1. Evaluation Summary:

    Maternal exposure to a certain phthalate (DEHP) has been shown to cause spermatogenesis defects in the male progeny, and in their offspring. In their paper, Tando et al have investigated the molecular consequences of this maternal exposure on fetal and adult male germ cells by studying DNA methylation and gene expression by large-scale approaches. They found three genes previously known to be involved in spermatogenesis that are deregulated following maternal exposure to DEHP and which could contribute to the observed spermatogenesis defects.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, Tando et al. investigated the effects of maternal exposure to Di (2-ethylhexyl) phthalate (DEHP) on DNA methylation levels in fetal male germ cells and spermatogenic cells in adult offspring. DNA methylation analysis showed DNA hypermethylation in promoter regions of spermatogenesis-related genes and RNA-sequencing analysis confirmed that expression of the corresponding genes is down-regulated in DEHP-exposed subjects compared to control. Findings from the study suggests DNA hypermethylation and the consequential down-regulation of spermatogenesis-related genes as a molecular mechanism underlying previously reported effects of maternal DEHP exposure on spermatogenesis defects.

    Comment:

    Authors used the FPKM framework to estimate gene expression levels from the RNA-seq data (line 448). Use of FPKM for differential gene expression analysis has been shown to be problematic due to its limitations in terms of inter-sample variability [PMID: 22988256, PMID: 32284352]. As the normalization is sample-specific for the FPKM framework, FPKMs are not suitable for across sample comparisons. Currently, TMM or VST normalized counts are widely accepted more suitable approaches for DEG analysis [PMID: 22988256, PMID: 32284352]. It would be very important that the authors re-analyze the RNA-seq data using TMM or VST normalized counts.

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  3. Reviewer #2 (Public Review):

    The conclusion that prenatal exposure to DEHP induced epi-mutations in the germ cells is highly relevant and sustained by the work. The limitation is that the genome may have change and this is not controlled. Indeed, if epi-mutations appear in the germ cells subsequent to DEHP exposure, they may allow transposition mechanisms, notably during reprogramming of the germ cells. Such scenario may directly change the germ cells genomes.

    The major strengths of the work are 1) to have extracted the male germ cells of the fetus directly after exposure, which is challenging, and at adulthood, providing information on how changes may persist across development, 2) the functional validation with the CpG-free plasmid.

    The weakness is 1) the smallest possible replicates number (n=2 only) for the majors part of the experiments that were conducted, a strong limitation to mention. 2) The experimental design regarding a breeding scheme is not understandable enough, this make the work difficult to follow.

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  4. Reviewer #3 (Public Review):

    Tando et al started their study by controlling that the progeny of female mice exposed to DEHP present spermatogenesis defects as previously shown in the literature. They then collected fetal germ cells and adult germ cells at different stages (i.e. spermatogonia, spermatocytes and round spermatids) and perform RRBS and RNA-seq analyses to identify differentially methylated regions and deregulated genes.

    The manuscript is very clear and well-written, the figures nicely presented. The chosen technical approaches are appropriate but the number of replicates (2 for each type of samples) is too small. It seems that the differences between CTL and exposed groups (both for methylation and expression) are quite subtle and indeed heatmap representation does not show clustering of replicates as one could hope for: See figure 2A, in particular adult SPG, or Figure supplement 4. Besides, for one type of sample (E19.5 DEHP RNAseq analysis) only one sample could be analyzed. The statistical analyses used for RNAseq and RRBS are not detailed enough and the lists of DMRs and DEG (differentially methylated regions and deregulated genes) which were derived from these analyses therefore appear quite uncertain.

    Following these high throughput analyses, the authors focused on 9 genes which were found hypermethylated in fetal germ cells and adult spermatogonia, and which are known to be involved in spermatogenesis. They performed targeted methylation analyses and expression analyses on F1 spermatogonia and found hypermethylation and downregulation for 3 of them: Hist1h2ba, Sycp1, and Taf7l. These data are convincing because performed on more samples (4 and 6 replicates). Luciferase assay confirmed that hypermethylation of theses gene promoters induces downgulation.

    The authors also mentioned in their article an effect on the F2 spermatogonia. Yet no significant changes in methylation or expression were found on these samples. Importantly, the changes which were found in F1 spermatogonia were not conserved in more differentiated germ cells, in agreement with the fact these "epi mutations" are not maintained and transmitted to the next generation.

    In conclusion, the topic and methodological approaches are very interesting and relevant but a global effect of maternal DEHP exposure on methylation correlated with gene deregulation could not be demonstrated, probably because of the reduced number of samples which were analyzed. The 3 spermatogenesis genes which were identified are nevertheless good candidates to explain the observed defects.

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