Molecular Mechanisms of Leydig Cell Regeneration

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Cells lost to normal wear and tear in many adult tissues are replaced with the activity of stem cells. In response to injury, however, cells can use entirely different strategies to repair and regenerate tissues. Injured tissues can be regenerated through the activation of stem cells or from progenitor cells that, unlike stem cells, are incapable of asymmetric division and self-renewal. The dramatic cellular changes that occur during regeneration are thought to arise from altered signaling in the tissue microenvironment, or niche, but the mechanisms regulating regeneration are largely unknown. A classical model of regeneration after injury is the adult rat testis. In the testis, somatic cells create a microenvironment that contributes to the germ cell niche. Critical somatic support cells include Leydig cells, the major androgen producing cells in males, which are required for male health and fertility. Although Leydig cells are a quiescent cell type, and not thought to divide under normal conditions, they are chemically ablated by ethane dimethane sulfonate (EDS). Several weeks after chemical ablation, new Leydig cells repopulate the testis. The mechanism of Leydig cell regeneration and the progenitors from which they arise remain poorly understood, partially due to the lack of genetic tools available in rats. In vitro work has hinted at candidate markers that may identify progenitors of Leydig cell regeneration. The goal of this project is to determine the cellular mechanism and molecular mechanism of Leydig cell regeneration. Mouse genetics make it possible to interrogate the cellular mechanism of regeneration by use of an inducible genetic lineage tracing system. In preliminary data, a subset of somatic testis interstitial cells trace into new Leydig cells after ablation in mice. We will continue using this model to determine the both identity of the progenitor cells and whether this cell type is self-renewing. We will also determine the molecular mechanisms driving regeneration with the use of sc-RNA sequencing before, during, and after regeneration. Candidate genes implicated in signaling and Leydig cell development that are differentially expressed in regenerating cells will be tested for functional roles during regeneration using small molecule pathway inhibitors and available genetic tools. While small molecules are an accessible way to gain crude information about whether a signaling pathway is involved, sc-RNA sequencing will illuminate exactly which cells produce signals that initiate the progenitor response. Determining the cellular and molecular mechanisms of Leydig cell regeneration will significantly contribute to the understanding of adult tissue regeneration in vivo with broader implications for male fertility and health.