17β-estradiol and estrogen receptor alpha protect mouse ovarian follicle development by repressing atresia

Abstract

Mice administered an inhibin antiserum, equine chorionic gonadotropin (eCG) mixture called CARD HyperOva (OVA)/human chorionic gonadotropin (hCG), ovulate more oocytes than those administered eCG/hCG. In this study, the mechanism by which more oocytes are ovulated was investigated. Apoptotic cells were not observed in ovaries 24 and 48 h after OVA injection, and INHBA expression was absent in the growing follicles with apoptotic cells, whereas granulosa cells in follicles expressing INHBA also expressed estrogen receptor α (ESR1). ESR1 and CYP19A1 expression and 17β-estradiol (E₂) in sera significantly increased in OVA-injected mice. Esr1 and Cyp19a1 expression and E₂ concentration increased in ovaries cultured with activin A. The ovulation number increased in mice administered diethylstilbestrol. Taken together, these results suggest that ESR1 and E₂ are involved in the inhibition of follicular atresia, which increases ovulation and oocyte number. This study may provide valuable information on the molecular mechanisms underlying mouse follicle selection.

Keywords: Endocrinology; Molecular physiology; Rodent reproduction.

Graphical abstract

Figure 1

Detection of apoptotic cells and FSH levels in the ovaries and sera of mice administered eCG or OVA (A–F) Apoptotic cells were detected using the TUNEL assay with sections prepared from the ovaries from mice 12 h (A and B), 24 h (C and D), and 48 h (E and F) after eCG or OVA injection. Enlarged images in the box in (A–C) and (E) are shown in (A′–C′) and (E′), respectively. The area surrounded by the line and dotted line in (A′–C′) and (E′) represent follicle and oocyte (Oo), respectively. Arrowheads indicate the follicles with apoptotic GCs. Scale bars: 200 μm (A, C, E); 100 μm (B, D, F); 50 μm (A′, B′, C′, E′). (G) The apoptotic index was expressed as the percentage of the TUNEL-positive cells area per whole area of the follicle. ∗∗p < 0.01 (ANOVA and Tukey-Kramer test, n = 7). (H) Plasma FSH levels were measured using ELISA in mice 12 and 48 h after eCG or OVA injection (t test, n = 4).

Figure 2

INHA and INHBA expression in ovaries and activin A and inhibin A levels in sera of mice administered eCG or OVA (A) INHA and INHBA expression were detected by western blotting using extracts prepared from the ovaries from mice 12 h and 48 h after eCG or OVA injection. (B and C) ACTB was used as a loading control. Plasma inhibin A and/or inhibin B (B) and activin A (C) levels were measured with ELISA using sera collected from mice administered eCG or OVA. ∗∗p < 0.01, ∗p < 0.05 (t test, n = 4).

Figure 3

Detection of INHA and INHBA expression and apoptotic cells in ovaries of mice administered eCG or OVA (A–L) INHA (A, D, G, and J) and INHBA (C, F, I, and L) localization and apoptotic cells (B, E, H, and K) were detected using immunohistochemistry or TUNEL assay, respectively, using sequential sections prepared from mouse ovaries collected 12 and 48 h after eCG or OVA injection. Follicles with positive signals of TUNEL staining are indicated within white dotted lines. Follicles corresponding to those with positive signals of TUNEL staining are indicated within black dotted lines. Scale bars, 100 μm.

Figure 4

Expression of steroidogenic enzyme genes and E₂ levels in ovaries and sera of mice administered eCG or OVA (A and B) RT-qPCR analysis of steroidogenic enzyme gene expression using cDNA prepared from the ovaries of mice collected 12 h (A) and 48 h (B) after eCG or OVA injection. ∗p < 0.05 (t test, n = 5). (C) CYP19A1 expression was detected by western blotting of extracts prepared from mouse ovaries collected 12 and 48 h after eCG or OVA injection. ACTB was used as a loading control (two upper panels). The signal intensity of the bands in the upper panels was quantified using densitometric analysis, and the ratio of CYP19A1 to ACTB expression was calculated as the relative expression (lower panels). ∗p < 0.05 and ∗∗p < 0.01 (t test, n = 4). (D) E₂ and testosterone levels were measured in the sera collected from mice 12, 24, and 48 h after eCG or OVA injection using ELISA. ∗p < 0.05, ∗∗p < 0.01 (t test, n = 6). (E) E₂ and testosterone levels were measured in the ovaries of mice collected 12 and 48 h after eCG or OVA injection using ELISA. ∗p < 0.05 (t test, n = 7).

Figure 5

Expression of caspase genes in ovaries of mice administered eCG or OVA (A and B) RT-qPCR analysis of caspase gene expression using cDNA prepared from the ovaries of mice collected 12 h (A) and 48 h (B) after eCG or OVA injection. ∗p < 0.05, ∗∗p < 0.01 (t test, n = 5).

Figure 6

Effect of E₂ or DES on the number of ovulated oocytes (A) The injection timing of eCG, hCG, E₂, and DES is shown. (B) The number of ovulated oocytes in mice administered E₂, DES, or EtOH together with eCG and hCG is shown. Each dot represents the number of ovulated oocytes from one individual mouse. The median value is indicated by a horizontal black line in each lane. ∗p < 0.05, ∗∗p < 0.01 (t test, n = 7–20). (C–F) The images of the ovulated oocytes are shown.

Figure 7

Expression of Esr1/ESR1 and Esr2 in the ovaries of mice administered eCG or OVA (A) The expression of Esr1 and Esr2 was measured using RT-qPCR in the ovaries from mice administered eCG or OVA. ∗∗p < 0.01 (t test, n = 5). (B) The purity of an antigen against ESR1 was confirmed by SDS-PAGE/Coomassie Brilliant Blue (CBB) staining. The specificity of the anti-mouse ESR1 antibody was evaluated by western blotting using the antibody (WB) and an absorbed antibody (WB [Abs]). (C) Expression of ESR1 was examined by western blotting in the ovaries from mice administered eCG or OVA. ACTB is used as a loading control (lower panel). The signal intensity of the bands was densitometrically quantified, and the ratio of the relative expression of ESR1 to ACTB was determined (right panel). ∗∗p < 0.01 (t test, n = 5).

Figure 8

INHBA and ESR1 localization in the ovaries of mice administered eCG or OVA INHBA (left panels) and ESR1 (right panels) localization were detected using immunohistochemistry using sequential sections prepared from ovaries harvested 12 and 48 h after mice were administered eCG or OVA. Follicles positive for INHBA and ESR1 in the GCs are indicated within black dotted lines. Scale bars, 100 μm.

Figure 9

Effect of activin A on Cyp19a1 and Esr expression and E₂ production in cultured mouse ovaries (A, C, and D) The expression of Cyp19a1 (A), Esr1 (C), and Esr2 (D) were examined using RT-qPCR using cDNA prepared from ovaries cultured with or without activin A. ∗p < 0.05 (t test, n = 3). (B) E₂ levels in the culture media were measured using ELISA. ∗∗p < 0.01 (t test, n = 3).

Figure 10

Overview of the mechanisms that induce or suppress follicular atresia in the ovaries of mice administered eCG or OVA For details, see the text.