A novel system used by pre-implantation mammalian embryos to amplify environmentally-induced changes in sperm miRNA content after fertilization.

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Summary Transgenerational epigenetic inheritance involves transmission of the effects of experiences to offspring across generations via epigenetic changes in germ cells. It is well established in lower organisms like C. elegans and plants, and growing evidence implies a similar phenomenon occurs in mammals including humans. Much support for this process in rodents derives from experiments on male mice exposed to chronic psychological stress, where stress-type specific changes in the levels of specific sperm miRNAs have been implicated in transmitting stress-associated traits across generations. However, sperm contain much lower levels of miRNAs than those of most cells, including those in early embryos. This fact implies a system must exist in embryos to magnify and extend environmental changes in their sperm content after fertilization. In fact, an auto-amplification systems exists and is necessary for transgenerational epigenetic inheritance in lower organisms. However, an analogous system has not been reported in mammals. This grant is driven by our recent discovery of what appears to be a novel amplification system for the sperm miR-34/449 family. These mRNAs are of particular interest in this context because we implicated them in the paternal transmission of enhanced anxiety and defective sociability displayed specifically to female offspring of male mice exposed to Chronic Social Instability (CSI) stress. We found that CSI stress reduces levels of miR-34b/c and miR-449a/b not only in sperm of male mice exposed to CSI stress but also in early embryos derived from them through at least the blastocyst stage of early embryogenesis, even though the levels of these miRNAs in sperm are much lower than that normally present in preimplantation embryos. The amplification system we uncovered is based on our findings that miR- 34c normally positively regulates the expression of its own gene and that for miR-449 in preimplantation mouse embryos, and that this system appears to be suppressed in embryos from CSI stressed males. Surprisingly, we do not detect changes in the primary transcripts or partially processed forms of miR-34c in these embryos, suggesting sperm miR-34, and possibly miR-449, regulates the stability of embryo produced members of this miRNA family. The potential relevance of this system to humans is supported by our findings that; a) both miR- 34 and miR-449 levels are also reduced in sperm of men who experienced severe abusive and/or dysfunctional family life when young; and b) a similar amplification system appears to exist in human embryonic stem cells. The goal of this proposal is to test this hypothesis by measuring the half-lives of miR-34 and miR-449 and their extent of their “tailing” and “trimming”, which are part of the degradation process. We anticipate that these findings will justify more long-term funding for experiments that reveal the how these miRNAs control their own degradation, since this process may necessary for specific forms of transgenerational epigenetic inheritance in both mice and men.