Analysis of the pluripotent and germline marker gene expression, and the state of X chromosome reactivation of primordial germ cells in pig gonads

The gonadal primordial germ cells (PGCs) possess a unique state of pluripotency and X chromosome activity. However, extensive evidence indicates developmental variability in PGCs across different species. This study aims to evaluate the pluripotency status, specific gene expression patterns, and X chromosome reactivation (XCR) of pig gonadal PGCs. Single-cell RNA-seq revealed significant heterogeneity within the population of gonadal PGCs. Notably, these PGCs expressed high levels of pluripotency markers OCT4, PRDM14, and NANOG, while lacking SOX2 expression. Through the screening of marker genes and subsequent protein expression validation, we identified growth differentiation factor 3 (GDF3) as a specific surface marker for pig gonadal PGCs, facilitating their efficient purification for further study. Furthermore, analysis of gonadal PGCs demonstrated complete XCR. This was evidenced by the absence of repressive histone modifications (H3K27me3, H3K9me3, and H2AK119ub), the lack of X inactive specific transcript (XIST) RNA FISH signal, and the doubled expression of Xlinked genes. Additionally, these PGCs expressed high levels of genes associated with epigenetic modification, chromatin remodeling, and XIST-associated RNA-binding. These factors likely play a crucial role in regulating pluripotency and X chromosome activity. In summary, this study reveals the heterogeneity in pig gonadal PGCs and identifies GDF3 as a specific surface marker. It also elucidates the expression patterns of pluripotency transcription factors and the events involved in XCR.