Paternal benzo[a]pyrene exposure affects gene expression in the early developing mouse embryo

Abstract

The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. 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.

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.

FIG. 2.

Relative gene expression at various developmental stages for (A) genes involved in DNA damage repair; (B) genes involved in cell-cycle regulation; (C) genes involved in embryo development; and (D) genes involved in methylation and acetylation pathways. Expression in embryos of exposed fathers is shown relative to expression in control embryos.

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.