snRNA-seq profiling of the hypothalamus from O, O.T and Y samples.

(A) UMAP visualization of nuclei colored by 10 cell types: neuron (Neu), astrocyte (Astro), oligodendrocyte (Oligo), oligodendrocyte precursor cell (OPC), tanycyte (Tany), ependymocyte (Epen), microglia (Micro), fibroblast (Fibro), pars tuberalis cell (PTC), and endothelial cell (Endo), from hypothalamus of aged rats (O), 17α-estradiol-treated aged rats (O.T) and young rats (Y). (B) Heatmap showing the classic markers of 10 major cell types in hypothalamus. (C) Cell-type compositions by groups (left panel) or by major cell types with the total cell numbers shown above each column. (D) Circos plot depicting the number of ligand–receptor pairs between Neu and other cell types (color strips) for each group. (E) Dot plot showing significant ligand–receptor interactions between Neurons for each group. Boxes showing the unique ligand–receptor interactions between Neuron.O (black boxes) or between Neuron.O.T (blue boxes). (F) Dot plot of enriched GO biological process terms across three groups of neurons via GSEA analysis. (G) The top 15 changed pathways/gene sets according to the ranks of AUC values in selected pathways related to neuronal synapses and axons from Gene Ontology (GO) biological process, GO molecular function and GO cellular component.

Two opposing regulatory signaling networks in neuron metabolism.

(A) Dot plot of the selected pathways representing the prominent changes of overall expression levels across Neuron.O, Neuron.O.T and Neuron.Y in metabolism, signaling and synaptic activity. (B) Correlation heatmap showing transcription factors (TFs) that correlated with the two opposing regulatory signaling networks in the mixed neurons of O, O.T and Y. (C) The shared unique markers of each quarter (c1-c4) in 6 pathways in hypothalamic neurons (O, O.T, and Y). The markers were then collected as c1.up.signature (19 genes) and c4.up.signature (12 genes). (D) The aging-related cell proportions of each quarter shown by 4 pathways. (E) The correlation of c1.up.signature and c2.up.signature with the two opposing regulatory signaling networks.

Screening of neuron subtypes by supervised clustering that responded distinctly to aging and 17α-estradiol treatment.

(A) Diagram outlining the features of supervised clustering of neurons in hypothalamus compared with the traditional unsupervised clustering. (B) The top 10 and bottom 10 neurons of 121 neurons according to the mean expression values of eight signaling pathways from stress, apoptosis, metabolism, immune response, and senescence in Neuron.O. The expression levels of these neuron subtypes from Neuron.O.T and Neuron.Y were also indicated. (C) Word clouds displaying the frequency of neurons that were among the top 10 in each of the 16 signaling pathways as shown in Table S2. The 16 pathways were from oxidative stress, apoptosis, metabolism, immune response, and senescence. (D) Venn diagram showing the number of neuron subtypes exclusively in each group or shared by the groups in (C). The neurons with frequency >3 were selected for calculation in (C). (E) The top 15 and bottom 15 neurons among 121 neurons according to the mean expression levels of c1.up.signature and c4.up.signature in Neuron.O. (F) The top 15 and bottom 15 neurons among 121 neurons according to the mean expression levels of c1.up.signature and c4.up.signature in Neuron.O.T.

The responses of Crh neurons to long-term 17α-estradiol treatment.

(A) The two intersected neurons among the top 10 neurons according to the mean expression levels of the three senescence-related pathways. (B) The expression profiles of selected pathways from the two opposing signaling networks in Neuron Crh, Nppa and Nppc. Neuron Crh and Nppa were the only two types of neurons shared by the three top 10 neurons in (A). (C) The downregulated and upregulated DEGs expressed by mitochondria or in pathway adherens junction between O.T and O in Crh neurons. (D) The top 25 TF activities in Crh and Gnrh1 neurons. (E) Enzyme immunoassay of the serum levels of Crh, cortisol, and aldosterone in Y, O and O.T. Two-tail unpaired T-test was performed. p values were labeled.

The response of Oxt neurons to 17α-estradiol and the causal effects of Oxt on other endocrine factors.

(A), (B) The top 10 (arrows) and bottom 10 (arrows) types of neurons from 121 subtypes ranked in cell proportions among three groups. (C) Dot plots showing the expression profiles of the selected pathways from the two opposing signaling pathways in four types of food uptake-related neurons, which decreased or increased among the top 10 ranks in (A) or (B). Blue arrows: c1.up.signature and c4.up.signature. (D) Volcanic plots showing the DEGs between Neuron.O.T and Neuron.O in the pathway synaptic membrane. (E) Enzyme immunoassay of the plasma levels of Oxt in three groups. (F) Top 25 TF activities in neuron Oxt. (G) Significant causal effects (p < 0.05, IVW) between exposure OXT (id: prot-a-2159) and 203 endocrine-related outcomes, which were not significant in reverse MR analysis. Significant heterogeneity (Q_pval < 0.05). Significant horizontal pleiotropy (pval < 0.05).

The response of HPG axis in the males to 17α-estradiol and the causal effects of Gnrh on other endocrine factors.

(A) The expression profiles of pathways from the two opposing signaling networks in Gnrh1-, Esr2-, Esr1-or Ar-positive neurons. (B) Enzyme immunoassay of the serum levels of Gnrh, bioavailable testosterone (T), and estrogen (E) in Y, O and O.T samples. Two-tail unpaired T-test was performed. (C) Inflammation of seminiferous tubules in testes of O and O.T. Left two panels: representative Hematoxylin–Eosin (HE) staining of testis inflammation in O and the normal seminiferous tubules of O.T. Right panel: the mean testis inflammation index of O and O.T. (D) The top 25 TF activities in Gnrh1 neurons in three groups. (E) The activities of 14 pathways in Gnrh1-, Esr2-, Esr1-or Ar-positive neurons. (F) Significant causal effects (IVW, p < 0.05) between exposure GNRH1 (id: prot-a-1233) and 203 endocrine-related outcomes, which were not significant in reverse MR analysis. (G) Items with significant causal effects (IVW, p < 0.05) in both directions of MR analysis between GNRH1 (id: prot-a-1233) and 203 endocrine-related outcomes.