Role of Estrogens in the Size of Neuronal Somata of Paravaginal Ganglia in Ovariectomized Rabbits

Abstract

We aimed to determine the role of estrogens in modulating the size of neuronal somata of paravaginal ganglia. Rabbits were allocated into control (C), ovariectomized (OVX), and OVX treated with estradiol benzoate (OVX + EB) groups to evaluate the neuronal soma area; total serum estradiol (E2) and testosterone (T) levels; the percentage of immunoreactive (ir) neurons anti-aromatase, anti-estrogen receptor (ERα, ERβ) and anti-androgen receptor (AR); the intensity of the immunostaining anti-glial cell line-derived neurotrophic factor (GDNF) and the GDNF family receptor alpha type 1 (GFRα1); and the number of satellite glial cells (SGCs) per neuron. There was a decrease in the neuronal soma size for the OVX group, which was associated with low T, high percentages of aromatase-ir and neuritic AR-ir neurons, and a strong immunostaining anti-GDNF and anti-GFRα1. The decrease in the neuronal soma size was prevented by the EB treatment that increased the E2 without affecting the T levels. Moreover, there was a high percentage of neuritic AR-ir neurons, a strong GDNF immunostaining in the SGC, and an increase in the SGCs per neuron. Present findings show that estrogens modulate the soma size of neurons of the paravaginal ganglia, likely involving the participation of the SGC.

Figure 1

Neuronal soma size of paravaginal ganglia for the C (a), OVX (b), and OVX + EB (c) groups. (a–c) Representative photomicrographs of pelvic vaginal sections stained with Masson's trichrome. (d) Ganglionic area per field, (e) number of ganglionic neurons after Abercrombie's correction, and (f) neuronal soma area. Data are mean ± SEM (n = 6 per group). One-way ANOVA followed by Newman–Keuls tests were carried out to determine significant differences between groups. ^∗∗∗P < 0.001 (compared to the C group); ^&P < 0.001 (compared to the OVX group). n, neuron; nn, neuronal nucleus; SGCn, satellite glial cell nucleus. Bar, 20 µm.

Figure 2

Serum concentrations of total estradiol (E2, (a)) and testosterone (T, (b)) vary between the C, OVX, and OVX + EB groups. (c) The ratio of E2 to T (as logarithm) was calculated to estimate the extent of extragonadal aromatization. Data are the mean ± SEM (n = 6 per group). (d) Aromatase expression in the ovary (O) and vagina (V) for control rabbits; Ponceau's Red staining was used to corroborate equal amounts of protein were loaded. Paravaginal neurons from the C (e), OVX (f), and OVX + EB (g) groups express aromatase as showed by immunohistochemistry. (h) Percentages of aromatase-ir neurons are means ± SEM (n = 6 per group). One-way ANOVA followed by Newman–Keuls post hoc tests were carried out to determine significant differences between groups. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 (compared to the C group); ^&P < 0.001 (compared to the OVX group). −n, negative neurons; +n, positive neurons; +SGC, positive satellite glial cell. Bar, 20 µm.

Figure 3

Expression of estrogen receptors (ERα and ERβ) in paravaginal neurons of C (a, d), OVX (b, e), and OVX + EB (c, f) groups. Representative photomicrographs showing the nuclear ERα-ir (a–c) and ERβ-ir neurons (d–f). The percentages of ERα-ir (g) and ERβ-ir neurons (h) were similar between groups. Data are means ± SEM (n = 6 per group). One-way ANOVA followed by Newman–Keuls post hoc tests were carried out to determine significant differences between groups. +nf, positive neural fiber; +nn, positive neuronal nucleus; −nn, negative neuronal nucleus; +SGCn, positive satellite glial cell nucleus; −SGCn, negative satellite glial cell nucleus. Bar, 20 µm.

Figure 4

Expression of the androgen receptor (AR) in the paravaginal neurons of the C (a), OVX (b), and OVX + EB (c) groups. Representative photomicrographs showing the nuclear and neuritic AR immunoreactivity. (d) The percentage of neuritic AR-ir neurons is expressed as the mean ± SEM (n = 6 per group). One-way ANOVA followed by Newman–Keuls post hoc tests were carried out to determine significant differences between groups. ^∗∗P < 0.01, ^∗∗∗P < 0.001 (compared to the C group). +n, positive neuronal cytoplasm; +nn, positive neuronal nucleus; −nn, negative neuronal nucleus; +nrt, positive neurite; +SGC, positive satellite glial cell. Bar, 20 µm.

Figure 5

Expression of GDNF and GFRα1 in neurons and satellite glial cells (SGCs) of the paravaginal ganglia. Representative photomicrographs showing the GDNF (a–c) and anti-GFRα1 (d–f) in the paravaginal ganglia from the C (a, d), OVX (b, e), and OVX + EB (c, f) groups (n = 6 per group). Inset, magnification of fields indicated by dashed squares. SGC, satellite glial cell; SGCn, satellite glial cell nucleus. Bar, 20 µm.

Figure 6

Satellite glial cells (SGCs) of the paravaginal ganglia. Representative photomicrographs showing the GFAP immunoreactivity surrounding neuronal somata for the C (a), OVX (b), and OVX + EB (c) groups. Inset, magnification of fields indicated by dashed squares. Percentage of GFAP-ir in the ensheathed neurons (d), number of SGCs per neuron as estimated from Masson-stained sections (e), and frequency distribution of attached SGC per neuron (f). Data are means ± SEM (n = 5 for both the C and OVX groups, and n = 6 for the OVX + EB group). One-way ANOVA followed by Newman–Keuls post hoc tests were carried out to determine significant differences between groups. ^∗∗P < 0.01 (compared to the C group). Bar, 20 µm.