Immunolocalization of G protein-coupled estrogen receptor in the rat epididymis

Abstract

Background: Estrogen plays an important role in male reproduction, and males lacking estrogen signaling in the reproductive tissues are infertile. Estrogen signaling is mediated via two nuclear receptors, ERα and ERβ, but it was recently found that a G protein-coupled estrogen receptor (GPER) is present in the testis. It is believed that GPER is a membrane form of the estrogen receptor and mediates non-classical estrogen signaling. However, the cellular localization of GPER in the epididymis is unknown. Therefore, the objective of this study was to determine the cellular and regional expression of GPER in the rat epididymis.

Findings: To localize expression, immunohistochemistry (IHC) was performed using fixed epididymal tissue. Three strains and ages of rats were used to identify whether GPER expression is strain or age specific. Our results are the first to demonstrate immunostaining of GPER in epididymal epithelial cells. Expression was highest near the apical membrane followed by the cytoplasm, consistent with a membrane bound receptor. The highest expression in adult rats was observed in corpus followed by cauda. Western blotting analysis of epididymal tissues from Sprague Dawley rats confirmed specificity of the antibody and regional expression.

Conclusions: Expression of GPER in the corpus and cauda suggests a role for non-classical estrogen signaling in sperm maturation in the corpus, and sperm protection/storage in the cauda. GPER expression pre-pubertally suggests that estrogen may have a role in epithelial cell development in addition to regulation of adult function.

Fig. 1

Immunolocalization of GPER in adult Wistar rats. GPER immunostaining is observed primarily in the principal cell cytoplasm (red color) within the corpus and cauda. Immunostaining is strongest near the apical membrane (arrows). No immunostaining was observed when slides were incubated with pre-absorbed primary antibody. Bar = 100 μm

Fig. 2

Immunolocalization of GPER in adult Brown Norway and Sprague Dawley rats. GPER immunostaining is observed primarily in the principal cell cytoplasm (red color) within the corpus and cauda. Immunostaining is strongest near the apical membrane (arrows). Bar = 100 μm

Fig. 3

Immunolocalization of GPER in the corpus of adult Wistar, Brown Norway and Sprague Dawley rats at higher magnification. GPER immunostaining is observed in the cytoplasm (red color) and immunostaining is strongest near the apical membrane (arrows). Bar = 20 μm

Fig. 4

Immunolocalization of GPER in four week old pre-pubertal and eight week old peri-pubertal Sprague Dawley rats. In pre-pubertal rats, GPER immunostaining is observed primarily in the epithelial cell cytoplasm (red color). Immunostaining is strongest in the cauda and near the apical membrane (arrows). In peri-pubertal rats, GPER immunostaining is observed primarily in the epithelial/principal cell cytoplasm within the corpus and cauda. Immunostaining is strongest near the apical membrane (arrows). Bar = 100 μm

Fig. 5

Western blot for GPER in the IS/caput, corpus and cauda from Sprague Dawley rats. a Proteins of appropriate molecular weight for GPER were observed at 43 kD and 55 kD. Equal amounts of protein were loaded into each lane which was confirmed by actin immunoblotting. b No protein bands were observed when membranes were incubated with pre-absorbed primary antibody. Images shown are representative of results from three separate animals