SAT-001 Impact of VCD-induced Menopause on Gonadotrope Transcriptomics Reveals Estrogen-dependent Genes in the GnRH Signaling Pathway

Abstract Disclosure: T.K. Miles: None. A.K. Odle: None. S.D. Byrum: None. A.N. Lagasse: None. A. Herdman: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. The VCD mouse model of menopause closely mimics the natural progression of perimenopause in humans, as the ovaries remain intact yet are depleted of follicles. While the model is well characterized by low serum estrogen and high gonadotropin levels, little is known about the impact of a prolonged low-estrogen state on gonadotrope transcriptomics. Female FVB/129S mice (3 months; n=13) were injected intraperitoneally (IP) with 4-vinylcyclohexene diepoxide (VCD) at a dose of 160 mg/kg/day in sesame oil daily for 20 days; age-matched controls (n=14) received vehicle only. By day 62 after the start of injections (menopause), serum luteinizing hormone (LH) levels in the VCD-Rx females were 12.7X higher than controls (1666 vs 130 pg/ml, p<0.0001), and normal cycling had ceased. By day 138, serum estradiol levels (Creative Proteomics, Mass Spectrometry) were lower than cycling control female levels (40 pg/ml vs 81 pg/ml proestrus PM p<0.0001). Serum gonadotropin levels were 15X (LH) or 8X (Follicle Stimulating Hormone-FSH) control levels (LH=220 vs 1452 pg/ml; FSH=9425 vs 1173 pg/ml; p<0.0001). Single-cell RNA sequencing was conducted on 10,000 cells from six freshly dispersed pituitaries per group. Bioinformatics clustering, using the 10X genomics platform, identified two distinct gonadotrope clusters. Differentially expressed genes (DEGs) were identified by comparing VCD-Rx vs controls, using a threshold of p <0.055 and Log2FC±0.58 (1.5-fold). Ingenuity Pathway Analysis (IPA) comparing GON1 and GON2 clusters revealed increased DEGs in canonical protein translation pathways and a 2.55X higher expression of gonadotropin-releasing hormone receptor (Gnrhr) mRNA in the GON1 cluster (p=0.01), which correlated with high serum gonadotropins. Yet, Fshb levels were downregulated 1.5-fold in the GON1 cluster, 7.7-fold in the GON2 cluster and the alpha subunit (Cga) was downregulated 2-fold in the GON2 cluster. Lhb was unchanged. IPA canonical pathway analysis showed that the GnRH pathway is reduced (z= -1.414) in the GON1 cluster but increased (z=1.732) in the GON2 cluster. A similar difference was observed in DEGs representing the G-protein coupled receptor signaling pathway (GON1= -1.213; GON2=2.041). Notably, specific downregulated DEGs associated with the suppressed GnRH pathway in the GON1 cluster include Map3 Kinase, Creb3l2, Phospholipase C, and three G protein subunit gamma genes. Stat3 was also downregulated 4-fold in the GON1 cluster but upregulated 2.4-fold in the GON2 cluster. This differential expression suggests heterogeneity in the secretory potential of the gonadotropes, with only the GON2 cluster expressing the increased DEGs needed to sustain the high castration-level gonadotropin secretion. These studies show that the VCD model can be used to identify potential estrogen-dependent targets in gonadotropes. Presentation: Saturday, July 12, 2025