Single-nucleus and spatial transcriptomics of human ovary: Molecular insights into signaling pathways underlying primary ovarian insufficiency in classic galactosemia

Classic galactosemia (CG) is a rare inborn error of galactose metabolism caused by mutations in GALT gene. Primary ovarian insufficiency (POI) is a later complication that affects 80% of women with CG due to significant decline in ovarian follicle reserve. The definite mechanisms underlying the early onset of POI in CG patients is not fully understood. We utilized single-nucleus RNA sequencing (snRNA-seq) to generate a single-nucleus transcriptomic atlas of human ovaries from pre-pubertal girls with CG to investigate dynamic gene expression profiles in ovarian follicles and stromal cells. Our snRNA-seq analysis revealed activation of several key genes involved in endoplasmic reticulum-stress and oxidative stress pathways which can promote apoptosis and autophagy of follicles. PTEN/PI3K/AKT signaling pathway crucial for primordial follicle activation and survival was dysregulated as evident by upregulated PTEN transcripts, marked reduction in phospho-AKT and increased CASP9 immunostaining intensity in the ovarian follicles of CG group. Nuclei were isolated from flash-frozen ovarian tissues from five classic galactosemia and four control patients using a 10X genomics chromium nuclei isolation kit. Nuclei suspension samples were loaded onto chromium microfluidic chip used to generate single-cell gel bead emulsions using the 10x Genomics Chromium controller. Later cDNA amplification was performed, and libraries were prepared as per the manufacturer’s protocol. Libraries were pooled and sequenced on S2 flow cell (paired-end 100 bp) using an Illumina NovaSeq 6000 platform.