Transcriptomic and epigenomic profiling of young and aged spermatogonial stem cells reveals molecular targets regulating differentiation.

Spermatogonial stem cells (SSC), the foundation of spermatogenesis and male fertility, possess lifelong self-renewal activity. Aging leads to the decline in stem cell function and increased risk of paternal age-related genetic diseases. In the present study, we performed a comparative genomic analysis of mouse SSC-enriched undifferentiated spermatogonia (Oct4-GFP+/KIT-) and differentiating progenitors (Oct4-GFP+/KIT+) isolated from young and aged testes. Our transcriptome data revealed enormous complexity of expressed coding and non-coding RNAs and alternative splicing regulation during SSC differentiation. Further comparison between young and aged undifferentiated spermatogonia suggested these differentiation programs were affected by aging. We identified aberrant expression of genes associated with meiosis and TGF-β signaling, alteration in alternative splicing regulation and differential expression of specific lncRNAs such as Fendrr. Epigenetic profiling revealed reduced H3K27me3 deposition at numerous pro-differentiation genes during SSC differentiation as well as aberrant H3K27me3 distribution at genes in Wnt and TGF-β signaling upon aging. Finally, aged undifferentiated spermatogonia exhibited gene body hypomethylation, which is accompanied by an elevated 5hmC level. We believe this in-depth molecular analysis will serve as a reference for future analysis of SSC aging. Author summary: Sperms are derived from a primitive population of undifferentiated spermatogonia called spermatogonial stem cells (SSCs), which lay the foundation of male fertility. It is widely known that the aging process results in decline in SSC function and quantity. However, the molecular mechanisms and gene expression changes underlying SSC differentiation and aging remain elusive. Here, we revealed the alterations in gene expression programs of both coding and non-coding RNAs, and alternative splicing regulation during SSC aging and differentiation by profiling the transcriptomes of SSC-enriched undifferentiated spermatogonia in mice. For instance, we found long non-coding RNA Fendrr is highly expressed in undifferentiated cells, suggesting its role in maintaining stem cell function. We further explored the changes at epigenetic level by profiling histone modifications and DNA methylation patterns, which revealed distinct distribution patterns of histone modification H3K27me3 in Wnt and TGF-β signaling upon aging. We also found that aged undifferentiated spermatogonia exhibited a decrease in average 5mC methylation level in gene bodies accompanied by an increase in 5hmC level, which is similar to the observations in humans. Therefore, we believe our work will serve as a reference for future investigation of human reproductive aging, and particularly the mechanisms of SSC aging. [ABSTRACT FROM AUTHOR]