Sufficient nutrition is critical for reproduction. We have previously shown that leptin, a circulating indicator of fat stores, signals to pituitary gonadotropes to maintain gonadotropin releasing hormone receptor (GnRHR) protein levels in female mice. We hypothesized that this process is post-transcriptional, happening primarily through regulation of the RNA-binding protein Musashi (MSI). We showed that MSI binds to Gnrhr and inhibits translation, and a gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi1/2-null) leads to increased GnRHR protein levels. This culminates in dysregulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH). We have recently identified other gonadotrope and pituitary targets of MSI. We therefore suspected that MSI plays a role in both the maturation of gonadotropes and the normal cyclic regulation of gonadotropes. We hypothesized that the deletion of MSI would lead to downstream effects on (1) the composition of the gonadotrope population and (2) the molecular landscape of these cells. Using our adult, diestrous Gon-Msi1/2-null females, we performed single-cell RNA-sequencing on methanol-fixed dispersed pituitary cells. Libraries were made from two control pools and two mutant pools (n=3 pituitaries/pool) using 10x Genomics v3.1 Single-Cell Gene Expression technology and initially sequenced on an Illumina Next-seq mid-output flow-cell, yielding 5,000 reads/cell. Subsequent high-output sequencing obtained 25,000 reads/cell. We recovered single-cell mRNA transcript information from 18,206 control pituitary cells and 16,255 Gon-Msi1/2-null cells. Our analyses revealed that the Gon-Msi1/2-null pools had a higher % of cells expressing Fshb, as well as an expected significant drop in Msi2-expressing gonadotropes and no change in Lhb-expressing cells. We have recently identified Fshb as an MSI target in silico, and qRT-PCR of female pituitary lysate immunoprecipitated with anti-MSI1 shows a 7-fold enrichment in Fshb mRNA. We identified differentially expressed genes comparing the control and Gon-Msi1/2-null gonadotrope clusters. Using Gene Ontology analyses, the Gon-Msi1/2-null gonadotrope cluster appears to have aberrant expression of mRNAs involved in protein folding and cellular responses to nutrients. Our high-output sequencing has allowed us to achieve 25,000 reads/cell and will provide greater resolution of the role of Musashi in control of gonadotrope function. Taken together, our data indicate that Musashi influences the molecular landscape and subsequent physiology of the female gonadotrope. We have identified potential gonadotrope-specific MSI targets, including pathways that may underlie the dysregulated gonadotropin production and secretion seen in our Gon-Msi1/2-null females. Future studies will compare pubertal and adult females, as well as females from different estrous cycle stages.
