SUMMARY Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermato- genic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.

Exposure to environmental factors can induce the epigenetic transgenerational inheritance of disease. Alterations to the epigenome termed “epimutations” include “primary epimutations” which are epigenetic alterations in the absence of genetic change and “secondary epimutations” which form following an initial genetic change. To determine if secondary epimutations contribute to transgenerational transmission of disease following in utero exposure to the endocrine disruptor vinclozolin, we exposed pregnant female rats carrying the lacI mutation-reporter transgene to vinclozolin and assessed the frequency of mutations in kidney tissue and sperm recovered from F1 and F3 generation progeny. Our results confirm that vinclozolin induces primary epimutations rather than secondary epimutations, but also suggest that some primary epimutations can predispose a subsequent accelerated accumulation of genetic mutations in F3 generation descendants that have the potential to contribute to transgenerational phenotypes. We therefore propose the existence of “tertiary epimutations” which are initial primary epimutations that promote genome instability leading to an accelerated accumulation of genetic mutations.

There is now considerable evidence indicating the potential for endocrine disrupting chemicals to alter the epigenome and for subsets of these epigenomic changes or “epimutations” to be heritably transmitted to offspring in subsequent generations. While there have been many studies indicating how exposure to endocrine disrupting chemicals can disrupt various organs associated with the body’s endocrine systems, there is relatively limited information regarding the relative susceptibility of different specific organs, tissues, or cell types to endocrine disrupting chemical-induced epimutagenesis. Here we review available information about different organs, tissues, cell types, and/or cell lines which have been shown to be susceptible to specific endocrine disrupting chemical-induced epimutations. In addition, we discuss possible mechanisms that may be involved, or impacted by this tissue- or cell type-specific, differential susceptibility to different endocrine disrupting chemicals. Finally, we summarize available information indicating that certain periods of development display elevated susceptibility to endocrine disrupting chemical exposure and we describe how this may affect the extent to which germline epimutations can be transmitted inter- or transgenerationally. We conclude that cell type-specific differential susceptibility to endocrine disrupting chemical-induced epimutagenesis is likely to directly impact the extent to, or manner in, which endocrine disrupting chemical exposure initially induces epigenetic changes to DNA methylation and/or histone modifications, and how these endocrine disrupting chemical-induced epimutations can then subsequently impact gene expression, potentially leading to the development of heritable disease states.

Analyzing whole-genome bisulfite and related sequencing datasets is a time-intensive process due to the complexity and size of the input raw sequencing files and lengthy read alignment step requiring correction for conversion of all unmethylated Cs to Ts genome-wide. The objective of this study was to modify the read alignment algorithm associated with the wg-blimp pipeline to shorten the time required to complete this phase while retaining overall read alignment accuracy. Here we report an update to the recently published pipeline wg-blimp (whole-genome bisulfite sequencing methylation analysis pipeline) achieved by replacing the use of the bwa-meth aligner with the faster gemBS aligner. This improvement to the wg-blimp pipeline has led to a > 7x acceleration in the processing speed of samples when scaled to larger publicly available FASTQ datasets containing 80-160 million reads while maintaining nearly identical accuracy of properly mapped reads when compared to data from the previous pipeline. The modifications to the wg-blimp pipeline reported here merge the speed and accuracy of the gemBS aligner with the comprehensive analysis and data visualization assets of the wg-blimp pipeline to provide a significantly accelerated workflow that can produce high-quality data much more rapidly without compromising read accuracy at the expense of increasing RAM requirements up to 48GB.

Background Analyzing whole-genome bisulfite sequencing (WGBS) datasets is a time-intensive process due to the complexity and size of the input raw sequencing files and lengthy read alignment step during downstream data processing. This is particularly challenging with WGBS data because the conversion of all unmethylated Cs to Ts genome-wide renders read alignment a cumbersome computational process that can take up to a full work week of computing time. The objective of the study described here was to modify the read alignment algorithm associated with the wg-blimp pipeline to shorten the time required to complete this phase while retaining overall read alignment accuracy. Results Here we report improvements upon the recently published pipeline wg-blimp (whole-genome bisulfite sequencing methylation analysis pipeline) achieved by replacing the use of the bwa-meth aligner with the faster gemBS aligner. This improvement to the wg-blimp pipeline has led to a > 7x acceleration in the processing speed of samples when scaled to larger publicly available FASTQ datasets containing 80–160 million (M) reads. Importantly, this acceleration was achieved while maintaining nearly identical accuracy of properly mapped reads when compared to data from the original pipeline. Conclusion The modifications to the wg-blimp pipeline reported here merge the speed and accuracy of the gemBS aligner with the comprehensive analysis and data visualization assets of the wg-blimp pipeline to provide a significantly accelerated pipeline that can produce high-quality data much more rapidly without compromising read accuracy.