatogenic cell apoptosis and subfertility. This apparent epigenetic mechanism involves altered DNA methylation and permanent re‐programming of the male germ‐line. A series of genes with altered DNA methylation and imprinting are being identified. Observations reviewed demonstrate that a transient embryonic in utero exposure to an endocrine disruptor influences the embryonic testis transcriptome and through epigenetic effects (e.g., DNA methylation) results in abnormal germ‐cell differentiation that subsequently influences adult spermatogenic capacity and male fertility, and that this phenotype is transgenerational through the germ‐line. The novel observations of transgenerational epigenetic endocrine disruptor actions on male reproduction critically impact the potential hazards of these compounds as environmental toxins. The literature reviewed provides insight into the molecular and cellular control of embryonic testis development, male sex determination, and the programming of the male germ‐line.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice.