# Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

## TL;DR

This study identifies protein markers for human undifferentiated spermatogonia and shows RAS signaling helps maintain their stem cell state.

## Contribution

The study introduces new protein markers for human spermatogonial subsets and reveals RAS signaling's role in their regulation.

## Key findings

- PIWIL4, EGR4, PPP1R36, and NANOS3 label distinct undifferentiated spermatogonia subsets.
- RAS signaling maintains the primitive state of undifferentiated spermatogonia.
- FSD1 is a pan-marker for purifying the entire undifferentiated spermatogonia population.

## Abstract

Spermatogonial stem cells (SSCs) are essential for long-term spermatogenesis and hold therapeutic potential for treating male infertility. While rodent SSCs are well characterized, human SSCs remain poorly understood. Here, we screen antibodies against proteins encoded by genes enriched in specific subsets of human undifferentiated spermatogonia (uSPG) identified by single-cell RNA sequencing. We characterize four markers labeling distinct uSPG subsets: PIWIL4 marks primitive, quiescent uSPG; EGR4 marks uSPG at a proliferative crossroads; and PPP1R36 and NANOS3 label distinct proliferative subsets poised for differentiation. The most and least advanced subsets—PIWIL4+ and NANOS3+ cells—do not overlap. Comparative transcriptomics uncover candidate pathways involved in uSPG fate transitions, including RAS signaling. Using FSD1, a pan-uSPG cell-surface marker identified here, we purify the entire uSPG population and demonstrate that RAS signaling maintains the primitive uSPG state. These findings provide a framework for human uSPG identity, with broad implications for reproductive biology and regenerative medicine.

Capponi et al. define protein markers that distinguish human undifferentiated spermatogonial subsets and provide functional evidence that RAS signaling maintains their primitive state. This work establishes a framework for understanding human undifferentiated spermatogonial identity and regulation.