biostudies-arrayexpressMarie-Justine GuerquinHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13835Fetal human fetal testes (8-11 weeks post-fertilization [WPF]) were cut into small pieces (~1 mm3 ) and 4-6 pieces/testis (depending of the age or size of the gonad) were insert into the muscle of the left back of two twin female mice. One week after surgery, one mouse was exposed by drinking water to a mixture of BPA (1 µM, CAS no. 80-09-1, Merck) and DEHP (1 µM, CAS no. 117-81-7, Merck) and the other was exposed by drinking water to the vehicle (absolute ethanol [ETOH] diluted at 1/1000000 in water). Mice were exposed during 6-11 weeks (corresponding to the 18 WPF equivalent age) and at the end of exposure, testes were collected and each pieces were cleaned from extracellular matrix and murine muscular tissue. One part of the pieces were used for transcriptome analysesbiostudies-arrayexpressLibrary Construction - RNA sequencing was carried out in triplicate under condition (CTL and DE/BP). RNA-Seq libraries are prepared by Integragen SA (Evry, France) with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina according to supplier recommendations (NEB). The capture is then performed on cDNA libraries with the Twist Human Core Exome +Custom IntegraGen Enrichment System according to supplier recommendations (Twist Bioscience). Then a capture of the transcriptome coding regions is performed and the resulting library is suitable for subsequent cluster generation and sequencing. Briefly, the RNA is fragmented into small pieces using divalent cations under elevated temperatures. CDNA is generated from the cleaved RNA fragments with random priming during first and second strand synthesis and sequencing adapters are ligated to the resulting double-stranded cDNA fragments and enriched by 7 PCR cycles. The transcriptome coding regions are then captured from this library using sequence-specific probes to create the final library. For that purpose 500 ng of purified libraries are hybridized to the Twist oligo probe capture library for 16 hr in a singleplex reaction. After hybridization, washing, and elution, the eluted fraction is PCR-amplified with 8 cycles, purified and quantified by QPCR to obtain sufficient DNA templates for downstream applications.Sample Collection - At the end of exposure, fetal human testis were collected and each pieces were cleaned from extracellular matrix and murine muscular tissue. Then, testicular pieces were pooled by condition and stored in RLT buffer from RNeasy minikit (Qiagen).Nucleic Acid Extraction - Total DNA and RNA from xenografted testes were simultaneously isolated using the AllPrep DNA/RNA microkit (ref : 80284, Qiagen, France) according to manufacturer’s instructions. DNA and RNA concentrations and quality were assayed with Qbit fluorometer (Thermofisher) and Agilent bioanalyser. After DNA and RNA quantitation, pools of DNA or RNA per condition were performed containing from 3 to 8 testis per pool.Sample Treatment - Xenografted CTL mice (control) were orally exposed (drinking water) to vehicle (ETOH, 1/1000000) and DE/BP mice were exposed to a mixture of DEHP and BPA at 1µM each.Sequencing - Each eluted-enriched DNA sample is then sequenced on an Illumina HiSeq4000 as 2 x 75b reads. Image analysis and base calling is performed with Illumina Real Time Analysis (2.7.7) with default parameters.Growth Protocol - human fetal testis (from 8-11 WPF) were cut into small pieces (~1 mm3 ) and 4-6 pieces/testis (depending of the age or size of the gonad) were insert into the muscle of the left back of two twin female mice. One week after surgery, mice were orally exposed to chemicals during 6-11 weeks (corresponding to the 18 WPF equivalent age)OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw sequencing data was quality‐controlled with the FastQC program. Low‐quality reads were trimmed or removed using Trimmomatic (minimum length: 50 bp). Paired-end‐reads were aligned to the human reference genome (hg38 build) with STAR software. Mapping results were quality‐checked using RNA‐SeQCData Transformation - Normalization and differential analysis were performed with the edgeR package.UnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000<h4>ABSTRACT</h4> Testicular cancer is an increasing burden in modern societies and the most common malignancy among young adult males. Environment contaminants, especially endocrine disrupting compounds (EDC), may play a significant role in the development of these cancers through epigenetic alterations occurring during fetal and neonatal development. As long-term studies in humans and suitable experimental models with the potential to develop testicular cancer are lacking, no causal link can be established between endocrine disruptor exposure and testicular cancer incidence. Therefore, we developed an experimental model that recapitulates the differentiation of germ cells from primordial germ cells (pluripotency) into spermatocytes (meiosis) by using xenografted human fetal testis combined with germ cell transplantation into adult testis compartments. Using this model, we demonstrate that long-term fetal exposure (until 12 weeks) to a mixture of Di-2-ethylhexylphthalate (DEHP) and Bisphenol A (BPA), two most prevalent plasticizers, could interfere with fetal germ cell differentiation, leading to carcinogenesis and seminomas. Transcriptome, methylome, and histological analyses reveal that BPA/DEHP exposure induced some significant hallmarks of germ cell tumors to occur: persistent pluripotent and proliferative germ cells, global hypomethylation of CpGs in germ cells, abnormal expression of meiotic markers and fibrotic signatures in fetal testis. Additionally, we found that EDC-exposed fetal germ cells were more likely to develop seminoma in a context that allows spermatogenesis to begin. This study proposes the first experimental evidence that EDC exposure can cause long-term, irreversible lesions in fetal germ cells, which then lead to testicular tumorigenesis in adults.RNA-seq of coding RNAHomo sapiensEvidence of the ability of endocrine disrupting compounds to induce testicular germ cell cancer in humansNour Nicolas, Delphine Moison, Amandine Jampy, Quentin Masson, Nathalie Dechamps, Sébastien Messiaen, Sonia Abdallah, Stéphanie Pozzi-Gaudin, Alexandra Benachi, Damien Ulveling, Claire Francastel, Virginie Rouiller-Fabre, Gabriel Livera, Marie-Justine GuerquinMarie-Justine GuerquinfalseRNAseq of human fetal testis treated to a mixture of DEHP/BPAFetal human fetal testes (8-11 weeks post-fertilization [WPF]) were cut into small pieces (~1 mm3 ) and 4-6 pieces/testis (depending of the age or size of the gonad) were insert into the muscle of the left back of two twin female mice. One week after surgery, one mouse was exposed by drinking water to a mixture of BPA (1 µM, CAS no. 80-09-1, Merck) and DEHP (1 µM, CAS no. 117-81-7, Merck) and the other was exposed by drinking water to the vehicle (absolute ethanol [ETOH] diluted at 1/1000000 in water). Mice were exposed during 6-11 weeks (corresponding to the 18 WPF equivalent age) and at the end of exposure, testes were collected and each pieces were cleaned from extracellular matrix and murine muscular tissue. One part of the pieces were used for transcriptome analyses2026-01-01T00:00:00Z2026-01-01T02:01:59.808Z2024-02-20T22:58:57.064ZE-MTAB-13835ERP157631EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1101/2023.12.22.573063