Research Paper Volume 12, Issue 7 pp 6089—6108

Estradiol-induced senescence of hypothalamic astrocytes contributes to aging-related reproductive function declines in female mice

Figure 2. Estradiol induces senescence of astrocytes in the hypothalamus. (A) The flow chart of mouse castration and estradiol intervention. (B) Representative microscopies showing SA-β-gal staining in the control (n=5), OVX (n=5) and OVX+E2 groups (n=5), black arrows representing SA-β-Gal–positive cells (left one). Dual-label immunohistochemistry showing astrocytes by GFAP staining (brown) and by SA-β-Gal staining (blue), black arrows representing SA-β-Gal–positive astrocytes (left two). Dual-label immunofluorescence showing astrocytes (green) with γ-H2AX (red), white arrows representing γ-H2AX–positive astrocytes (right two). Dual-label immunofluorescence showing astrocytes (green) with p16 (red), white arrows representing p16–positive astrocytes (right one). Scale bar= 100μm. (C) The flow chart of estradiol intervention in primary cultured astrocytes. (D) Dual-label immunohistochemistry showing astrocytes by GFAP staining (brown) and by SA-β-Gal staining (blue) with three different estradiol concentrations (10-10M, 10-8M, 10-6M) (upper), black arrows representing SA-β-Gal–positive astrocytes. Scale bar= 100 μm. Dual-label immunofluorescence showing astrocytes (green) and γ-H2AX (red) with different estradiol concentrations, white arrows representing γ-H2AX–positive astrocytes. Scale bar=50μm. (E) Detection of p16 and p21 mRNA levels under different estradiol concentrations. Estradiol increased the expression of p16 and p21 with different estradiol concentrations in hypothalamic astrocytes (n = 3-4). The experiments used two-way analysis of variance. The p-value was determined by One-way ANOVA: *p<0.05, ** p< 0.01.OVX, i.e. ovariectomy, OVX+E2, i.e. ovariectomy plus estradiol replacement, 10-6M E2, i.e.10-6M estradiol concentrations.