RESEARCH ARTICLE
Uludag Univ., J. Fac. Vet. Med. 2018: 37 (2) 119-125 
DOI: 10.30782/uluvfd.415592
Effect Of Tamoxifen Treatment On The Epidermal Growth Factor 
Receptor Expression In The Mouse Ovarian Tissue
Ender Deniz Asmaz1, Berrin Zık 1*
1 Department of Histology Embryology, Faculty of Veterinary Medicine, Uludag University, 16059, Bursa, Turkey 
Received 02.05.2018   Revised: 15.06.2018 Accepted 03.07.2018
Abstract
The objective of this study was to investigate the effect of Tamoxifen (TAM) treatment on epidermal growth factor receptor (EGFR) expres-
sion in the pubertal mice ovary. In this study, 80 female mice (8 week-old) were used. Animals divided four groups; non-injected (control 
A), injected with TAM’s vehicle solution (control B). The mice in groups TAM 0.5 and TAM 1.5 were treated with TAM at a dose 0.5 and 
1.5 mg/mouse/day respectively. TAM was dissolved with 10% ethanol: 90% corn oil. Mice were given daily subcutaneously injections for 5 
days. Ovarian sections were immunostained with EGFR antibody and trichrome staining for examine the general structure. We observed 
that follicular atresia was increased, follicular cysts were formed in stroma, and interstitial cells were increased with TAM dose treatment. In 
the present study, there were no significant differences in the EGFR expression in mouse ovary of all groups. EGFR expression was not deter-
mined in the granulosa cells. While strong EGFR immunoreaction was observed in the interstitial cells and oocyte cytoplasm, weak EGFR 
immunoreactions was observed in theca cells of follicles.
As a result, it was observed that the administered doses of TAM have not significant influence on EGFR expression.
Key Words: EGFR, Immunohistochemistry, Ovary, Tamoxifen
Introduction kinase and ultimately triggers multiple signaling pathways 
(Hudson et al. 2009). The signaling pathways activated by 
EGFR is a member of the human epidermal receptor EGFR dimerization play a key role in variety cancer cell 
(HER) family, comprising four type I transmembrane ty- behaviors, including regulation of cell proliferation, adhe-
rosine kinase receptors: EGFR (HER1, erbB-1), erbB-2 sion, migration, invasion, angiogenesis and resistance to 
(HER2 or Neu), HER3 and HER4. EGFR is the expression apoptosis (Yarden and Pines 2002).
product of oncogene c-erbB1 and is structurally subdivi- In addition to, EGFR has been identified as a strong prog-
ded into an extracellular ligand-binding domain, a single nostic indicator in head and neck, breast, ovarian, cervical, 
lipophilic transmembrane domain, and an intracellular ty- bladder, and esophageal cancers (Herbst and Langer 2002). 
rosine kinase domain (Uberall et al. 2008). Upon binding High EGFR expression has been shown to correlate with 
of the ligand to the extracellular domain, EGFR is activated poor survival in a range of tumors including non-small 
and undergoes homodimerization or heterodimerization, cell lung cancer, breast, head and neck, gastric, colorectal, 
which in turn induces the activation of intrinsic tyrosine esophageal, prostate, bladder, renal, pancreatic, and ovari-
*Corresponding author: Uludağ Üniversitesi, Veteriner Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı,  16059, Bursa, Turkey
e-mail: bzik@uludag.edu.tr
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an cancers (Arteaga, 2002, Atalay et al. 2003, Bernstein et expression, which plays an important role in folliculoge-
al. 1999, Salomon et al. 1995). Overexpression of the EGFR nesis. Therefore, in this study, it was aimed to determine 
protein has been detected in 9%–62% of human ovarian whether or not the EGFR which plays an important role in 
cancers (Lassus et al. 2006, Stadlmann et al. 2006). On the folliculogenesis and follicle development of different doses 
other hand, EGFR plays a role in oocyte maturation, in the of TAM in mouse ovary.  
folliculogenesis and also stimulates the proliferation and 
differentiation of granulosa cells in vitro and in vivo (Vlo- Materials and Methods
davsky and Brown1978,  Jones et al. 1982,  Knecht and Catt Animals and experimental protocol
1983, Ashkenazi et al. 2005). Eighty female BALB/C mice (8 week-old) obtained from 
The selective estrogen receptor modulator (SERM) ta- the Experimental Animals Breeding and Research Center, 
moxifen has been used treatment for patients with ER-po- Turkey, were used throughout the experiments. The ani-
sitive breast cancer in both the early and the advanced/ mals were housed five per cage in temperature (20-24 °C), 
metastatic stages (Shang and Brown, 2002). Acting as both humidity (60-70%), and lighting (12 h light/dark cycles) 
an estrogen agonist and antagonist, tamoxifen is the most controlled conditions and were provided with feed and wa-
frequently used drug in endocrine therapy of breast can- ter ad libitum. The experimental protocols were approved 
cer (Sunderland and Osborne 1991, Goldrisch and Gelber by the Animal Care and Use Committee of Uludag Univer-
1991). It may act as a full estrogen agonist, partial agonist sity and were in accordance with the National Institute of 
or antagonist depending on the dose, species, sex or target Health Guide for the Care and Use of Laboratory Animals 
organ (MacGregor and Jordan 1998). Its anti estrogenic ef- (2012- 09/04). 
fect appears to be related to reduce estrogen receptor levels Mice were randomly divided into 4 groups. (1) Control 
(Spinelli et al. 1991, Gorodeski et al. 1992) or to inhibit the group (control A, n=20), remained without any treatment. 
binding of estradiol (E2) to the estrogen receptor (Jordan (2) Control B group, (vehicle treated, n=20) received an
and Hoerner 1975). Although tamoxifen acts primarily as equal volume of solvent, (3) 0.5 TAM group, (n = 20) re-
an antiestrogen, it also exerts a mild estrogenic effect. It is ceived 0.5 mg/mouse/day of TAM and (4) 1.5 TAM group
known to be effective and safe with minimal side effects (n = 20), received 1.5 mg/mouse/day of TAM. TAM was
(Jolles et al. 1990). However, recently it has been found to dissolved in 10% ethanol: 90% corn oil and the solution
be associated with various endometrial pathologic condi- prepared for this dissolution was used for control B (0.1 ml
tions and endometrial carcinoma (Bernstein et al. 1999, / mouse). The dissolved TAM was injected into the animals
Clarke et al. 1998, Fisher et al. 1994). Several reports sug- for 5 days in a total of 0.1 ml subcutaneously at a dose of 0.5
gest that there may be an association between the tamoxi- mg/mouse and 1.5 mg/mouse. TAM was freshly prepared
fen exposure and the development of ovarian cysts or even every day before injection. At the end of the treatment, the
an increased risk of ovarian cancer (Jordan and O’Malley, animals in the proestrus period which is characterized by
2007, Liu et al. 1993). Cancer treatments significantly inc- nucleated and some cornified epithelial cells are present in
rease survival, but also cause significant side effects such as the vaginal smear were separated and weighed and anest-
ovarian failure and sterility in young patients (Blumenfeld hetized by ether inhalation (Ekambaram et al. 2017). They
et al. 2002, Fong et al. 2008, Meirow and Nugent 2001). were sacrificed by cervical dislocation and then right ova-
TAM agents commonly used in the premenopausal and ries of each mouse were dissected aseptically.
menapausal women for adjuvant chemotherapy following The ovaries were fixed in 10% neutral buffered formalin
breast cancer (Dowsett et al. 2010). TAM treatment may and embedded in paraffin blocks. Five μm thick sections
also increase the occurrence of ovarian cancer especial- were cut from paraffin blocks, mounted on slides, and
ly among premenopausal women (Rutqvist et al. 1995). dried overnight. After dewaxing and rehydration, sections
Mostly studies has been investigated that the expression of were stained with immunohistochemistry (IHC) for exp-
EGFR in vitro breast cancer cells and in the breast cancer ression EGFR and by the Crossman’s modified trichrome
patients who used TAM (Ma et al. 2004, Atalay et al. 2003, stain (Crossman, 1937) for ovary morphology.
Lichtner, 2003). However, in the ovary there is limited in- Immunohistochemistry analysis
formation on the effect of different doses of TAM on EGFR After dewaxing and rehydration, slides were carried out
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antigen retrieval by boiling sections in microwave oven at of atretic follicles with depending on the dose on the surfa-
750 W in sodium citrate buffer (1 M, pH 6.1). After coo- ce of the ovary were observed (Fig. 1B). As a result of eva-
ling, slides were rinsed with PBS and endogenous peroxi- luation, no atresia was observed in primordial and primary 
dase activity was blocked by 10 min incubation at room follicles in all groups, but atretic follicles were observed in 
temperature in 3% H2O2 solution in distilled water. After some secondary, preantral and antral follicles, especially in 
blocking with non-immune serum into kit for 30 minu- experimental group compared to control group.
te to reduce nonspecific antibody binding, sections were 
incubated with primary antibodies, a rabbit polyclonal an-
tibody to EGFR (sc 03, Santa Cruz, CA, USA) diluted to 
1:100 for overnight at 4°C. The sections were stained using 
ImPRESS IgG-peroxidase kits (Vector Labs) (cat. No. MP-
7401), according to the supplier’s instructions. Finally, 
3.3’-diaminobenzidine (DAB) was used for colour deve-
lopment. After counterstaining with haematoxylin, speci-
mens were dehydrated and mounted. Slides processed wit- Fig.1.  Overview of the ovary; A; Control A group, B; 1.5 TAM group.  Healthy follicle (thin arrow), Atretic follicle ( arrow head), cystic structure (thick arrow), 
hout primary antibodies were included for each staining as corpus luteum (CL).  Trichrome staining, Bar: 200µm.
negative control. Immunohistochemical Findings
Quantitative evaluated according to the staining intensity EGFR immunoreactivity was detected in the ovaries of 
as follows: no staining (negative, −), slight brown (weak, control and experimental groups. Generally, EGFR showed 
+), brown-yellow (moderate, ++), and dark brown (strong, an intracytoplasmic staining in the cell with different stai-
+++). The accumulated score of the positive staining rep- ning intensities. EGFR showed a negative immunoreaction 
resented the relative expression of the protein (Fromowitz 
et al. 1987, Ergin et al. 2008). 
Results
Histological Findings
During experimental study, the dead was not observed in 
the animals of control and experimental group. When the 
ovarian tissues of control and experimental groups were Fig. 2. A; EGFR expression in control A group ovary B; EGFR expression in 1.5 
TAM group ovary. Strong EGFR immunoreactivity in interstitial cells (star), 
examined, there were not found pathological findings. In Negative EGFR expression in granulosa cells (arrow), IHC staining. Bar: 200µm.
the TAM treatment groups, it was observed that the cortex in the germinative epithelium, which is formed of single la-
and medulla distinction on the surface of the ovary was yer columnar cells in the ovaries of the control and experi-
significant, the interstitial area was wide, and especially mental groups (Fig. 2A, B). In addition, strong immunopo-
antral follicles were less in experimental groups than in the sitive reaction was detected in oocyte cytoplasm of follicles 
control groups. Furthermore, it was determined that the in all groups (Fig. 3). EGFR expression was not determined 
corpus luteum was very low on the surface of the ovarium in the granulosa cells of healthy and atretic follicles in the 
in the TAM treatment groups (Fig. 1A, B). In some of the ovaries of all groups. A strong EGFR expression was deter-
healthy follicles, a small number of granulosa cells showed mined in the interstitial cells as well as oocyte cytoplasm in 
an apoptotic structure with spilling into the antrum, but the all groups (Fig. 2B). In theca cells of the follicles in the 
most of the granulosa cells in the follicle were well organi- all groups, a weakly intensity EGFR expression was obser-
zed and the cell membrane integrity was not deteriorated. ved (Fig. 3). Depending on the developmental stages of the 
Based on this morphological evaluation, in the experimen- corpus luteum was observed moderate intensity of EGFR 
tal groups according to the control group, the presence of expression in the ovary of the control group. Also corpus 
small cystic structures in the medulla region and presence luteum was observed to moderate intensity of EGFR 
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they have an intense EGFR immunoreactivity in the pri-
mordial follicles before and after cryopreservation in the 
human ovary. These findings are in conflict with our fin-
dings which we determined there is no expression of EGFR 
in primordial follicles. Additionally, Moreno-Cuevas and 
Khan-Dawood (1997) also mentioned that this difference 
in EGFR expression in the primordial follicles maybe due 
to sacrificing of animals during different estrus periods. 
In the present study, the strong immunoreactivity was de-
termined in the oocyte cytoplasm of follicles (from pri-
mary follicles to antral follicles) in the all groups and no 
different between groups. On the contrary, EGFR expressi-
on disappears in the granulosa cells of healthy and atretic 
Fig. 3. Strong EGFR expression in oocyte cytoplasm (arrow) and  weak EGFR follicles in the all groups. But on the other hand, unlike our 
immunoreaction in theca cells (arrow head). IHC staining Bar: 100µm. study, Garnett et al. (2002) determined that weak immu-
noreaction through from primordial follicle to large pre-
expression in the ovary of the 0.5-dose TAM-treated test antral follicle in the granulosa cells, but in the antral follic-
group, although not as frequently observed as in the cont- les have strong EGFR immunoreactivity. This differences 
rol group. In general, EGFR expression was not showed about expression or localization of EGFR reaction may 
significantly difference in oocyte cytoplasm, granulosa cel- depending on different animal species or  different kind 
ls, interstitial cells, theca cells and corpus luteum of control of primary antibodies. In the present study, a strong EGFR 
and experimental groups (Table) immunoreactivity was detected in the interstitial area both 
in the control group and experimental groups. Garnett et 
al. (2002) also reported that EGFR expression has a mode-
rate to strong immunoreactions in interstitial cells of the 
hamster ovary. In our study in theca cells of all groups, a 
weakly intensity EGFR expression was observed. Accor-
Table. Semiquantitative observations of the EGFR immunoreactivity in the ding to Onagbesan et al. (1994) study, EGFR and EGF were 
mouse ovarium. also detected in theca cells on the chicken ovary by immu-
nohistochemistry and their levels decreased with follicle 
Discussion and Conclusion maturation (Onagbesan et al. 1994). But in our study we 
In previous studies, it was determined EGFR’s expressions have not observed such a decrease in expression level due 
by using immunohistochemistry method in human, rat, to follicular maturation. 
and hamster ovaries (St. Arnaud et al. 1983, Roy and Gre- As that of Ayyagari and Khan-Dawood’s (1987) study, we 
enwald, 1990, Stewart, 1992). determined expression of EGFR in the corpus luteum, but 
Similar to our findings, previously papers (Ayyagari and there was no significant difference in EGFR expression 
Khan-Dawood, 1987, Feng et al. 1987, Garnett et al. 2002, in the corpus luteum between the control group and the 
Maruo et al. 1993, Qu et al. 2000) expression of EGFR was low-dose TAM group. EGFR signaling causes increased 
detected in oocyte cytoplasm, interstitial cells, theca cells, proliferation, decreased apoptosis, and enhanced tumor 
and corpus luteum. Maruo et al. (1993) showed that in hu- cell motility and neo-angiogenesis (Arteaga, 2002). Furt-
man ovary, there was neither EGF staining nor EGF recep- hermore, EGF and its receptor in ovaries during follicu-
tor staining in primordial follicles. These results are con- lar growth and regression was investigated. It was repor-
sistent with our results. Just like them, in our study there ted that EGF and EGFR was also regulate the function of 
wasn’t EGFR expression in the follicle epithelial cells and follicular development (Das et al. 1991, Reeka et al. 1998). 
oocyte cytoplasm of primordial follicle in the all groups. In previous studies it was reported that TAM administra-
But on the other hand, Qu et al. (2000) have reported that tion suppresses ovarian follicle development but does not 
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affect follicle reserve, so that women who have received Crossman GA. Modification of Mallory’s Connective Tis-
chemotherapy during premenopause may use TAM for sue stain with a discussion of the principles ınvolved. Anat 
continuation of fertility and ovarian function (Ting and Rec, 69(1): 33-38, 1937.
Petrof, 2010). It has also been reported that tamoxifen is 
antioxidant and anti-apoptotic due to estrogen-like action Das K, Stout LE, Hensleigh HC, Tagatz GE, Phipps WR, 
(Duncan et al. 1994, Nathan and Chaudhuri, 1998).  In our Leung BS. Direct positive effect of epidermal growth factor 
study, there was no difference in EGFR expression among on the cytoplasmic maturation of mouse and human oocy-
the groups after TAM administration. It was observed that tes. Fertil Steril, 55: 1000–1004, 1991.
the administered doses of TAM have not significant influ-
ence on EGFR expression. Dowsett M1, Cuzick J, Wale C, Forbes J, Mallon EA, Sal-
Acknowledgments: This work was supported by a grant ter J, Quinn E, Dunbier A, Baum M, Buzdar A, Howell 
from TÜBİTAK-TOVAG (The scientific and technological A, Bugarini R, Baehner FL. Shak S. Prediction of risk of 
research council of Turkey; project no: 112 O 574). distant recurrence using the 21-gene recurrence score in 
node-negative and node-positive postmenopausal patients 
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