Under UMB3C, the focus has been on reproduction, genotoxic effects and the underpinning explanatory mechanisms. We are following the offspring of (mice and zebrafish) parents exposed to radiation, to test the hypothesis that exposure during the formation of gametes affects the offspring development and phenotype, and to link these effects to changes in gene expression. Previous results obtained for selenium deficient male mice exposed to gamma radiation demonstrated reproduction failure when low antioxidant status is combined with stressors.
Previous results obtained for AB wild type zebrafish exposed to gamma radiation demonstrated reprotoxic effects, lower embryo production and genomic instability in offspring after exposure to dose-rates ≥10 mGy/h during gametogenesis. These effects included damages to female reproductive organs, and genotoxic effects such as persistently increased micronuclei and DNA damage long time post-irradiation. In offspring of F1 of exposed parents, oxidative stress persisted in embryos long term after parental irradiation, including gene expression patterns pointing to disrupted reproductive and developmental pathways, global DNA methylation differences, histone modifications and persistent altered non-coding RNA expression. The parentally exposed embryos (not exposed themselves) showed developmental changes in the eyes with malformed eye membranes and lens suggesting that parental exposure induce developmental effects in the offspring.
Figure 1
A. Control. Normal eye morphology )3 dpf=, with clear lensm, clearly defined layers of cells surrounded by the retinal layer
B. F1-10 E. Cells present in the lens
C. F1-10 G. Presence of cells in the lens and poor differentiation of the cells outside the lens.
D. F1-10GE. Cells in the lens, undifferentiated cellular layer of the eye.
Gamma radiation dose rate of 40 mGy/h during gametogenesis in zebrafish caused the complete disappearance of reproductive organs, while during embryogenesis the reproductive organs were replaced by fat tissue. Dose rate of 10 mGy/h during embryogenesis and up to 21 days caused a 90% reduction in the number of females in the population, including a lower embryo production.
A further hypothesis we have worked with is that primordial germ cells (PGC) are most susceptible to damage by radiation, and that ‘late effects’ such as developmental malformations, or reproductive defects, originate from damage to PGCs cell populations. The zebrafish model are used with the objective to study developmental effects of gamma radiation during sensitive life stages including gametogenesis, embryogenesis and the prepubertal using two lines of zebrafish : a transgenic (vas:egfp) with fluorescent label of germline-specific primordial germ cells (PGC) and an AB wild type (wt) strain.
To determine how germline development is affected in: (i) F0 embryos exposed between 2.5- 5.5 hpf and juvenile F0 fish exposed between 0-21 dpf; and (ii) F1- F3 offspring from exposed parental fish. The transgenic line allows imaging of migrating PGCs during germline development and isolation of the PGC population for single cell type analyses after FACS mediated cell sorting of homogenized embryos and larvae. Analyses of 5.5 hpf embryos and information on the early programming of the zygotic epigenome give insights into mechanisms behind later phenotypic observations.
Subgoal 1. The ATAC-seq analysis of embryos from FO exposed parents revealed a dose response effect with 346, 1774 and 8483 loci showing altered access in chromatin structure after 0.1, 1, and 10 mGy/h (Fig. 1). 1-2 % of fish developed distinct phenotypes with tumors and pigment disorganization, and GFP fluorescence was decreased in offspring embryos from both groups of exposed parental fish
