Visium Spatial Transcriptomics Reveal Sex Differences in Supraoptic Nucleus Gene Expression of Adult Rats Related to Cell Signaling and Ribosomal Pathways

Purpose: There are many well-known sexually dimorphic regions of the hypothalamus; however, sex differences in gene expression in the supraoptic nucleus (SON), a region crucial in the regulation of body fluid homeostasis, has been relatively unexplored. Our previous spatial transcriptomics study revealed gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. Our current study aims to interrogate the sex differences in SON gene expression using two unique methods of differential gene expression (DGE), gene ontology, and pathway analyses. The first DGE approach used Loupe Browser, an application developed by 10x Genomics specifically for their transcriptomics workflow, while the second approach used DESeq2, a more traditional DGE analysis method. Methods: Gonadally-intact adult male (n=4) and female (n=4) Sprague-Dawley rats were anesthetized with isoflurane (2-3% in 95% O2) and their brains were removed and flash frozen. Each brain was sectioned at 10μm thickness and sections (~4x4mm) containing the SON and other brain structures were mounted in capture areas on Visium slides containing probes that bind mRNA. All sections underwent the following workflow: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics’ Loupe Browser application and other bioinformatic tools. Results: Using Loupe Browser, DGE analysis of the SON identified 116 genes (e.g., Avp and Oxt) common to both sexes, 31 genes unique to the males, and 73 genes unique to the females. DGE analysis using DESeq2 revealed 183 significant differentially expressed genes 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) Gi 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. In contrast, GO/pathway analyses using significant genes identified via DESeq2 comparing female vs. male groups revealed GO terms/pathways related to ribosomal structure/function. Conclusions: The two DGE analysis approaches elucidated different aspects of sex differences in SON gene expression. Loupe Browser-based analysis identified genes related more to cell signaling pathways, while DESeq2 identified genes associated with ribosomal structure/function. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.