OR20-03 Leptin-mediated Regulation Of Gene Regulatory Networks In Gonadotropes

Disclosure: J. Banik: None. A. Lagasse: None. A. Haney: None. S. Byrum: None. U. Boehm: None. G.V. Childs: None. M.C. MacNicol: None. A.M. MacNicol: None. A.K. Odle: None. The female reproductive system is energetically expensive and requires tight control of the hypothalamic-pituitary-gonadal (HPG) axis to promote gonadal priming and ovulation. Thus, a crosstalk between the metabolic and reproductive systems exists to regulate the rate-limiting steps of reproduction. Leptin is an adipokine hormone that is produced and secreted proportionally from white fat cells, and leptin receptors (Lepr) are present in the hypothalamus, pituitary, and gonads. Leptin binding to hypothalamic LepR functions as a sensor of nutritional status and, thus, orchestrates energy expenditure during reproductive events. We hypothesized that leptin is crucial to influence fertility in females via differential gene regulation during the estrus cycle in pituitary gonadotropes. To test this hypothesis, we utilized Cre-LoxP technology to selectively ablate leptin receptors within female gonadotropes (CreGnRHR(+/-) Lepr(fl/fl) TdTomato(+/+), referred to as “Gon-Lepr-null”) and performed single-cell RNA-sequencing (scRNA-seq) to identify leptin-dependent targets within the gonadotrope transcriptome. We observed a significant decrease in cell percentage within the gonadotrope cluster, and differential analysis reported a significant decrease in Fshb expression in the Lepr-null mutant gonadotropes. Moreover, the lack of leptin signaling in gonadotropes also caused a shift in the lactotrope-specific transcript signature in the Gon-Lepr-null mutants. We employed SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data, to delineate altered upstream regulators contributing to the reduction of Fshb expression in gonadotropes and the shift in the lactotrope transcriptome signature in Gon-Lepr-null mutants versus control animals. Regulons are co-regulated gene sets that share binding sites for a common transcription factor driver, and we identified 381 and 399 regulons specific to controls and mutants, respectively. Examination of the gonadotrope regulons revealed Gata2, Nhlh2, NeuroD1, and Sox11 to be drivers of Fshb expression in control animals only. Differential expression analysis showed significant downregulation of Gata2 and Sox11 in Lepr-null mutant gonadotropes, suggesting Fshb is a leptin-dependent target of Gata2 and Sox11. Moreover, Gata2, Nhlh2, NeuroD1, and Sox11 target 117 genes in controls and 263 genes in mutants, implying a more restricted ability of Gata2, Nhlh2, NeuroD1, and/or Sox11 to target gene expression in the presence of leptin signaling in controls, as an abrogation of leptin signaling leads to an expansion of the regulatory network in Lepr-null mutant gonadotropes. Overall, our analysis reveals how leptin signaling modulates the gonadotrope transcriptome to optimize pituitary reproductive functions at the molecular level. Presentation: Saturday, June 17, 2023