(230) The PRAME Gene Family Regulates Meiotic Initiation Revealed by Single-Cell Transcriptomics of Single and Double Knockout Mouse Testes

The PRAME Gene Family Regulates Meiotic Initiation Revealed by Single-Cell Transcriptomics of Single and Double Knockout Mouse Testes 

Michael Riggs1; Mingyao Yang1; Chandlar Kern1; Camilla H.K. Hughes1; Tae Hyun Kim1; Yuguo Lei2; Wan-Sheng Liu1 

1. Department of Animal Science, The Pennsylvania State University, University Park, PA, United States 

2. Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA, United States 


Abstract Text: The PRAME (Preferentially Expressed Antigen in Melanoma) gene family encodes proteins that are cancer-testis antigens (CTAs), with established roles in immune responses and reproduction. PRAME proteins act as repressors of retinoic acid (RA) signaling, a key pathway regulating cell proliferation and differentiation in germ cells and tumors. Despite high expression, the role of PRAME members during meiosis remains unknown. Recent characterization of Pramel1 and Pramex1 knockout (KO) mouse models—two members of the Prame family—revealed reduced male fecundity, mediated through altered RA signaling. In this study, single-cell RNA sequencing was performed on postnatal day 7 (P7) testes from wild-type (WT), Pramel1 (L1 gKO), Pramex1 (X1 gKO), and Pramel1/Pramex1 double KO (dKO) mice (n=2 per genotype). P7 was selected to capture the developmental transition from undifferentiated to differentiating spermatogonia, and to detect the earliest molecular signs of meiotic initiation. cDNA libraries were prepared using the Chromium Next GEM Single Cell 3’ Kit v3.1 (10X Genomics) and sequenced on an Illumina NextSeq 2000 platform. We targeted 5,000 cells and 225 million reads per sample to ensure sufficient coverage of rare germ cell populations and key meiotic stages. Raw reads were processed using Cell Ranger (v9.0.0) with the GRCm39 (2024-A) mouse reference genome for alignment and quantification. Downstream analysis in R (v4.4.2) using Seurat (v5.2.1) applied stringent quality control thresholds, including nFeature_RNA ≥ 300 (minimum genes per cell), nCount_RNA 1,619–30,000 (total RNA molecules per cell), log10GenesPerUMI ≥ 0.8 (library complexity), and mitochondrial content < 15% (cell quality indicator). After filtering, 18,552 genes and 27,355 cells were retained for analysis. Pairwise differential gene expression (DEG) analysis was performed on the germ cell subsets, applying an adjusted p-value threshold of 0.05 and a log₂ fold change cutoff of ≥ 2.5 and ≤ –2.5. DEGs were subjected to gene ontology (GO) enrichment using the PANTHER (v19.0) classification system to identify affected biological processes. In the combined KO vs WT comparison, only four significant GO terms were identified, with homologous chromosome pairing at meiosis being the most enriched, while the others reflected general cellular processes. In the L1 gKO vs WT comparison, 1,417 DEGs were identified (855 upregulated, 562 downregulated). Upregulated genes were associated with diverse cellular functions without a reproductive focus, while the downregulated subset showed strong enrichment for meiotic pathways, dominating 14 of the top 15 GO terms. The dKO vs WT comparison yielded 221 downregulated DEGs. The top five GO terms were unrelated to reproductive biology, although meiosis-associated terms such as homologous chromosome pairing and male meiotic nuclear division appeared lower in the rankings. In X1 gKO vs WT, 1,044 DEGs were detected (626 upregulated, 418 downregulated). Downregulated genes showed strong enrichment for meiotic processes alongside pathways involved in vesicle trafficking and synaptic function. Additional meiosis-related GO terms, including synaptonemal complex assembly and chiasma assembly, were also significantly enriched. Upregulated genes lacked a cohesive biological theme, spanning broad cellular processes. Collectively, this transcriptomic analysis advances our understanding of Prame’s role in germ cell differentiation and meiotic initiation, highlighting complex gene regulatory dynamics and providing a foundation for further mechanistic studies into its function in male reproductive biology.