DECODING MALE GERM CELL DEVELOPMENT:  CHARTING THE TRANSCRIPTOMIC AND EPIGENETIC LANDSCAPE

Hayden Micheal McSwiggin

Mammalian spermatogenesis is a fascinatingly complex process in which dynamic transcriptional activity and extensive epigenetic remodeling transforms a spermatogonial stem cell into a mature, motile spermatozoon capable of fertilization. This transformation requires the coordinated regulation of mitotic proliferation, meiotic division, and spermiogenesis – the morphological reshaping of round spermatids into elongated sperm with tightly condensed chromatin and functional flagella. To resolve this process with high spatial and temporal resolution, we developed a stage-specific single-cell atlas of spermatogenesis by microdissecting seminiferous tubules into defined stage pools and performing single-nucleus RNA sequencing. This dataset represents the most comprehensive stage-informed transcriptional map of the male germline to date. Using this atlas, we identified five transcriptionally distinct Sertoli cell states that exhibit stage-specific expression patterns and signaling profiles, suggesting critical roles in coordinating germ cell development. We further uncovered that meiotic sex chromosome inactivation (MSCI) initiates earlier than previously appreciated and is accompanied by a compensatory upregulation of autosomal genes, highlighting a finely tuned balance between chromosomal silencing and autosomal activation. In parallel, we constructed a 6-base (5mC/5hmC) epigenetic blueprint of the developing testis and purified germ cell types, revealing that global methylation levels increase by approximately 20% during postnatal spermatogenesis across two discrete waves. The first major wave at the prospermatogonia-to-spermatogonia transition and a second wave spanning spermatogonial differentiation through epididymal maturation. We also identified transition-specific DMRs enriched at meiotic recombination hotspots at the onset of meiosis, consistent with TET-mediated active demethylation priming recombination sites for PRDM9 recognition. Additionally, key spermiogenic loci including Prm1 and Tnp1 accumulated progressive promoter hypermethylation during spermiogenesis while maintaining high transcript levels, revealing a dissociation between promoter methylation state and transcriptional output that challenges canonical models of methylation-driven gene repression. Finally, to examine whether paternal metabolic dysfunction alters the molecular composition of human spermatozoa, we profiled sperm small non-coding RNA (sncRNA), mRNA, and lncRNA content in obese/inactive (OI) and lean/active (LA) men enrolled in a structured 12-week lifestyle intervention. We identified 1,744 differentially expressed sncRNA transcripts in OI men at baseline, spanning ten biologically distinct RNA classes, with rRNA-derived and mitochondrial tRNA-derived small RNA as the dominant contributors. The mRNA and lncRNA fractions were largely preserved, consistent with the transcriptionally silent state of mature spermatozoa. Despite clinically meaningful improvement in metabolic parameters across all four intervention arms, the obesity-associated sperm sncRNA signature persisted through the 48-week follow-up period, indicating that short-term lifestyle modification is insufficient to reverse an established sperm RNA program and raising important considerations for preconception counseling.

DECODING MALE GERM CELL DEVELOPMENT: CHARTING THE TRANSCRIPTOMIC AND EPIGENETIC LANDSCAPE

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