Blocking a Methylation Enzyme Keeps Sperm Stem Cells Young Across Species

Spermatogonial stem cells, the rare cells in the testis that sustain sperm production for life, have long resisted a basic laboratory demand: keeping them multiplying in a dish without drifting into a more mature, differentiated state. A new study published in Cell Regeneration reports that blocking a single epigenetic enzyme, protein arginine methyltransferase 5, or PRMT5, can hold these cells in their undifferentiated, proliferative state in mice, monkeys and humans. The finding, from researchers working with mouse cell lines, mouse testicular tissue, and small samples of human and cynomolgus monkey testicular biopsies, points to a chemical strategy that may eventually help expand the scarce stem cells needed for experimental fertility treatments.

The team started from a simple observation drawn from publicly available single-cell transcriptome datasets of human, rhesus macaque and mouse testes. As spermatogonia move from an undifferentiated stage toward differentiation, a specific set of DNA and histone methyltransferases, including DNMT1, EHMT2, EZH2, PRMT3 and PRMT5, becomes more highly expressed. Gene-set scores for histone H4 arginine 3 methylation and maintenance of DNA methylation also rose in the differentiating cells. That pattern suggested a hypothesis: if these upregulated epigenetic regulators help drive differentiation, then pharmacologically inhibiting them might freeze the cells in a younger, self-renewing state.

To test that idea, the researchers ran a small-molecule screen in cultured mouse spermatogonial stem cells under conditions that push them to differentiate. The negative control was simply withdrawal of GDNF, the glial cell line-derived neurotrophic factor that is well established as critical for mouse stem cell self-renewal. Cells were plated into 96-well plates on feeder cells, treated with compounds from a chemical library at 10 micromolar, and assessed after six days by measuring green fluorescent protein signal as a proxy for clone number. Hits were defined as compounds that raised proliferation more than three standard deviations above the control mean. Among them, the PRMT5 inhibitor EPZ015666 stood out as the most potent, and a dose-response experiment identified 1 micromolar as the optimal concentration used in all subsequent culture work.

The validation experiments in mouse cells were consistent across several readouts. In the first culture passage, EPZ-treated cultures contained significantly more cells double-positive for ID4, a marker of undifferentiated mouse spermatogonial stem cells, and KI67, a proliferation marker, than vehicle controls. At the same time, the treated cultures had significantly fewer DDX4-positive cells expressing DMRT1, a transcription factor known to promote spermatogonial differentiation. Over successive passages the contrast widened: by the third passage, around day 18, EPZ-treated cells still expressed the key undifferentiated marker PLZF, which was nearly absent in controls, and the treated clones remained larger and more numerous through three consecutive passages. By the fifth passage, at day 30, cell numbers were still gradually rising in the EPZ group while the control cultures had almost disappeared.

The effect also appeared in living animals. Mice given oral EPZ015666 showed a significant increase in PLZF-positive undifferentiated spermatogonia in their testes compared with vehicle-treated controls, along with a marked reduction in KIT-positive differentiating spermatogonia. In a mouse testicular tissue culture system, seven days of EPZ treatment increased the percentage of ID4-positive stem cells among DDX4-positive germ cells and decreased STRA8-positive early spermatocytes. Crucially, the inhibition proved reversible. When EPZ was withdrawn after seven days and the tissues were cultured for another two weeks, the balance flipped back: ID4-positive cells fell and STRA8-positive cells rose again, showing that the block on differentiation lifts once the drug is removed.

Functional competence survived the treatment. Green fluorescent protein-labeled mouse stem cells cultured with EPZ for ten passages, about 60 days, were transplanted into the seminiferous tubules of infertile recipient mice. Two months later the donor cells had colonized the tubules and could be found across germ cell stages, from undifferentiated spermatogonia to spermatocytes and spermatids. The team then isolated GFP-positive spermatozoa from the recipients and injected them into oocytes. Of 14 embryos produced, 12 two-cell embryos were transferred into pseudopregnant mice, three pups were born naturally, and those offspring grew up fertile and produced progeny of their own.

The researchers also probed the drug in a model relevant to chemotherapy injury. Busulfan, a chemotherapy agent known to deplete undifferentiated spermatogonia and impair the niche-supporting Sertoli and Leydig cells, was given to adult mice. When EPZ015666 was administered orally starting ten days after busulfan, the testes examined on day 20 showed a significant increase in PLZF-positive undifferentiated spermatogonia compared with vehicle-treated animals. When the drug was given simultaneously with busulfan, the percentage of undifferentiated spermatogonia among Sertoli cells was also higher by day ten. These results suggest the compound can help preserve or restore the undifferentiated cell pool after and during chemotherapy-induced damage, at least in mice.

Mechanistically, the study argues that PRMT5’s methyltransferase activity is what drives differentiation. Transient and stable knockdown of Prmt5 in cultured mouse stem cells, using siRNA and a CRISPR-Cas13d system, mimicked the drug: proliferation increased under GDNF-free conditions, ID4-positive KI67-positive cells rose, and differentiation genes including Stat3, Dmrt1 and Kit fell. In stem cells genetically engineered so that Prmt5 could be deleted with 4-hydroxytamoxifen, loss of the protein reduced DMRT1 expression. A rescue experiment then separated the enzyme’s structural and catalytic roles: re-expressing wild-type PRMT5 restored DMRT1, whereas a catalytically dead E444Q mutant restored PRMT5 protein levels but failed to restore DMRT1. Transcriptome analysis of the mutant-rescued cells showed downregulation of differentiation markers and co-expression modules enriched for meiotic spindle-related genes, supporting the conclusion that EPZ015666 acts by blocking the enzymatic active site of PRMT5 rather than by reducing its abundance.

The most clinically suggestive results came from primate material. The team enriched spermatogonia from monkey and human testicular biopsies using magnetic sorting for the conserved surface marker ITGA6, confirming that roughly 40 percent of the sorted monkey cells were UTF1-positive undifferentiated spermatogonia and that the human fraction contained UCHL1- and ID4-positive undifferentiated cells. In culture, EPZ015666 increased UTF1-positive undifferentiated cells and reduced DMRT1-positive differentiating cells in monkey samples, and in human samples it produced significantly larger clones, more UCHL1-positive undifferentiated cells and fewer DMRT1-positive differentiating ones. Cultured human testicular tissue fragments from donors with obstructive azoospermia also showed a higher percentage of UTF1-positive undifferentiated spermatogonia after 14 days of EPZ treatment.

The authors are careful about what these results do and do not show. The primate experiments were preliminary, based on only two biologically independent samples per group in the culture assays, and the mouse in vivo work used small groups of three animals or fewer. The team notes that whether the increased cell numbers reflect a direct effect on proliferation or a downstream consequence of blocking differentiation remains unresolved, and the precise molecular mechanism by which PRMT5’s methyltransferase activity controls differentiation is still unknown. Prior genetic studies also show that PRMT5 is indispensable for germ cell development, with knockout in male germ cells causing meiotic defects and infertility, which underscores that any future use of the inhibitor would need careful dosing and timing. Even with those caveats, the study establishes a concrete, reversible chemical handle, validated across three species, for a problem that has stalled the field: keeping human spermatogonial stem cells undifferentiated and expanding in vitro, a prerequisite for cell-based approaches aimed at restoring fertility in boys and men whose stem cell reserves have been damaged by treatments such as chemotherapy.

Subject of Research: Inhibition of the epigenetic enzyme PRMT5 to maintain undifferentiated spermatogonial stem cells across mouse, monkey and human systems

Article Title: PRMT5 inhibition promotes cross-species spermatogonia expansion and suppresses differentiation

Article References: Yao, Z., Wang, W., Tang, K., Yang, Y., Ren, S., Weng, Y., Ma, L., Chen, D., Gu, H., Wan, J., Liu, Z., Zheng, Y., Li, C., Luo, F., Zhao, X.-Y., & Fan, Y. (2026). PRMT5 inhibition promotes cross-species spermatogonia expansion and suppresses differentiation. Cell Regeneration, 15(1), Article 22. https://doi.org/10.1186/s13619-026-00293-x

Image Credits: AI Generated

DOI: 10.1186/s13619-026-00293-x

Keywords: spermatogonial stem cells, PRMT5, EPZ015666, male infertility, epigenetics, GDNF, spermatogenesis, small-molecule screen, testicular tissue culture, busulfan, cell transplantation, Cell Regeneration