Single-cell sequencing reveals alterations in the ovarian immune microenvironment regulated by 17β-estradiol in neonatal mice

Abstract

The immunomodulatory function of estrogen within the ovary remains a subject of ongoing debate, and the neonatal ovarian immune microenvironment, particularly its modulation by estrogen, has not been comprehensively characterized. In this study, the effects of 17β-estradiol (E ₂), a key regulator of immune function, were investigated using single-cell transcriptomic profiling of C57BL/6J neonatal mouse ovaries after E ₂ treatment. Results revealed dynamic alterations in the proportion of immune cell types after E ₂ treatment, accompanied by changes in cytokine and chemokine expression. Detailed analyses of gene expression, cell states, and developmental trajectories across distinct cell types indicated that E ₂ treatment influenced cell differentiation and development. Notably, E ₂ treatment reduced the abundance of macrophages and promoted a phenotypic transition from M1 to M2 macrophages. These findings demonstrate that the neonatal mouse ovarian immune microenvironment is sensitive to estrogenic modulation, which governs both the distribution and functional specialization of resident immune cells, offering novel mechanistic insights into the immunomodulatory roles of estrogen across various immune cell types.

Keywords: 17β-estradiol; Immune cells; Immune microenvironment; Ovary; Transcriptional alterations.

Figure 1

E₂ treatment alters the ovarian immune cell landscape in neonatal mice A: UMAP plot of single-cell transcriptome from 4 day post-partum (dpp) mouse ovaries, colored by cell cluster. B: Proportions of major ovarian cell types between CTRL and E₂-treated groups. C: Proportions of ovarian immune cells in 4 dpp, 3-month-old (3 M), and 9-month-old (9 M) mice. D: UMAP plot of immune cell subpopulations between CTRL and E₂-treated groups. E: UMAP plot showing eight immune cell clusters. F: Distribution of immune cell clusters between CTRL and E₂-treated groups. G: UMAP plot of five annotated immune cell subtypes between CTRL and E₂-treated groups. H: Proportion of five immune cell subtypes between CTRL and E₂-treated groups. I: Representative immunofluorescence staining of macrophages (F4/80) in ovarian sections from CTRL and E₂-treated groups. Scale bar: 100 μm (left), 50 μm (others). J: Analysis of F4/80-positive cells in ovarian sections from CTRL and E₂-treated groups. K, L: Detection and analysis of F4/80 protein expression in ovaries. CTRL: Control group. E₂: 17β-estradiol group. Data are shown as mean±SEM. All experiments were repeated at least three times (^**: P<0.01).

Figure 2

E₂ treatment suppresses immune cell proliferation and disrupts their cell fate dynamics A: Volcano plot showing DEGs in immune cell subpopulations between CTRL and E₂-treated groups. B: mRNA expression levels of Ccl2, Ccl4, Cd68, Il10, and Il6 in 4 dpp ovaries from CTRL and E₂-treated groups. C: Immunohistochemical staining of IgG and IL-1β in ovarian sections from CTRL and E₂-treated groups. Scale bar: 50 μm. D: Top 10 GO (BP) terms enriched in immune cell DEGs between CTRL and E₂-treated groups. E: Top 10 KEGG pathways enriched in immune cell DEGs. F: Cell cycle phase distribution in immune cells from CTRL and E₂-treated groups. G: Distribution of five cell states across pseudotime between CTRL and E₂-treated groups. H: Pseudotime trajectory of immune cells in CTRL and E₂-treated groups. I: Proportion of cells in each state between CTRL and E₂-treated groups. J: CytoTRACE analysis of differentiation potential in immune cells. K: Heatmap of DEGs distinguishing two cell fates at branch point 2. L: Top five GO terms (BP) in each gene set. CTRL: Control group. E₂: 17β-estradiol group. Data are shown as means±SEM. All experiments were repeated at least three times (ns: Not significant; ^*: P<0.05; ^**: P<0.01).

Figure 3

E₂ treatment alters B cell transcription and disrupts their cell state dynamics A: Volcano plot showing DEGs in B cell subpopulations between CTRL and E₂-treated groups. B: Top 10 GO (BP) terms enriched in B cell DEGs between CTRL and E₂-treated group. C: Top 10 KEGG pathways enriched in B cell DEGs. D, E: GSEA of representative pathways (ribosome, steroid hormone biosynthesis, ovarian steroidogenesis, and linoleic acid metabolism) in B cells between CTRL and E₂-treated groups. F: Developmental trajectory of B cells between CTRL and E₂-treated groups. G: Distribution of three B cell states between CTRL and E₂-treated groups. H: Pseudotime trajectory of B cells between CTRL and E₂-treated groups. I: Proportion of B cells in each state. J: Heatmap of DEG expression in B cells across pseudotime. K: Top five GO terms (BP) enriched in DEGs of each cluster. CTRL: Control group. E₂: 17β-estradiol group.

Figure 4

E₂ treatment alters innate lymphoid cell transcription and disrupts their cell fate dynamics A: Volcano plot showing DEGs in innate lymphoid cell subpopulations between CTRL and E₂-treated groups. B: Top 10 GO (BP) terms enriched in innate lymphoid cell DEGs between CTRL and E₂-treated groups. C: Top 10 KEGG pathways enriched in innate lymphoid cell DEGs. D, E: GSEA of representative pathways (oxidative phosphorylation, nitrogen metabolism, cell cycle, and linoleic acid metabolism) in innate lymphoid cells between CTRL and E₂-treated groups. F: Developmental trajectory of innate lymphoid cells between CTRL and E₂-treated groups. G: Distribution of three innate lymphoid cell states between CTRL and E₂-treated groups. H: Pseudotime trajectory of innate lymphoid cells between CTRL and E₂-treated groups. I: Proportion of innate lymphoid cells in each state. J–M: Expression of representative DEGs (Hbb-bs, S100a8, S100a9, and Igkc) in innate lymphoid cells across pseudotime. CTRL: Control group. E₂: 17β-estradiol group.

Figure 5

E₂ treatment alters mitotic immune cell transcription and disrupts their cell state dynamics A: Volcano plot showing DEGs in mitotic immune cell subpopulations between CTRL and E₂-treated groups. B: Top 10 GO (BP) terms enriched in mitotic immune cell DEGs between CTRL and E₂-treated group. C: Top 10 KEGG pathways enriched in mitotic immune cell DEGs. D: Cell cycle analysis of mitotic immune cells between CTRL and E₂-treated groups. E, F: GSEA of representative pathways (oxidative phosphorylation, ribosome, primary immunodeficiency, and B cell receptor signaling pathway) in mitotic immune cells between CTRL and E₂-treated groups. G: Developmental trajectory of mitotic immune cells between CTRL and E₂-treated groups. H: Distribution of three cell states between CTRL and E₂-treated groups. I: Pseudotime trajectory of mitotic immune cells between CTRL and E₂-treated groups. J: Proportion of mitotic immune cells in each state. K: Heatmap of DEG expression in mitotic immune cells across pseudotime. L: Top five GO terms (BP) enriched in DEGs in each cluster. CTRL: Control group. E₂: 17β-estradiol group.

Figure 6

E₂ treatment alters dendritic cell transcription and disrupts their cell state dynamics A: Volcano plot showing DEGs in dendritic cell subpopulations between CTRL and E₂-treated groups. B: Top 10 GO (BP) terms enriched in dendritic cell DEGs between CTRL and E₂-treated groups. C: Top 10 KEGG pathways enriched in dendritic cell DEGs. D, E: GSEA of representative pathways (ribosome, nitrogen metabolism, intestinal immune network for IgA production, and fat digestion and absorption) in dendritic cells between CTRL and E₂-treated groups. F: Developmental trajectory of dendritic cells between CTRL and E₂-treated groups. G: Distribution of three states in dendritic cells between CTRL and E₂-treated groups. H: Pseudotime trajectory of dendritic cells. I: Proportion of dendritic cells in each state. CTRL: Control group. E₂: 17β-estradiol group.

Figure 7

E₂ treatment alters macrophage transcription and inhibits M1-to-M2 transition A: Volcano plot showing DEGs in macrophage subpopulations between CTRL and E₂-treated groups. B: Top 10 GO (BP) terms enriched in macrophage DEGs between CTRL and E₂-treated groups. C: Top 10 KEGG pathways enriched in macrophage DEGs. D: UMAP plot of macrophages between CTRL and E₂-treated groups. E: UMAP plot of four clusters in macrophages between CTRL and E₂-treated groups. F: Proportion of each macrophage cluster between CTRL and E₂-treated groups. G: UMAP plot of M1 and M2 macrophages between CTRL and E₂-treated groups. H: Proportion of M1 and M2 macrophages. I: Representative immunofluorescence of M1 macrophage staining (iNOS) in ovarian sections between CTRL and E₂-treated groups. Scale bar: 50 μm. J: Number of iNOS-positive cells per section between CTRL and E₂-treated groups. K: IgG and CD163 staining of ovarian sections between CTRL and E₂-treated groups. Scale bar: 100 μm (Middle); 50 μm (others). L: Analysis of CD163-positive area in each section between CTRL and E₂-treated groups. CTRL: Control group. E₂: 17β-estradiol group. Data are shown as mean±SEM. All experiments were repeated at least three times (^*: P<0.05; ^**: P<0.01).

Figure 8

E₂ treatment alters macrophage differentiation trajectories and alters cell fate in M1 and M2 subtypes A: Developmental trajectory of macrophage subpopulations between CTRL and E₂-treated groups across pseudotime. B: Three cell states of macrophages across pseudotime. C: Pseudotime trajectory of macrophage subpopulations. D: Proportion of macrophages in each state between CTRL and E₂-treated groups. E: Heatmap of DEG expression in macrophages from two cell fates. F: Top five GO terms (BP) enriched in DEGs in each gene set. G: Developmental trajectory of M1 macrophages between CTRL and E₂-treated groups. H: Distribution of five states in M1 macrophage subpopulation. I: Pseudotime trajectory of M1 macrophages. J: Proportion of M1 macrophages in each state. K: Developmental trajectory of M2 macrophages between CTRL and E₂-treated groups. L: Three cell states of M2 macrophages between CTRL and E₂-treated groups. M: Pseudotime trajectory of M2 macrophages. N: Proportion of M2 macrophages in each state. CTRL: Control group. E₂: 17β-estradiol group.

Figure 9

Effects of 17β-estradiol on ovarian immune system in neonatal mouse ovaries E₂ treatment led to increase proportions of several innate immune cells, including innate lymphoid cells, dendritic cells, and mitotic immune cells, within the total immune cell population. Conversely, proportions of B cells and macrophages decreased, with a reduction in M1 macrophages and an increase in M2 macrophages. Additionally, E₂ exposure induced marked cellular heterogeneity and altered the expression profiles of multiple cytokines and chemokines, reflecting broad immunomodulatory effects on the neonatal mouse ovarian immune microenvironment.