Animal Reproduction Science 121S (2010) S168–S170

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Abstract

Immunohistochemical localization of estrogen � and � receptors in
adult stallion epididymis�

I. Martina,∗, C.M. Meloa, M.M.P. Rodriguesb, G.A. Monteiroa, P.N. Guasti a,

L.C.O. Magalhãesa, R. Laufer-Amorimb, F.O. Papaa

a Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, Unesp, Botucatu, Distrito de Rubião Jr,
s/n, SP, Cep 18618-000, Brazil
b Department of Clinical Veterinary Medicine, School of Veterinary Medicine and Animal Science, Unesp, Botucatu, Distrito de Rubião Jr, s/n, SP, Cep
18618-000, Brazil
1. Introduction

Steroid hormones like testosterone and estradiol are
primarily produced in the testes, delivered to the epi-
didymides by the systemic circulation, and exert their
effects by binding to specific receptors in the target tis-
sue (Hess et al., 2002). There is evidence that estrogens are
also important for epididymal function (Hess et al., 1997),
mainly in species like the horse, where the testes produce
large amounts of estrogens (Stewart and Roser, 1998). The
importance of estrogens for proper epididymal function is
most clearly demonstrated in estrogen receptor knockout
mice which have significantly impaired fluid reabsorption
from the efferent ducts, abnormal epididymal morphol-
ogy and abnormal epididymal sperm (Eddy et al., 1996).
Testosterone and estradiol may regulate the epididymal
secretion of many proteins and other factors important
for sperm maturation (Parlevliet et al., 2006). The present
study investigated whether the stallion epididymis is an
estrogen responsive tissue and also characterized changes
in the localization of the estrogen receptor subtypes �
(ER�) and � (ER�) among the different regions of the epi-

didymis.

� This paper is part of the supplement entitled “Proceedings of the
Tenth International Symposium on Equine Reproduction”, Guest Edited
by Margaret J. Evans.

∗ Corresponding author. Tel.: +55 14 38116249; fax: +55 14 38116249.
E-mail address: ianmartin@uol.com.br (I. Martin).

0378-4320/$ – see front matter
doi:10.1016/j.anireprosci.2010.04.107
2. Materials and methods

The caput, corpus and cauda of the epididymis were
collected from seven adult stallions, 5 years old, castrated
during the breeding season. The tissue specimens, approx-
imately 5 mm in diameter were washed in saline, placed in
plastic cassettes for inclusion, fixed in 10% buffered forma-
lin for 24 h and stored in 70% ethanol until embedding in
paraffin. Four micrometers tissue sections were mounted
onto glass slides previously treated with Organosilane
(Sigma Chemical Co., USA). Sections were deparaffinized
with xylene and rehydrated in graded alcohol. The slides
were then washed in tap water for 10 min. For antigen
retrieval, sections were microwaved for three periods of
5 min in sodium citrate 10 mM (pH 6.0) solution (ER�)
or in a Pascal pressure chamber (Dako, USA) also in
sodium citrate solution (ER�). After heating, slides were
allowed to cool for 20 min and then washed 10 times
in distilled water. Endogenous peroxidase activity was
quenched with 8% peroxidase solution for 20 min (two
periods of 10 min) followed by 10 baths in distilled water
and incubation with a 3% milk solution for 1 h at room
temperature for blocking. Slides were then washed in Tris
buffered solution 10 mM (pH 7.4), encircled using DakoPen
(Dako, USA) and incubated with the primary mouse anti-
human-ER� monoclonal antibody (clone 1D5, Dako, USA),
diluted 1:50 in antibody diluent (Antibody Diluent with
Background Reducing Components, Dako, USA) or with

the primary mouse anti-human-ER� monoclonal antibody
(clone PPG5/10, Dako, USA), diluted 1:100 in antibody dilu-
ent. Both were incubated in a humidified chamber for 18 h
at 4 ◦C. Then, slides were washed in Tris buffered solu-
tion and incubated with the secondary antibody (Advance,

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I. Martin et al. / Animal Reproduction Science 121S (2010) S168–S170 S169

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ig. 1. Immunolocalization of ER� in caput (A), corpus (B) and cauda (C) o
auda (F) of the stallion epididymides. Negative control, inset in C. Immun
f 400×.

ako, USA) for 30 min with each reagent in a humidified
hamber, according to manufacturer instructions. Each tis-
ue section was washed in Tris buffered solution, and DAB
hromogen (3,3′-diaminobenzidine, Dako, USA) was added
s a chromogen staining substrate for 5 min. Reaction was
topped by rinsing in Tris buffered solution. Tissue sec-
ions were counterstained with Mayer’s hematoxylin for
ither 3 min (ER�) or 1 min (ER�), dehydrated and pre-
erved using Permount (Fisher Scientific, USA) mounting
edium. For negative controls, another section was incu-

ated with mouse immunoglobulin (N-Universal Negative
ontrol Mouse, Dako, USA) instead of primary antibody.

For the evaluation of immunoreactivity, stained sec-
ions were observed and photographed using a Leica DML
ptical microscope (Leica Microsystems, DMLB, Germany)
t magnifications of 400×, with an image analyzer program
Leica QWin Standard V2.5). Both positively and negatively
taining nuclei were evaluated in all slides.

. Results

Immunolocalization for ER� was observed in caput, cor-
us and cauda of the epididymides. The great majority of
pithelial cells (basal and principal cells) were strongly
ositive. Smooth muscle presented more sporadic staining
ith more positive cells observed in the caput, half of the

ells positive in the corpus and more negative cells in the
auda. Connective tissue presented the majority of positive
ells in the caput and half of the cells positive in corpus and
auda. Spermatozoa present in the lumen were always neg-
tive. Endothelial cells were positive in all three evaluated

egions and vascular smooth muscle cells presented more
ositive cells in the caput and half of the cells positive in
he corpus and cauda (Fig. 1).

Immunolocalization for ER� was observed only in caput,
ith corpus and cauda remaining negative. Immunos-
llion epididymis. Immunolocalization for ER� in caput (D), corpus (E) and
hemistry, DAB, counterstaining with Mayer’s hematoxylin, magnification

tainning for ER� was always weaker when compared to
ER�. ER� was localized in basal and principal cells of the
caput, was absent in the majority of the smooth mus-
cle and connective tissue with some cells presenting a
weak immunostaining. Spermatozoa present in the lumen,
vascular smooth muscle cells and endothelial cells were
negative. No staining was seen in negative control (Fig. 1).

4. Discussion

Results show that the estradiol receptor is present in
the epididymides of the stallion and that subtype � is more
expressed in all regions of the epididymis. These findings
reinforce that estrogens are involved in epididymal func-
tion (Hess et al., 1997) and corroborate the findings of
Parlevliet et al. (2006) with respect to ER�. Hejmej et al.
(2005) also evaluated the intensity of the staining; although
our findings were similar for ER�, the intensity of the
staining was stronger in the present than previous study.
Findings in the present study differ from those of Parlevliet
et al. (2006) and Hejmej et al. (2005) with respect to ER�,
because no immunostaining was found in the corpus and
cauda of epididymides.

In conclusion, these results suggest that the stallion epi-
didymis is responsive to estrogens and that this hormone is
important for proper epididymal function. Regional differ-
ences in estrogen regulation are probably due to differences
in receptor expression.
Acknowledgement

To Fapesp for financial support.

Conflict of interest
None.



uction S

Immunolocalization of estrogen and androgen receptors and steroid
concentrations in the stallion epididymis. Theriogenology 66,
S170 I. Martin et al. / Animal Reprod

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