key: cord-0256505-wmsroi58 authors: Gapp, Katharina; Parada, Guillermo; Gross, Fridolin; Corcoba, Alberto; Grau, Evelyn; Hemberg, Martin; Bohacek, Johannes; Miska, Eric A. title: Single paternal Dexamethasone challenge programs offspring metabolism and reveals circRNAs as novel candidates in RNA-mediated inheritance date: 2021-03-10 journal: bioRxiv DOI: 10.1101/2021.02.10.429888 sha: 23b600ac42640d2fa6c8b8510b10e8bc58772cb4 doc_id: 256505 cord_uid: wmsroi58 Single traumatic events that elicit an exaggerated stress response can lead to the development of neuropsychiatric conditions. Studies in mice suggests germline RNA as a mediator of effects of chronic environmental exposures to the progeny. The effects of an acute paternal stress exposure on the germline and their potential consequences on offspring remain unknown. We find that acute administration of an agonist for the stress- sensitive Glucocorticoid receptor, using the common corticosteroid Dexamethasone, affects the RNA payload of post-meiotic transcriptionally silent, mature sperm as soon as 3 hours post exposure. It further impacts early embryonic transcriptional trajectories, as determined by single embryo sequencing, and metabolism in the offspring. Importantly, we show persistent regulation of tRNA fragments in sperm and the descendant 2-cell- embryos, suggesting actual transmission from sperm to embryo. Lastly, we unravel environmentally induced alterations in the previously underconsidered class of sperm circRNAs, and their targets in the early embryo, highlighting this class as a novel candidate in RNA-mediated inheritance. were analysed jointly as to test for (1) effects of Dex injection independent of sampling time potentially relevant small RNA changes mostly require either sperm to reside in testis at 1 exposure time or rely on a prolonged residency in the exposed organism. Interestingly, Based on the two reports on effects of single foot shock on offspring weight and the impact of 2 a single GR activation on germline small RNA payload, we hypothesized that this acute 3 impact on the receptor is sufficient to elicit transgenerational effects. We thus injected Dex 4 once intraperitoneally, then harvested sperm 14 days post injection, and performed IVF using 5 naïve oocytes to generate offspring for phenotyping ( Figure 3A ). Dex treatment did not affect 6 sperm count, fertility rate or resulting litter-sizes (Supplemenatary Fig.10 -12). The weight and size of pups was measured every 2 to 4 weeks starting at weaning (3 weeks sex-dependent effect of paternal Dex injection on glucose tolerance, with impaired tolerance 1 in females and decreased glucose levels in males in response to glucose challenge. In addition, blood glucose levels were assessed during the insulin tolerance test. Overall The small quantity of paternal RNAs in the zygote relative to the large pool of maternal RNAs 1 poses serious obstacles to their accurate quantification (36). While initial reports on small 2 RNA transmission relied on comparative sequencing or microarrays analyses of unfertilized 3 oocytes and fertilized zygotes (61), today we are aware that such comparisons can be 4 deceiving, as they rely heavily on both assessment method (eg. microarray restricted to a 5 selective set versus unbiased genome-wide sequencing) and sequencing depth (54, 62). An 6 example are inconsistent results regarding miRNAs that are exclusively supplied from the 7 sperm, such as miR-34c, -99a, -214 (63, 64). Alternative approaches have used indirect 8 measures, e.g. assessing mRNA targets of paternally derived small RNAs (24, 64-66). We 9 attempted to directly examine the relative difference between the small RNA landscape in 10 early embryos resulting from IVF of naïve oocytes with sperm from either Dex or vehicle 11 injected males ( Figure 4A ). We used small-RNA sequencing to compare 2-cell embryos 12 derived from Dex treated or control fathers. We detected an average of 29 % mappable 13 reads including 21% multimappers. While we only detected subtle changes in miRNAs of 14 Dex exposed progeny ( Supplementary Fig. 6 . CircAtlas(83) revealed several potential 11 miRNA sponge-targets to be captured by the altered circRNAs. Some of these miRNAs are Finally, it might be useful to consider testing the translatability of our findings to humans. Here we investigated the effects of a single Dex administration soon after the injection in , where uptake of tsRNAs and miRNAs via epididymisomes has been suggested to lead 9 to differential sperm payload, yet required sperm to transit from caput to cauda to bring about Animal lengths were measured using a standard ruler and weighed for assessing body 1 weight. Body mass index was calculated using the following formula: weight (g)/(length 2 (cm)^2). Organs were dissected after sacrifice and weighed immediately on a scale using "g" as a unit 6 with an accuracy of 2 decimals (accurate down to 10 mg). For the data set collected 14 days after Dex injection, library preparation included the 4 insertion of 2 random tetranucleotides between read and adapters. By including only unique 5 sequences in the analysis we removed duplicates due to PCR amplification. , assuming a negative binomial distribution and a lower detection limit of 0.5. We 2 performed differential gene expression analyses between the total treated and control 3 embryos, and also between the treated and control embryos inside of C1 and C2 clusters. 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