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. 2019 Nov 19;9:17034. doi: 10.1038/s41598-019-53579-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Altered DNA methylation and hydroxymethylation levels in sperm from JWH-133 exposed males and in the placenta of their progeny. (A) Histogram reporting gene expression analysis of the DNA methyltransferases (Dnmts) and Tet hydroxylase performed by RT-qPCR on sperm RNA from JWH-133 exposed males (n = 6) with respect to controls (n = 6) that is set as 1 (Dnmt1: 1 ± 0.19; Dnmt3a: 1 ± 0.27; Dnmt3b: 1 ± 0.15; Dnmt3l: 1 ± 0.52; Tet1: 1 ± 0.41; Tet2: 1 ± 0.11; Tet3: 1 ± 0.09). b-Actin is used as reference gene. (B) MeDIP/hMeDIP analysis performed on pooled DNA obtained from sperm of six JWH-133 exposed males and six controls showing the enrichment of 5mC and 5hmC on imprinted genes. (C) MeDIP/hMeDIP analysis performed on pooled DNA obtained from placentas (n = 6) of three females crossed with JWH-133 exposed or control males. IgGs are used as controls and data obtained are shown as percentage of input DNA. Selected imprinted genes are Peg10 and Plagl1, paternally expressed and H19, maternally expressed. Results are reported as mean ± SD and statistical significance was obtained by Student’s t-Test; (**p < 0.01;***p < 0.001).