Spatial transcriptomics reveal basal sex differences in supraoptic nucleus gene expression of adult rats related to cell signaling and ribosomal pathways

BACKGROUND: The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that secrete the hormones vasopressin and oxytocin. Sex differences in SON gene expression have been relatively unexplored. Our study used spatially resolved transcriptomics to visualize gene expression profiles in the SON of adult male (n = 4) and female (n = 4) Sprague-Dawley rats using Visium Spatial Gene Expression (10x Genomics). METHODS: Briefly, 10-mum coronal sections (~ 4 x 4 mm) containing the SON were collected from each rat and processed using Visium slides and recommended protocols. Data were analyzed using 10x Genomics' Space Ranger and Loupe Browser applications and other bioinformatic tools. Two unique differential expression (DE) analysis methods, Loupe Browser and DESeq2, were used. RESULTS: Loupe Browser DE analysis of the SON identified 116 significant differentially expressed genes (DEGs) common to both sexes (e.g., Avp and Oxt), 31 significant DEGs unique to the males, and 73 significant DEGs unique to the females. DESeq2 analysis revealed 183 significant DEGs between the two groups. Gene Ontology (GO) enrichment and pathway analyses using significant genes identified via Loupe Browser revealed GO terms and pathways related to (1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females, (2) G(i) signaling/G-protein mediated events for the significant genes unique to males, and (3) potassium ion transport/voltage-gated potassium channels for the significant genes unique to females, as some examples. GO/pathway analyses using significant genes identified via DESeq2 comparing female vs. male groups revealed GO terms/pathways related to ribosomal structure/function. Ingenuity Pathway Analysis (IPA) identified additional sex differences in canonical pathways (e.g., 'Mitochondrial Dysfunction', 'Oxidative Phosphorylation') and upstream regulators (e.g., CSF3, NFKB complex, TNF, GRIN3A). CONCLUSION: There was little overlap in the IPA results for the two different DE methods. These results suggest sex differences in SON gene expression that are associated with cell signaling and ribosomal pathways. The brain releases the hormones oxytocin and vasopressin from the supraoptic nucleus. Oxytocin is involved in maternal behaviors, lactation, and childbirth. Vasopressin is involved in sex-based differences in social behavior and body fluid regulation. However, how the brain contributes to sex-based differences in vasopressin and oxytocin release is poorly understood. This study aimed to address this knowledge gap using spatial transcriptomics to test for sex-based differences in gene expression in the supraoptic nucleus. Spatial transcriptomics combines anatomy with gene sequencing technology, allowing us to identify groups of genes that are expressed in specific locations in the brain. We applied this approach to brain sections containing the supraoptic nucleus from four adult male and four adult female rats. Using a data analysis workflow specifically for spatial transcriptomics, we identified genes that are significantly expressed in the supraoptic nuclei of both males and females (116 genes), primarily males (31 genes), and primarily females (73 genes). Genes enriched in the supraoptic nucleus of both males and females are related to the synthesis and release of peptides like vasopressin and oxytocin. Genes specific to the male supraoptic nucleus are broadly related to cell signaling, while the female-specific genes are related to ion transporters/channels. Results from a more traditional data analysis workflow identified sex-based differences in the expression of genes related to cell metabolism and protein synthesis. Together these results may provide a mechanistic foundation that can be used to better understand how differences in gene expression related to biological sex influence brain function.