Since the germ line is required for transmitting genetic information between generations, a permanent epigenetic modification of the germ line can result in transgenerational phenomena (Box 2
). Epigenetic programming of the germ line occurs during the migration of the primordial germ cells in the embryo. The migrating primordial germ cells in the genital ridge undergo an erasure of methylation of the DNA during migration and colonize the early bipotential gonad prior to gonadal sex determination [77
]. Once gonadal sex determination is initiated, the primordial germ cells develop female or male germ cell lineage and re-methylate the DNA in a male or female specific manner. Therefore, the germ cell epigenetic programming during gonadal sex determination is a sensitive period to environmental factors [77
] (Box 3
Box 2. Transgenerational Phenotype Definition
The majority of the actions of environmental factors or toxicants involve direct exposures of somatic tissues that are important for the exposed individual’s disease, but will not be transmitted to the next generation. In contrast, transgenerational phenotypes and toxicology by definition excludes direct exposure and must be transmitted through multiple generations [1
]. For example, exposure of a gestating female provides direct exposure of the F0 generation female, the F1 generation embryo, and the germ-line that will generate the F2 generation [108
]. Therefore, a phenotype in the F3 generation is required to have a transgenerational phenomenon or phenotype. The effects observed in the F0 and F1 generations are due to direct exposures, as well as that in the F2 generation germ line [1
]. The ability of a direct exposure to influence multiple generations is defined as a multiple generation phenotype and not a transgenerational phenotype. In contrast, a transgenerational phenotype requires the absence of a direct exposure and transmission of the phenotype to minimally the F3 generation [108
Box 3. Germ Cell Developmental Epigenetics
An important factor to consider with a transgenerational phenotype is the action of environmental factors on the germ line and gonadal development. During embryonic development in mammalian species, the primordial germ cells migrate down the genital ridge towards the developing gonad prior to sex determination [77
]. At the time of gonadal sex determination, the germ cell develops into a male or female germ cell lineage at the initial stages of gonadal sex determination. The female germ line then enters meiosis in the developing embryonic ovary, while male germ cells continue to proliferate until immediately prior to birth and then resume proliferation after birth until puberty [77
]. In the adult, the female germ line undergoes oogenesis during follicle development to generate oocytes. The male germ line, in turn, develops from spermatogonial stem cells and undergoes spermatogenesis for the production of spermatozoa in the testis. The critical period for epigenetic regulation and modification of the germ line is during the period of primordial germ cell migration and gonadal sex determination. The permanent alteration in the epigenetic programming of the germ line appears to be the mechanism involved in the transgenerational phenotype [1
Although there are alterations in the male and female germ line epigenomes (i.e. DNA methylation) during gametogenesis in the adult gonads [79
], the embryonic period of gonadal sex determination is the most sensitive to environmental insults. During spermatogenesis, the male germ cell replaces the majority of histones with protamines, DNA condensation occurs to eliminate chromatin structure, and the genome is silenced for reduced expression of non-coding RNAs [80
]. Although a small percentage of histones are maintained in developmentally important loci [81
], the role of histones in sperm remains to be established. Therefore, the primary epigenetic process that is transmitted through the male germ line is DNA methylation.
One of the first studies to demonstrate the ability of an environmental factor to modify the epigenetic programming of the male germ line used the endocrine disruptor vinclozolin. When embryonic rats were exposed through maternal administration to vinclozolin, an anti-androgenic environmental endocrine disruptor, during gonadal sex determination, adult onset disease occurred in the first generation and persisted for four subsequent generations [33
] (). This phenomenon was found to be due to male germ line changes in DNA methylation, which resulted in heritable changes in transcription in a number of tissues, such as the testis [82
], brain [70
] and prostate [83
]. The pathology of adult onset disease from vinclozolin exposure during embryonic life included testicular, prostate and renal abnormalities and incidence of tumors [33
]. A modification of the sperm epigenome appears to have occurred following vinclozolin exposure at the time of gonadal sex determination, and this enabled transgenerational transmission to subsequent generations to promote adult onset disease [1
] (). This was one of the first reports of an environmental factor promoting epigenetic transgenerational inheritance.
A follow-up study by a company that produces vinclozolin (BASF, Germany) found that oral administration of the same dose used intraperitoneally (IP) [33
] did not have transgenerational effects nor major effects in the F1 generation [86
]. Previously, we found that a 4-fold decrease in the dose eliminated the vinclozolin effect [84
]. For most compounds, oral gavage treatment generally has an order of magnitude lower circulating dose than an IP injection, such that the lack of effect was likely due to insufficient dosing [33
]. In regards to toxicology, this study suggests vinclozolin may not be a significant risk factor at the dose used [86
]. However, in our studies, we used vinclozolin as a pharmacologic agent to promote the transgenerational phenotype and to study mechanism [33
] and did not perform risk assessment or classic toxicology experiments. A second study repeated the vinclozolin experiment [87
] using a more inbred CD-Sprague Dawley (Charles River) rat line versus the outbred Harlan Sprague Dawley line [33
]. This study did not obtain a dramatic transgenerational phenotype [87
]. Previously, we reported that the inbred Fisher rat line did not respond as well as the outbred Harlan Sprague Dawley line [33
] and have recently found the CD-Sprague Dawley response is also not as robust. The hypothesis is proposed that the inbred status of the line may be a factor in the efficiency of promoting the phenotype. We recently repeated the original observation [33
] with the outbred Harlan Sprague Dawley line [88
]. In addition to the outbred status of the line, the exposure timing and duration have been found to be critical. The parameters required to obtain the transgenerational phenotype will likely help reveal aspects of the mechanisms involved. Several other recent studies confirm the ability of environmental agents to promote transgenerational phenotypes [89
], and a recent independent study confirmed the epigenetic transgenerational actions of vinclozolin [90
A number of epigenetic transgenerational phenomena and phenotypes have since been observed in various species and with various environmental factors involved (). The first non-Mendelian hereditary phenomenon reported in plants was called paramutation [44
] and later this transgenerational phenomenon was found to be epigenetic in nature and controlled by DNA methylation [91
]. This event was observed in mammals when a similar mode of inheritance was found in mice [92
]. Nutrition also promotes a transgenerational adult onset obesity phenotype, as described in the Agouti mouse model [61
], and there is also documentation of transgenerational responses to nutrition in humans [94
]. A transgenerational mechanism exists which appears to capture an alteration in nutrition in a sensitive period of perinatal development from the previous generation(s). This requires a mechanism for transmitting the change in environmental exposure (epigenetic) that then alters gene expression and phenotype in the next generation (). A nutritionally induced transgenerational response has been observed down the male line and implies that the sperm carries the ancestral exposure information. A study by Arai et. al. [95
] demonstrates the ability of an animal’s environment to modulate the signaling network that promotes long-term potentiation (LTP) in the hippocampus and to improve contextual fear memory formation across generations. In addition, environment also enhances LTP in their future offspring through adolescence, even if the offspring are not exposed. Stress-induced maternal programming also promotes behavioral changes transgenerationally [96
Epigenetic Transgenerational Events
Heritable disease states such as multiple sclerosis (MS) also appear to have an epigenetic origin [98
]. Epigenetic modifications differentiate among human leukocyte antigen class II risk haplotypes and are involved in the gender bias in MS [98
]. Processes such as embryonic stem cell culture to generate spermatogonial stem cells have been shown to epigenetically alter the germ line and promote abnormalities transgenerationally (F0–F4) in mice [99
]. As discussed, environmental toxicants such as vinclozolin [33
] and the plastic component BPA promote transgenerational disease. The plasticizer BPA also promotes testicular disease from F1 to F3 generations in rats [89
]. Further studies (Box 4
) are required to determine the critical time of exposure of environmental toxicants and to identify factors which result in germ line-transmitted adult onset diseases and those that have an epigenetic basis.
Box 4. Future Questions and Considerations
- The epigenetic and genetic mechanisms of how the germ line epigenome becomes permanently programmed to transmit a transgenerational phenotype needs to be determined.
- A correlation of epigenetic biomarkers with disease needs to be assessed for the potential future development of early stage diagnosis of disease.
- A correlation of epigenetic biomarkers with environmental exposures is needed to develop advanced risk and toxicology assessments.
- The paradigm that genetics is the primary molecular mechanism involved in biology and medicine needs to be modified to incorporate epigenetics as a critical regulatory factor as well.