Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and, as such, their isolation and transplantation have potential for successful therapeutic application for fertility preservation and treatment of male infertility. SSCs have a unique developmental path: they originate from bipotent primordial germ cells (PGCs) that colonize the developing male gonad as pre/prospermatogonia and subsequently mature into adult SSCs during the prepubertal years in humans. Our understanding of the maturation events of SSCs in prepubertal boys is in its infancy, yet this knowledge is a prerequisite for successful clinical use of SSCs in fertility-preserving therapies. The temporal details of SSC maturation dynamics and associated molecular mechanisms, as well as their implications on stem cell potential are generally unknown. Published work from my graduate thesis demonstrated that prepubertal SSCs rely on oxidative phosphorylation (OXPHOS) and undergo a metabolic transition to an adult-like metabolic phenotype in humans just before puberty. Metabolites from OXHPOS serve as important cofactors for epigenetic modifiers that are essential for demethylation during epigenetic reprogramming of PGCs. Reprogramming is characterized by a comprehensive demethylation and sex-specific de novo methylation of male germ cells, events that are critical for male fertility. While de novo methylation events occur quickly in the mouse and are finalized prior to birth, this phenotype does not seem to translate to higher mammals and humans. In fact, the exact de novo methylation dynamics in human SSCs are still unknown. Our goal is to assess the de novo methylation dynamics in pigs and humans for a comparative analysis of prepubertal male germ cell development. As porcine primordial germ cell development has similarities in transcriptional regulation and prepubertal pig samples are readily available, we will use two different ages (1-week and 8-week-old pigs) for preliminary experiments. Firstly, analysis of real-time quantitative PCR (RT-qPCR) for porcine samples and publicly available datasets of serial single cell RNA- Sequencing (scRNA-Seq) will be used to assess the expression of epigenetic modifiers in prepubertal undifferentiated spermatogonia, including SSCs in pigs and humans. Secondly, we aim to visualize and quantify DNA methylation in prepubertal spermatogonia in situ with anti-5-methylcytosine and 5-hydroxymethylcytosine antibodies in pig and human samples using immunostaining. Immunostaining for epigenetic modifiers, such as ten-eleven translocases (TETs) and de novo methylases (DNMTs) in situ will aim to validate the results obtained from transcriptomic analyses. High purity isolations of prepubertal human and pig undifferentiated spermatogonia, including SSCs, will be used to determine the epigenetic dynamics during prepubertal development using serial enzymatic low input methylation sequencing (EM-Seq). Defining critical parameters of DNA methylation across species will help us to decipher epigenetic states of SSC maturation and will be a pivotal piece in the advancement of our understanding of prepubertal development of SSCs that brings us further to harnessing their therapeutic potential.