Spatial Transcriptomics Reveal Potential Sex Differences in Gene Expression of the Supraoptic Nucleus

Abstract

The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well‐known sexually dimorphic regions of the hypothalamus, little is known about whether this is also true for the SON and whether there are sex differences in SON gene expression. Our study aims to address this knowledge gap by leveraging spatially‐resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially‐resolved gene expression data for the SON of adult male (n=3) and female (n=3) Sprague‐Dawley rats. Briefly, each brain was sectioned at 10μm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the workflow as follows: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics’ Space Ranger and Loupe Browser applications to overlay spatial gene expression data with reference images. Results show that 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. From the list of significant genes after performing differential expression analysis on the SON region using 10x Genomics’ Loup Browser, 74/157 genes were unique to the males, 228/311 genes were unique to the females, and 83 genes (e.g., Avp and Oxt) were common to both sexes. Preliminary Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) homeostatic processes, regulation of peptide hormone secretion, and ion transport for the common genes; 2) ribosomes and translation for the significant genes unique to males; and 3) neurotransmitter release cycle and synaptic transmission for the significant genes unique to females, as some examples. These spatially‐resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with processes and pathways important for osmoregulation and fluid balance. 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.