Changes in Estrogen Receptor ERβ (ESR2) Expression without Changes in the Estradiol Levels in the Prostate of Aging Rats

Abstract

Although the prostate is androgen-dependent, it is also influenced by estrogens, which act via the estrogen receptors ERα and ERβ. In the prostate, ERβ is highly expressed in the epithelium and appears to participate in the regulation of cell proliferation, apoptosis and differentiation. Evidence shows that ERβ is decreased in malignant prostate, suggesting that it plays an important role in protecting this tissue. Despite the relationship between reductions in ERβ and abnormal growth of the gland, little is known about the age-dependent variation of this receptor. Therefore, we aimed to investigate ERβ expression in the prostatic lobes of aging Wistar rats (3 to 24 months). Histopathological alterations, including hyperplasia, intraluminal concretions, nuclear atypia and prostate intraepithelial neoplasias (PIN), were observed in the prostates of aging rats. Epithelial proliferation led to cribriform architecture in some acini, especially in the ventral prostate (VP). In the VP, areas of epithelial atrophy were also observed. Furthermore, in the lateral prostate, there was frequent prostatitis. Immunohistochemistry revealed that the expression of ERβ is reduced in specific areas related to PIN, atrophic abnormalities and cellular atypia in the prostate epithelium of senile rats. Corroborating the involvement of the receptor with proliferative activity, the punctual reduction in ERβ paralleled the increase in cell proliferation especially in areas of PIN and nuclear atypies. The decrease in ERβ reactivity occurred in a hormonal milieu characterized by a constant concentration of estradiol and decreased plasmatic and tissue DHT. This paper is a pioneering study that reveals focal ERβ reduction in the prostate of aging rats and indicates a potential disorder in the ERβ pathway. These data corroborate previous data from humans and dogs that silencing of this receptor may be associated with premalignant or malignant conditions in the prostate.

Fig 1. Histopathology of the prostatic complex of rats at different ages.

Ventral (A-G); dorsal (H-K); lateral (L-O) and anterior (P-S) prostate. (A, H, L and P) Prostates of young adult rats showed normal histology. (B, I, M and Q) Senile rats showed increased unfolding of the epithelium (arrows) in all prostatic lobes. (B) Luminal concretions (c). (C) Epithelial proliferation resulted in cribriform architecture with intraluminal growing (*) in the ventral prostate. (D) Mucinous metaplasia of the epithelium (bounded area). (E) Epithelial atrophy (a) and area of a cell with nuclear enlargement and prominence of nucleoli (n). (F and G) Hyperproliferative epithelium with characteristic foci of prostatic intraepithelial neoplasia (PIN). (G) Cells with evident nuclear enlargement (n) in the PIN area. (J) Detail of the prostatic intraepithelial proliferation observed in the bounded area of the dorsal prostate in panel I. (K) Epithelial proliferation with papillary growth. Thickening of the stroma (**) occurred around the lesion. (N) Lateral prostate with epithelial hyperplasia (arrow) as well as foci of inflammatory cells infiltrating the epithelium (bounded area), lumen (#) and stroma (i). (O) PIN area next to an inflammatory cell infiltrate containing plasma cells (Pl). Cells sloughed into the lumen (sc). (R) Intense unfolding and vacuolization in the epithelium of the anterior prostate (v). (S) PIN area in the anterior prostate with some nuclear atypia (n). Arrowhead = perialveolar smooth muscle cell; Pl = plasma cells; bv = blood vessel; c = luminal concretions; Lu = lumen. Stained with H&E.

Fig 2. Immunostaining for ERβ is affected in specific areas of alterations related to aging.

(A–F) Immunostaining for ERβ in the ventral and (G–K) dorsal prostate of rats at different ages. (A and G) Young adult animals showing intense positivity for the receptor in the epithelial luminal (l) and basal cells (b). Black arrowhead = positive perialveolar smooth muscle cells. (B and H) Senile rats presenting unfolding of the epithelium with normal ERβ expression. (C–D and I–J) Areas of intraepithelial proliferation (*) in the ventral and dorsal prostate, respectively, showing reduced ERβ staining compared with the normal epithelium (Ne). Insert in C and I: immunostaining for CK HMW in the ventral (C) and dorsal (I) prostate showing that the dorsal prostate presents a greater number of basal cells in intraepithelial proliferating areas. (E and K) Drastic reduction of ERβ immunostaining in areas of intense cellular atypia. (F) Transition from normal epithelium (Ne) showing standard ERβ positivity to atrophic epithelium (Ae) presenting a marked decrease in receptor staining. (L and M) Negative immunostaining controls for the ventral and dorsal prostate, respectively. Lu: lumen. Bar in A and G (= B, C, E, F, H, I, J, K) = 50 μm; bar in D = 40 μm; bar in M (= L) = 30 μm.

Fig 3. Quantification of ERβ immunoreactivity in the ventral and dorsal prostates of rats at different ages.

(A–B) Compared to the normal adjacent epithelium, the areas of hyperplasia were similarly stained for ERβ, whereas those of PIN (C–D) and atrophy (E) showed decreased ERβ immunoreactivity. * = p ≤ 0.05; n = 4 per group.

Fig 4. Western blotting of ERβ in the rat prostates at different ages.

(A, C, E and G) Representative bands from the assay. β-actin was used as a reference. (B, D, F and H) Graphical representation of the band densitometric analysis. The data shown are representative of two to three different assays. VP = ventral prostate; DP = dorsal prostate; LP = lateral prostate and AP = anterior prostate. n = 5 per group.

Fig 5. ERβ mRNA levels in the rat ventral and dorsal prostates at different ages.

(A and C) Representative bands from the assay. Gapdh was used as a reference. (B and D) Graphical representation of the band densitometric analysis. The data shown are representative of two different assays. n = 5 per group.

Fig 6. Immunostaining and quantification of MCM7 positive cells in the rat ventral prostate at different ages.

(A) Immunopositivity for MCM7 in normal epithelium of young and senile rats. (B) Graphical representation of quantification of MCM7-positive cells in normal epithelium showing reduced number of positive epithelial cells as rat age. (C) MCM7 immunopositivity in PIN areas of aging prostates. (D) Graphical representation of MCM7-positive cells showing an increase of positive cells in PIN areas (arrows). * and # = p ≤ 0.05 compared to 3 and 6 months, respectively; n = 5 per group. Arrowhead = MCM7 positive cells. Insert: negative control. Bar = 50μm.

Fig 7. Colocalization of ERβ and the proliferation marker Ki67 in the ventral prostate of senile rats.

(A) Comparison between normal epithelium (Ne), atrophic epithelium (Ae) and PIN areas. In PIN areas the ERβ immunoreaction is reduced especially in cells with nuclear atypies (PIN2), whereas the proliferating cells (arrowhead) were more frequently found. (B) 3D reconstruction of the areas demarcated in A. Inserts correspond to negative controls of the respective channels. Bar = 50 μm.

Fig 8. Plasmatic and tissue hormonal levels of 17β-estradiol (E2) and dihydrotestosterone (DHT) of rats at different ages.

(A and B) Levels of E2 in the plasma and ventral prostate. (C and D) Levels of DHT in the plasma and ventral prostate. The data shown are representative of two to three different assays. VP = ventral prostate. * = p ≤ 0.05; n = 3 per group.