Paternal exposure and effects on microrna and mrna expression in developing embryo. Department of Chemical and Radiation Nur Duale

Transcription

1 Paternal exposure and effects on microrna and mrna expression in developing embryo Department of Chemical and Radiation Nur Duale

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3 Our research question Can paternal preconceptional exposure to environmental contaminants (benzo[a]pyrene, B[a]P) affect the expression of mirna and gene levels of the developing embryo? We investigate the effects of paternal preconceptional exposure to B[a]P on mirna and mrna expression in the developing mouse embryo.

4 Overview of experimental work Sperm from B[a]P treated male mice Testis Liver lung Oocytes from unexposed superovulated mice In vitro fertilization (IVF) BaP (i.p. 150 mg/kg b.w.) Single embryo mrna expression analysis: RT-qPCR (100 selected genes) Global mirna expression profiling (~20 embryos per treatment group): RTqPCR (~750 mature mirnas)

5 Venn-diagram showing number of mirnas at the three different developmental stages among control embryos and embryos of B[a]P exposed fathers cell 64 8-cell Two-cell 68 Eight-cell Blastocyst 33 Blastocyst 200 Control samples 253 BaP treated samples

6 Expression levels of the six up- and downregulated mirnas in developing embryos derived from B[a]P exposed fathers, relative to unexposed controls. Heatmap of 102 mirnas 2-, 8-cell and blastocyst stage

7 A matrix of 51 genes 90 B[a]P-exposed samples (embryos)

8 Anti-correlated mirna:mrna pairs

9 mirna-mrna interaction network of inversely correlated mirna-mrna pairs 16 mirnas 17 mrnas

10 Summary Paternal exposure to B[a]P affected the expression of numerous mirnas and genes. The target genes of dysregulated mirnas were in enriched in many different pathways relevant for the developing mouse embryo. This study represents a first exploration of paternally mediated transcriptomal changes in early embryo development, induced by a common environmental chemical. Gene and mirnas expression in early embryos may provide valuable knowledge about potential trans-generational effect of sub-lethal exposure to contaminants.

11 Plasma mirna as radiation exposure biomarkers: C57BL6 mice were exposed to different doses of gamma radiation (Total Body Irradiation, TBI) using a GammaCell 40 irradiator with a Cesium-137 source at a dose rate of 52 cgy/min. Control mice were sham-exposed. Radiation dose group: mice (n = 10/group) exposed to 0, 0.5, 2.0, and 10 Gy at 6 h and 24 h. Plasma mirna profiling using Taqman Rodent MicroRNA Array Card A and Card B (Applied Biosystems) containing all 518 mature mouse mirnas.

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13 mirnaexpression profiles that distinguish different levels of radiation exposure at 6 h. 32 mirnas distinguishing all radiation doses in comparison to the unirradiated mice at 6 h. Cui W, Ma J, Wang Y, Biswal S (2011) Plasma mirna as Biomarkers for Assessment of Total-Body Radiation Exposure Dosimetry. PLoS ONE 6(8): e doi: /journal.pone

14 mirna expression profiles that distinguish different levels of radiation exposure at 24 h 12 mirnas distinguishing all radiation doses in comparison to the unirradiated mice at 24 h.

15 Plasma mirna as biomarker mrna is intrinsically unstable. Unlike mrna-based biomarkers, mirna based biomarkers are relatively stable and their total number is small. The measurement of mirnas in plasma has the advantage of reduced noise compared to measurements of mrna from blood cells, which can have variation due to differences in the cell number due to infection and other secondary conditions unrelated to exposure.

16 Development of radiation response signature that can predict radiation doses Since we were able to develop a mirna signature reflective of radiation dosage, we attempted to find the expression signatures or metagenes that could accurately predict the exposure to various doses of radiation. We used the PAM statistical package to determine the minimal sets of mirnas that can predict specific radiation doses at 6 h and 24 h. As shown in Figure 6A, while training the 6 h time point samples, a 32 mirna model was identified; and its training error was 0% (Figure 6B). To cross validate the model, we used a 10-fold cross validation. As shown in Figure 6C, the 32 mirna metagene can distinguish all radiation doses in comparison to the unirradiated mice. The predictive error was 0% for the unirradiated and 10 Gy 6 h comparison and 10% for the 0.5 Gy 6 h and 2 Gy 6 h groups. A heatmap of these 32 mirnas is shown in Figure 6E. The overall accuracy, sensitivity, and specificity of the 32 mirna signature for the 6 h group are shown in Table 1. The 32 mirna signature achieved 97.5% overall accuracy in the 6 h groups; 100% sensitivity in the unirradiated and 10 Gy groups; 90% sensitivity in the 0.5 Gy and 2 Gy groups; and 97% specificity in the unirradiated and 10 Gy groups; and 100% specificity in the 0.5 Gy and 2 Gy groups.

17 Little attention has been given to how microrna expression profiles are affected by exposure to environmental contaminants. Here we investigate the effects of paternal preconceptional exposure to benzo[a]pyrene (B[a]P) on mirna expression in the developing mouse embryo. Male mice were exposed to B[a]P (150mg/kg i.p.) four days prior to in vitro fertilization experiments. Twenty embryos from each 2-, 8-cell and blastocyst stage were used for genome-wide mirna expression profiling. Paternal exposure to B[a]P affected the expression of several mirnas, and the target genes for some of dysregulated mirnas were in enriched in many different pathways that are likely to be relevant for the developing mouse embryo. By linking the mirna target genes to publicly available databases, we identified some mirna target genes that may serve as global markers of BaP-madieted genotoxic stress. The identification of dysregulated mirnas may provide valuable knowledge about potential trans-generational effects of sub-lethal exposure to contaminants. In conclusion preconceptional paternal exposure to B[a]P may affect mirna expression in the developing mouse embryo. More research is needed to fully appreciate the implications of early dysregulated mirna expression, but given the wide array of cellular processes targeted by mirnas, undesired consequences are expected. Gene and mirnas expression in early embryos may provide valuable knowledge about potential trans-generational effect of sub-lethal exposure to contaminants.

18 We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150 mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qpcr at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

19 In conclusion, paternal exposure to B[a]P leads to altered gene expression in preimplantation mouse embryos. In particular, a significant downregulation of several genes at the blastocyst stage was observed. At the 8-cell stage a clear differential response to paternal B[a]P exposure was found, with nearly 41% of the embryos forming a distinct cluster with significantly upregulated expression of 13 genes. Analysis of the embryonic gene expression suggests a dysregulation of genes related to DNA repair, cell cycle, Trp53, and MAPK signaling. It is well known that post-translational modifications may affect the functionality of transcribed gene products. It is at his point, however, not clear to what extent dysregulated gene expression at these early stages of embryo development may result in phenotypic changes, possibly predisposing theindividual to disease later in life. This is an intriguing question that clearly deserves further attention. This study represents a first exploration of paternally mediated transcriptomal changes in early embryo development, induced by a common environmental chemical.

20 FIG. 1. Unsupervised hierarchical clustering analysis of the relative expression of 51 genes after filtering and normalization of the data. The hierarchical clustering analysis is based on similarities in gene expression. Genes are coded according to their expression patterns (the expression scale spans from -6.0 to 4.5). Samples are horizontally labeled based on the developmental stage they belong to. Vertically, labeled genes indicate more than twofold significantly downregulated expression levels at the blastocyst stage. The covariance value was used as distance metric in this complete hierarchical linkage clustering analysis.

21 TABLE 1 Differentially Expressed Genes (Based on Twofold Cutoff Value) Between Control Embryos and Embryos of Exposed Fathers at Various Stages of Embryo Development

22 FIG. 3. mirna-mrna interaction network of 33 inversely correlated mirna-mrna pairs (19 mirnas and 17 mrnas). In this network, 16 out of 19 mirnas (mmu-mir-133b, mmu-mir-138, mmu-mir-181d, mmu-mir-197, mmu-mir-210, mmu-mir-297c, mmu-mir-298, mmu-mir-30b, mmu-mir-455, mmu-mir-503, mmu-mir-532-3p, mmu-mir-665, mmu-mir-696, mmu-mir- 709, mmu-mir-762 and mmu-mir-92a) were upregulated and the remaining three mirnas (mmu-mir-1906, mmu-mir-204 and mmu-mir-669b) were downregulated, in previously determined of B[a]P dysregulated mirnas (Brevik et al., 2012). These mirnas are inversely correlated with the 17 mrnas from this study. Connection lines represent mirna-mrna interaction; there are negative mirna-mrna pair correlation between the 16 upregulated mirnas and downregulated mrnas. There are also connection between genes, and inconsistently correlated mirna-mrna pairs.

23 Figure 4. Differential expression of plasma mirna after different radiation doses at 24 h. Cui W, Ma J, Wang Y, Biswal S (2011) Plasma mirna as Biomarkers for Assessment of Total-Body Radiation Exposure Dosimetry. PLoS ONE 6(8): e doi: /journal.pone

24 mirna biogenesis van Rooij E. Circulation Research 2011;108: