Biomedicine & Pharmacotherapy 100 (2018) 132–141

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Biomedicine & Pharmacotherapy

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

Differential expression of the receptors for thyroid hormone, thyroid T

stimulating hormone, vitamin D and retinoic acid and extracellular signal-
regulated kinase in uterus of rats under influence of sex-steroids
Abu Sadat Md Sayema,b, Nelli Giribabub, Kamarulzaman Karimb, Si Lay Khiangc,
⁎
Sekaran Muniandyd, Naguib Sallehb,
a
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Physiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Department of Obstetric & Gynaecology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia
d
Department of Biochemistry, MAHSA University College, Bandar Saujana Putra, 42610 Jenjarum, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Sex-steroids play important role in modulating uterine functions. We hypothesized that these hormones affect
TRα-1 expression of proteins in the uterus related to thyroid hormone action. Therefore, changes in expression levels of
TRβ-1 receptors for thyroid hormone (TRα-1 and TRβ-1), thyroid stimulating hormone (TSHR), vitamin D (VDR) and
TSHR retinoid acid (RAR) as well as extracellular signal-regulated kinase (ERK1/2) in uterus were investigated under
VDR
sex-steroid influence.
RAR
ERK1/2
Methods: Two rat models were used: (i) ovariectomised, sex-steroid replaced and (ii) intact, at different phases of
Sex-steroids oestrous cycle. A day after completion of sex-steroid treatment or following identification of oestrous cycle
Uterus phases, rats were sacrificed and expression and distribution of these proteins in uterus were identified by
Western blotting and immunohistochemistry, respectively.
Results: Expression of TRα-1, TRβ-1, TSHR, VDR, RAR and ERK1/2 in uterus was higher following estradiol (E2)
treatment and at estrus phase of oestrous cycle when E2 levels were high. A relatively lower expression was
observed following progesterone (P) treatment and at diestrus phases of oestrous cycle when P levels were high.
Under E2 influence, TRα, TRβ, TSHR, VDR, RAR and ERK1/2 were distributed in luminal and glandular epithelia
while under P influence, TSHR, VDR abn RAR were distributed in the stroma.
Conclusions: Differential expression and distribution of TRα-1, TRβ-1, TSHR, VDR, RAR and ERK1/2 in different
uterine compartments could explain differential action of thyroid hormone, TSH, vitamin D, and retinoic acid in
uterus under different sex-steroid conditions.

1. Introduction and ERK1/2 signaling protein [11]. These proteins participate in

Sex-steroids play important role in regulating the uterine functions.
Under the influence of E2, uterus undergoes a remarkable growth and
proliferation, while under the influence of P, extensive secretion and
stromal decidualization occurs in preparation for pregnancy [1–3]. E2
and P exert their action via binding to sex-steroid receptor, following
which the intracellular signallings are activated, leading to transcrip-
tion of genes that encode proteins involved in many uterine functions
[4,5]. Among the proteins which expression have been reported to be
influence by sex-steroids are the receptors for thyroid hormone, TRα
and TRβ [6,7], receptor for thyroid stimulating hormone, TSHR [8],
receptor for vitamin D, VDR [9], receptor for retinoic acid, RAR [10]
thyroid hormone actions.
Besides sex-steroids, thyroid hormone (TH) is also important for
many uterine functions [12,13]. This was evidence from deficiency of
thyroid hormone which can cause menstrual irregularities and failure of
the embryo to implant, that would eventually impair fertility [14].
There are two forms of tyroid hormones i.e. thyroxine (T4) and tri-
iodothyronine (T3) which mediate their action via binding to specific
thyroid hormone receptor (TR) [6,15] that isare located intracellularly
[16] or in the membrane [17]. Currently, two isoforms of TR i.e. TRα
and TRβ have been identified [18,19]. Expression of TR has been de-
tected in the endometrium in humans and rodents [6,7].
Additionally, thyroid stimulating hormone (TSH) also plays

⁎
Corresponding author.
E-mail addresses: naguib.salleh@gmail.com, naguib.salleh@yahoo.com.my, naguibsalleh@um.edu.my (N. Salleh).

https://doi.org/10.1016/j.biopha.2018.02.008
Received 1 June 2017; Received in revised form 1 February 2018; Accepted 2 February 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
A.S.M. Sayem et al.
important role in the uterus [20]. Among its uterine actions include
increases the expression of leukemia inhibitory factor (LIF) and its re-
ceptor (LIFR) in the endometrial stroma of humans [6] and primates
[21]. TSH is also involved in endometrial glucose transport, in view
that expression of Glut-1 mRNA was up-regulated by TSH in human
endometrial stromal cells and Ishikawa (uterine adenocarcinoma) cells
[6]. TSH might have direct actions on the uterine functions, since re-
ceptors for thyrotropin-releasing hormone (TRH) and TSH were dec-
tected in the monkey uterus following long term treatment with sex-
steroids [22]. TSH acts via binding to TSHR which has been found to be
expressed in the uterus in humans and rabbits [23,24]. Expression of
TSHR and TRs in the uterus was reported to be influence by sex-steroids
in which chronic administration of conjugated equine estrogen and
medroxyprogesterone acetate to ovariectomized cynomolgus macaques
caused up-regulation of TSHR and TR expression in uterine compart-
ments [8]. However, direct effects of individual sex-steroids i.e E2 and P
on TSHR and TR expression in the uterus have never been identified.
In the meantime, Vitamin D has been reported to play important
role in uterine functions including regulating uterine smooth muscle
contraction [25] and uterine cell proliferation [26]. Expression of VDR
and enzyme that convert vitamin D to its active form (1α-hydroxylase)
has been reported in the uterine tissue [27]. In uterus, VDR is found to
be expressed in the follicular and luteal phases of the menstrual cycle
[9]. VDR can be localized in both endometrium and myometrium in
humans [28]. VDR is a transcription factor located in the nuclei, and
this protein mediates the genomic effect of vitamin D (1,25(OH)2D3)
[29]. Lack of VDR expression in thyroid disorders has been linked to
infertility and pregnancy loss [30]. It was also reported that mice lack
VDR had impaired fertility [26].
Retinoic acid (RA) is a low molecular weight acid that is a lipophilic
metabolite in terms of Vitamin A. RA is crucially involved in main-
tenance of the female reproductive system functions, including cell
proliferation and differentiation [31]. RA has important role in reg-
ulating expression of matrix metalloproteinases (MMP) produced by
endometrial stromal cells during decidualization [32,33]. In addition,
RA has protective role on the uterus, since growth of cancer cells in the
endometrium was reduced following treatment with RA [34]. RA binds
to RAR, which is a nuclear receptor [35]. Expression of RAR has been
reported in the uterine stroma of mice [36] and uterine epithelium of
rats [10] and humans [37]. Expression of RAR in uterus was found to be
influence by ovarian steroids, as documented in rats [10] and humans
[37]. The functional heterodimer complex of TR and retinoid acid re-
ceptor (RAR) interact with specific thyroid hormone responsive element
(TRE) on DNA, initiating protein synthesis [38].
ERK1/2, a member of well-known mitogen-activated protein kinase
(MAPK), is reported to also be involve in many uterine functions, in-
cluding proliferation and decidualization [39,40]. ERK1/2 plays critical
role in embryo implantation, in mice and humans [41]. ERK1/2 is also
involve in mediating the non-genomic effects of thyroid hormone
[42–44]. Both E2 and P were reported to regulate ERK1/2 expression in
the smooth muscle of uterine artery [11].
In view of the important role of E2 and P for the uterus, it was
hypothesized that both hormones could affect uterine expression of TR
isoforms, TSHR, VDR, RAR and ERK1/2. As there was currently in-
adequate information pertaining to the effect of individual sex-steroids
on expression of these proteins in the uterus, our study aims to identify
changes in expression and distribution of these proteins under different
sex-steroid influence.
2. Materials and methods
2.1. Animals and hormones treatment
All experimental procedures were approved by the Animal Care and
Use Committee (IACUC), University of Malaya, Kuala Lumpur. Twelve
weeks old adult female Sprague–Dawley (SD) rats were housed in a
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Biomedicine & Pharmacotherapy 100 (2018) 132–141
clean and well-ventilated environment (12 h light and 12 h dark cycle,
temperature 24 ± 2 °C). All rats weighing 210 ± 20 g were kept in
cages with 5–6 animals in eachcage. Rats were given food pellet
(Harlan, Rossdoff, Germany) and tap water ad libitum. Ovariectomy was
performed under ketamine: xylazine (80 mg/kg: 8 mg/kg; in-
traperitoneal) anaesthesia in order to eliminate the variation in en-
dogenous sex-steroid levels [45,46]. Twenty-one (21) days after ovar-
iectomy, estradiol benzoate and progesterone (P), dissolved in peanut
oil and peanut oil (vehicle) only (Sigma Aldrich Co., St. Louis, USA),
were injected behind the neck scruff of these rats daily for three (3)
consecutive days. Rats were divided into two (3) groups with six ani-
mals per group and the treatments are as follows:
Group 1: peanut oil only (control: C)
Group 2: E2 s.c. at 1 μg/kg/day
Group 3: P s.c. at 4 mg/kg/day
Doses of E2 and P were selected based on previously reported dose
[47].
In another cohort of rats, estrus and diestrus phases were identified
by vaginal smear. Smears were performed in three consecutive cycles in
order to determine the cycle regularity. In this study, all rats used were
found to have regular cycle based on the appearance of the cells in the
smear during each stages (as described below).
For vaginal smear, secretions were collected immediately following
vaginal flushing with 10 μl normal saline (NaCl 0.9%). Secretions were
smeared on glass slides which were then observed under a light mi-
croscope (Olympus, Japan). Identification of estrous cycle phases was
based on the criteria as described by Marcondes et. al., [48]. Briefly,
three types of cells can be found in the smear. During proestrus and
estrus stages, smears contained predominantly nucleated epithelial and
anucleated cornified cells, respectively. Approximately the same pro-
portion of epithelial, leukocytes and cornified cells were observed in
smear obtained during metestrus stages, while smear from rats ar
diestrus contained predominantly leukocytes. Following estrous cycle
phase identification, intact rats were divided into the following groups:
Group 5: Estrus phase (Es)
Group 6: Diestrus phase (Ds)
Though the level of E2 increases at proestrus and decreases at the
end of estrus, the latter phase was selected for the study for endogenous
E2 effect on protein expression as optimum level for E2 was reported in
estrus phase. Additionally, length of the phases was relatively short
with the mean duration of each phase was estimated to be only a day.
Proestrus phase was not selected in view that the level of E2 is still
inclining and have yet to reach the peak. In the meantime, P secretion
becomes high during metestrus and diestrus and decreases afterwards
[49].
Following completion of sex-steroid treatment and identification of
estrous cycle phases, rats were euthanized by anesthetic overdosage
using ketamine: xylazine (100 mg/kg: 10 mg/kg) anaesthesia that was
administered intraperitoneally. This was then followed by cervical
dislocation. The mid-portion of uteri (whole) were removed for protein
expression analyses by Western blotting and protein distribution ana-
lysis by immunohistochemistry (IHC). Blood was withdrawn via direct
heart puncture immediately following sacrifice for determination of
plasma sex-steroid levels.
2.2. Measurement of plasma sex-steroid levels by enzyme-linked
immunosorbent assay (ELISA)
Blood, once collected into the plain tubes was left for 30 min and
allowed to clot at room temperature. In order to prepare the plasma,
blood was centrifuged at 2500 × g for 15 min. Then, plasma (clear
fluid) was aliquot and stored at −20 °C for measurement of sex-steroid
levels by using enzyme-linked immunoassay (ELISA) kit. ELISA proce-
dures follow the guidelines of the manufacturer(Cayman Chemical-
USA, Estradiol ELISA kit-582251 and Progesterone ELISA kit-582601).
The kits can measure E2 and P levels between the range from 6.6 to
A.S.M. Sayem et al.
Fig. 1. Plasma level of sex-steroids. (A) showing level of E2 following subcutaneous injection of 17-β E2 (1 μg/kg/day) and at different phases of oestrous cycle. (B) showing level of
progesterone (P) following subcutaneous P injection (4 mg/kg/day) and at different phases of oestrous cycle. Groups; C = Control, E2= 17-β estradiol, P = progesterone, Es = Estrus,
Ds = Diestrus.
4000 pg/ml and 7.8 to 1000 pg/ml, respectively. The levels of E2 and P
were expressed in pg/ml.
2.3. Immunohistochemistry (IHC) for visualization of protein distribution in
the endometrium
Immunohistochemistry was performed following the methods as
described [50]. Immediately after harvesting, the mid-portion of the
uterus was fixed overnight in 10% formalin prior to processing. Fol-
lowing then, uteri were dehydrated through increasing concentrations
of ethanol, cleared in chloroform and blocked in paraffin wax. Tissues
were then sectioned into 5-μm thickness, which were deparaffinized in
xylene and rehydrated in lowering concentrations of ethanol. Following
this, antigen was retrieved via incubating the sections in 0.01M sodium
citrate buffer (pH 6.0). 3% H2O2 in phosphate-buffered saline (PBS) was
used to neutralize the endogenous peroxidase. Sections were incubated
with 5% normal goat serum (sc-2043, Santa Cruz Biotechnology, Santa
Cruz, Candaa) that can block the non-specific bindings. This was done
prior to incubation with TRα1 (sc-10819), TRβ1 (sc-398007), TSHR (sc-
13936), VDR (sc-13133), RAR (sc-773) and ERK 1/2 (sc-135900) pri-
mary antibodies (Santa Cruz Biotechnology), at a dilution of 1:100 in
5% normal goat serum.
Sections were kept overnight at 4 °C and incubated with biotiny-
lated secondary antibody which was done after three times washing
with PBS, for 5 min each. Proteins were localized by ImmunoCruz™ ABC
Staining Kit (sc-2018, Santa Cruz Biotechnology). The sites of primary
antibody binding appeared as dark-brown stain. In order to visualize
the nuclei, sections were counterstained with hematoxylin, followed by
5 min rinsing in distilled water. Sections were washed with different
dilution of ethanol and xylene and finally were covered with a drop of
mounting medium.
2.4. Quantification of protein expression level by western blotting
Western blotting was performed following the methods as described
[51]. Firstly, whole mid-portion of the uterus, which was stored in
−80 °C immediately following harvesting, was crashed with liquid ni-
trogen and were then homogenized in PRO-PREP extraction solution
(Intron, Seoul, South Korea). Concentration ofs protein were de-
termined. 30 μg proteins, mixed with loading dye were injected onto
12% SDS-PAGE gel, transferred onto polyvinylidene difluoride (PVDF)
membranes, then incubated with 5% BSA for 60 min. Membranes were
incubated with TRα1 (sc-10819), TRβ1 (sc-398007), TSHR (sc-13936),
VDR (sc-13133), RAR (sc-773) and ERK 1/2 (sc-135900) primary an-
tibodies (Santa Cruz Biotechnology) at 4 °C overnight.
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Biomedicine & Pharmacotherapy 100 (2018) 132–141
The antibody dilution was 1:1000. Membranes were then washed
three times in PBS for 5 min each, followed by incubation with horse-
radish peroxidase conjugated secondary antibody (Santa Cruz, USA), at
a dilution of 1:5000, for 1 h. Membranes were then washed and sub-
jected to Opti-4CN™ Substrate Kit (Bio-Rad) to visualize the protein
bands. Photos of the bands were captured, and density of each band was
determined by using Image J software. The ratio of each protein
/GAPDH band densities was calculated and was considered as the ex-
pression level of the targets.
3. Statistical analysis
Statistical analysis was performed using Student t-test and one-way
ANOVA. P levels ≤ 0.05 was considered as significant. Tukeys post-hoc
statistical power analysis was performed and all values were > 0.05
which indicate that sample size is adequate.
4. Results
4.1. Plasma sex-steroid levels
Three days treatment with E2 to the ovariectomized rats resulted in
plasma E2 level to increase by approximately 2-fold (Fig. 1A). Plasma E2
level in P-treated ovariectomized rats was no significantly different as
compared to ovariectomized control rats, with the levels in both groups
were lower than E2-treated ovariectomized group. Plasma E2 level in
ovariectomized-only and ovariectomized, E2 and P-treated rats was
significantly higher than intact rats either at Es or Ds phase of the
oestrous cycle. Plasma E2 levels in intact rats at Es phase was higher
than Ds phase (p < .05).
Following three days treatment with P, plasma level of P in ovar-
iectomized rats increased by approximately 3-fold (Fig. 1B). No sig-
nificant different in plasma P level was observed between ovar-
iectomized control and ovariectomized rats treated with E2. Meanwhile,
plasma P levels in intact rats were higher than ovariectomized rats. In
intact rats, levels of P at Ds phase were higher than Es phase, ovar-
iectomized only and E2-treated ovariectomized rats. P levels in intact
rats were not significantly different when compared to ovariectomized,
P-treated rats. In intact rats, P levels at Es phase were higher than
ovariectomized rats treated with E2.
4.2. Levels of TRα-1 protein expression and its distribution in uterus
Levels of expression of TRα-1 protein in uterus in rats receiving E2-
treatment were higher than P-treatment (Fig. 2A). Higher TRα-1
A.S.M. Sayem et al.
Fig. 2. (A) Representative western blot band and analysis of expression level of TRα-1 protein in the uterus and (B) immunoperoxidase images showing distribution of TRα-1 in the
uterus. TRα-1 could be seen to be distributed in the endometrial luminal and glandular epithelia and myometrium. Larger images and smaller images were taken at magnification of 10×
and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to Ds. Data were expressed as mean ± standard error of
mean with n = 6 per group. Es: estrus, Ds = diestrus, E2= estradiol, P = progesterone. M = myometrium, E = endometrium, G = Gland, L = lumen. Molecular weight of TRα-
1 = 47 kDa and GAPDH = 36 kDa.
expression was observed in uterus at Es phase as compared to Ds phase.
Expression levels of TRα-1 protein in E2-treated rats were not sig-
nificantly different as compared to Es phase, but the levels were higher
than Ds phase (p < .05). No significant different in TRα-1 levels was
observed between P-treated rats and rats in Ds phase. In P-treated rats,
expression level of TRα-1 was lower than E2-treated rats and rats at Es
phase. However, in these rats, TRα-1levels were not significantly dif-
ferent when compared to rats at Ds phase.
A relatively higher TRα-1 distribution was obserdved in the luminal
and glandular epithelia, stroma and myometrium in E2-treated rats as
compared to P-treated rats (Fig. 2B). Similarly, TRα-1 distribution in
luminal and glandular epithelia, stroma and myometrium at Es phase
was relatively higher than Ds phase. TRα-1 could be seen to be dis-
tributed both intracellularly and at the plasma membrane of the en-
dometrial epithelium.
4.3. Levels of TRβ-1 protein expression and its distribution in uterus
The level of TRβ-1 in the uterus of E2-treated rats was significantly
higher when compared to P-treated rats and rats at Es phase. In intact
rats, TRβ-1 level was higher at Es phase than Ds phase (p < .05)
(Fig. 3A). In P-treated rats, levels of TRβ-1 expression were lower than
rats at Es phase however was not significantly different when compared
to rats at Ds phase (Fig. 3B).
A relatively higher TRβ-1 distribution was observed in luminal and
glandular epithelia and in the stroma of E2-treated rats than P-treated
rats. TRβ-1 distribution was also higher in luminal and glandular epi-
thelia and in the stroma of rats at Es phase as compared to Ds phase
(Fig. 3B). TRβ-1 could be seen to be distributed both intracellularly and
at the plasma membrane of the endometrial epithelium.
4.4. Levels of TSHR protein expression and its distribution in uterus
Expression level of TSHR protein in uterus of ovariectomized rats
treated with E2 was higher than ovariectomized rats treated with P
(Fig. 4A). In the meantime, expression level of TSHR in rats at Es phase
was higher than Ds phase (p < .05). Although the level of THSR in E2-
treated rats were not significantly different as compared to its level at
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Biomedicine & Pharmacotherapy 100 (2018) 132–141
Es phase, the former had significantly higher TSHR expression level as
compared to P-treated rats and rats at Ds phase (p < .05). However, in
P-treated rats, levels of TSHR expression were not significantly different
as compared to rats at Ds phase (Fig. 4A).
A relatively higher TSHR distribution was observed in the luminal
and glandular epithelia in E2-treated rats than P-treated rats (Fig. 4B).
However, higher TSHR distribution was observed in the stroma of P-
treated rats than E2-treated rats. TSHR distribution in the luminal and
glandular epithelia in rats at Es phase was relatively higher than rats at
Ds phase, however stromal distribution was relatively higher in the
latter as compared to the former. TSHR could be seen to be distributed
mainly at the plasma membrane.
4.5. Levels of VDR protein expression and its distribution in uterus
Expression level of VDR protein in the uterus of E2-treated rats was
higher than P-treated rats (Fig. 5A). Similarly, expression levels of VDR
protein in the uterus at Es phase were higher than Ds phase (p < .05).
However, levels of VDR protein in E2-treated rats were not significantly
different as compared to rats at Es phase but were higher than rats at Ds
phase (Fig. 5A). In P-treated rats, VDR expression level was not sig-
nificantly different as compared to rats at Ds phase.
A relatively higher VDR distribution was observed in the luminal
and glandular epithelia of E2-treated rats as compared to P-treated rats
(Fig. 5B). Similarly, VDR distribution in the luminal and glandular
epithelia at Es phase was relatively higher than Ds phase. However,
VDR distribution in the stroma was relatively higher in P-treated rats
when compared to E2-treated rats. Similarly, stromal distribution of
VDR was higher in rats at Ds phase as compared to rats at Es phase. VDR
could be seen intracellularly and at the plasma membrane.
4.6. Levels of RAR protein expression and its distribution in uterus
Expression levels of RAR protein in uterus were significantly higher
in E2-treated rats as compared to P-treated rats (p < .05) (Fig. 6A).
Rats at Es phase have higher uterine RAR expression level as compared
to Ds phase. Expression levels of RAR in E2-treated rats were sig-
nificantly higher than rats at Es and Ds phases (p < .05). However, in
A.S.M. Sayem et al. Biomedicine & Pharmacotherapy 100 (2018) 132–141

Fig. 3. (A) Representative western blot band and analysis of expression level TRβ-1 protein in the uterus. (B) Representative immunoperoxidase images showing distribution of TRβ-1.
TRβ-1 could be seen to be distributed in the stroma and luminal and glandular epithelia. No myometrial distribution could be seen. Larger images and smaller images were taken at
magnification of 10× and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to Ds. Data were expressed as
mean ± standard error of mean with n = 6 per group. Es: estrus, Ds = diestrus, E2= estradiol, P = progesterone, M = myometrium, E = endometrium, G = Gland, L = lumen.
Molecular weight of TRβ-1 = 55 kDa.

P-treated rats, RAR expression level was not significantly different as
compared rats at Ds phase.
A relatively higher RAR distribution was observed in the luminal
and glandular epithelia in E2-treated rats than P-treated rats (Fig. 6B).
Distribution of RAR protein in the luminal and glandular epithelia was
also higher at Es phase as compared to Ds phase. However, distribution
of this protein in the stroma was relatively higher in P-treated rats as
compared to E2-treated rats and was also higher in rats at Ds phase as
compared to Es phase. RAR could be seen to be distributed in-
tracellularly.
4.7. Levels of ERK1/2 protein expression and its distribution in uterus
Expression level of ERK1/2 protein in uterus of E2-treated rats was
higher than P-treated rats (p < .05) (Fig. 7A). A significantly higher
ERK1/2 protein expression level was observed in rats at Es phase as
compared to Ds phase (p < .05). In E2-treated rats, levels of ERK1/2
protein were significantly lower when compared to rats at Es phase but
higher when compared to rats at Ds phase. Lower ERK1/2 protein ex-
pression levels were observed in P-treated rats as compared to rats at Es
phase and in the latter was not significantly different when compared to
rats at Ds phase.

Fig. 4. (A) Representative western blot band and analysis of expression level of TSHR protein in the uterus. (B) Representative immunoperoxidase images showing distribution of TSHR in
the uterus. TSHR could be seen to be distributed in the endometrium, both in the stroma and luminal and glandular epithelia. No myometrial distribution could be seen. Larger images
and smaller images were taken at magnification of 10× and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to
Ds. Data were expressed as mean ± standard error of mean with n = 6 per group. Es: estrus, Ds = diestrus, E2= estradiol, P = progesterone, M = myometrium, E = endometrium,
G = Gland, L = lumen. Molecular weight of TSHR = 62 kDa.

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Fig. 5. (A) Representative western blot band and analysis of expression level of VDR protein in the uterus (B) Representative immunoperoxidase images showing distribution of VDR. VDR
could be seen to be distributed in the endometrium, both in the stroma and luminal and glandular epithelia, No myometrial distribution could be seen. Larger images and smaller images
were taken at magnification of 10× and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to Ds. Data were
expressed as mean ± standard error of mean with n = 6 per group. Es: estrus, Ds = diestrus, E2 = estradiol, P = progesterone. M = myometrium, E = endometrium, G = Gland,
L = lumen. Molecular weight of VDR = 60 kDa.

A relatively higher distribution of ERK1/2 was observed in the lu-
minal and glandular epithelia and stroma of E2-treated rats as compared
to P-treated rats (Fig. 7A). Similarly, distribution of ERK1/2 was rela-
tively higher in the luminal and glandular epithelia and in the stroma at
Es phase as compared to Ds phase. ERK1/2 protein could be seen to be
distributed intracellularly and at the plasma membrane.
5. Discussion
To the best of our knowledge, this study is the first to show the
individual effect of female sex hormones i.e E2 and P which were found
able to differentially affect expression and distribution of TRα-1, TRβ-1,
TSHR, VDR, RAR and ERK1/2 proteins in uterus. This study also
showed that in intact rats, differential distribution of these proteins
were observed at different phases of oestrous cycle. Under E2 influence,
expression level of TRα-1, TRβ-1, TSHR, VDR, RAR and ERK1/2 were
higher than under P influence which indicated that uterine effect of
thyroid hormone could be greater under E2 influence as compared to P
influence. It was also found that distribution of these proteins in uterine
compartments were markedly different under the different hormonal
condition. A relatively higher TRα-1, TRβ-1, TSHR, VDR, RAR and
ERK1/2 proteins were distributed in endometrial luminal and glandular

Fig. 6. (A) Representative western blot band and analysis of expression level RAR protein in the uterus. (B) Representative immunoperoxidase images showing distribution of RAR. RAR
could be seen to be distributed in the endometrium, both in stroma and luminal and glandular epithelia, myometrium. Larger images and smaller images were taken at magnification of
10× and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to Ds. Data were expressed as mean ± standard error
of mean with n = 6 per group. Es: estrus, Ds = diestrus, E2= estradiol, P = progesterone. M = myometrium, E = endometrium, G = Gland, L = lumen. Molecular weight of
RAR = 60 kDa.

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A.S.M. Sayem et al. Biomedicine & Pharmacotherapy 100 (2018) 132–141

Fig. 7. (A) Representative western blot band and analysis of expression level of ERK1/2 protein in the uterus. (B) Representative immunoperoxidase images showing distribution of
ERK1/2. ERK1/2 could be seen to be distributed in the endometrium, both in the stroma and luminal and glandular epithelia. No myometrial distribution could be seen. Larger images
and smaller images were taken at magnification of 10× and 40× respectively. Scale bar = 50 μm. In (B) *,#,Øp < .05 compared to E2, $, £p < .05 compared to P, €p < .05 compared to
Ds. Data were expressed as mean ± standard error of mean with n = 6 per group. Es: estrus, Ds = diestrus, E2 = estradiol, P = progesterone. M = myometrium, E = endometrium,
G = Gland, L = lumen. Molecular weight of ERK1/2 = 44/42 kDa, where 44 kDa has been used for analysis.

epithelia under E2 influence, however a relatively higher TSHR, VDR
and RAR were distributed in the stroma under P influence. Meanwhile,
only TRα-1 and TR-β were found to be distributed in the myometrium,
particularly under E2 influence.
Increased in expression of these proteins in the endometrial luminal
and glandular epithelia in particular under E2 influence could have
implications on growth of the endometrium under this hormonal con-
dition. Under E2 influence, growth and proliferation have been reported
in both endometrium and myometrium [52–55]. Besides, E2 was re-
ported to regulate stromal growth through the epithelium [56]. Via this
mechanism, thyroid hormone could exert its effect on the uterine
stroma. Under E2 influence, increased in growth and proliferation of the
uterus is associated with increased in the basal metabolic rate that can
be achieved via the action of thyroid hormone. Therefore, an increased
in the number of thyroid hormone receptors reflect increased uterine
metabolism. The important role of thyroid hormone on uterine growth
could be seen in a condition related to thyroid hormone deficiency i.e
hypothyroidism where an absolute decreased in the volume of epithe-
lial cells and nuclei were observed [57,58]. A study has shown that E2
stimulates mitogenic response in endometrial cells via upregulating
pyruvate kinase expression that coactivate estrogen receptor-α. This
integrates the metabolic reprogramming leading to a shift in the glucose
metabolism toward aerobic glycolysis [59]. These E2 effect could be
accomplished via the action of thyroid hormone.
Our findings which indicated differential expression of thyroid
hormone receptors; TRα1, TRβ1 and TSHR in different endometrial
compartments were supported by the reported differential expression of
these receptors in the glandular and luminal epithelial, and the stroma
throughout the menstrual cycle in humans [6]. Furthermore, similar
pattern of uterine distribution was observed following sex hormone
treatment in macaque [8]. A study has also shown that TSHR dis-
tribution in the endometrial luminal epithelia and TRα1 and TRβ1
distribution in the endometrial luminal and glandular epithelia in-
creased significantly on luteinizing hormone (LH) days 6–9 in humans,
co-incide with high E2 levels [60].
In this study, VDR expression was observed to be high in the epi-
thelium under the influence of E2. Our findings were consistent with a
report by Emam et al., [61] which shown that in the uteri of cows,
expression of VDR was restricted to the luminal and glandular epithelia
of endometrium under E2 influence which indicated that this receptor
plays important role in endometrial epithelial functions. Further, they
found that Vdr mRNA was expressed in the endometrium throughout
the estrous cycle with a relatively high expression at Es phase, under
the influence of E2, the findings that were similar to ours.
Immunohistochemical analysis revealed luminal and glandular
epithelia of endometrium strongly express VDR, particularly under E2
influence. Our findings were supported by others who showed similar
pattern of VDR expression in the endometrium [62]. VDR mediates the
action of Vitamin D which participates in many uterine events. For
example, transport of calcium across the uterine epithelium is vitamin
D dependent. The role of vitamin D in uterine calcium transport is
further supported by a study which showed that expression of vitamin D
calcium binding protein (CaBP or calbindin-D9k) in the uteri of 21-day-
old non-pregnant rats increased following administration of tamoxifen
or physiological doses of estrogens [63]. Activation of vitamin D by
cytochrome (CYP27A1 and CYP2R1) enzymes were also reported to be
involved in controlling endometrial epithelial growth [64]. Therefore,
up-regulated VDR expression as observed under E2 influence could play
a role in endometrial epithelial growth that occur under this hormonal
influence.
In this study, we have shown that RAR expression in the uterus was
enhanced under the influence of E2, particularly in the luminal and
glandular epithelia. Increased RAR expression suggests its involvement
in growth and proliferation of endometrial epithelia under the influence
of E2. In postmenopausal women receiving estrogen replacement
therapy, increased expression of RAR in the uterus was observed while
in premenopausal women, levels of RAR in the endometrium increased
during the proliferative phase, which is associated with high E2 level
[65]. Similar study by Fukunaka et al., [66] showed that RAR was
strongly expressed in the nuclei of endometrial epithelium in the pro-
liferative phase of the menstrual cycle.
We have shown that the level of ERK1/2 in uterus increased under
E2 influence and this protein was found mainly in the luminal and
glandular epithelia. ERK1/2 protein could mediate growth and pro-
liferative effects of E2. Our findings were consistent with a report by
Wang et al., [67] who showed that E2 regulates protein synthesis in the

138
A.S.M. Sayem et al.
uterine epithelial cells through activation of protein kinase C (PKC) that
in turn stimulates ERK1/2 to phosphorylate and activate the central
regulator of protein synthesis, mTOR. Besides mediating growth and
proliferative effects, ERK1/2 also mediates several E2 effects in the
endometrial epithelia such as MMP-2/9 expression [39] and expression
of early growth response 1 (Egr1), a zinc finger transcription factor that
regulates cell growth, differentiation and apoptosis in the uterus [68].
Meanwhile, under P influence, expression of TRα1, TRβ1, TSHR,
VDR, RAR and ERK1/2 in the epithelia was lower than under E2 in-
fluence, with exception of the stroma where expression of TSHR, VDR
and RAR were higher under P influence than under E2 influence.
Aghajanova et al., [60] showed that human endometrial stroma ex-
pressed mRNA for Tshr. Our findings indicated that under P influence,
thyroid hormone exerts lesser action on uterine epithelium. However,
high expression of TSHR, VDR and RAR in the stroma under P influence
indicated that thyroid stimulating hormone (TSH), vitamin D and re-
tinoid acid plays important role in stromal functions under this hor-
monal influence.
We have shown that P downregulates epithelial expression of TR
and TSHR but up-regulates expression of TSHR in the stroma which
were consistent with the findings by Hulchiy et al., [8] who reported
that in uterine stroma of macaque, TSHR expression was increased
following administration of conjugated quine estrogen and medrox-
yprogesterone acetate (CEE + MPA). The role of TSH in endometrial
physiology has been proposed. Aghajanova [60] reported that admin-
istration of TSH to cultured human endometrial stroma cells sig-
nificantly increase leukemia inhibitory factor (LIF) and LIF receptor
(Lifr) mNA levels. Furthermore, glucose transporter 1 (Glut-1) mRNA
levels were also enhanced by TSH in Ishikawa (uterine adenocarci-
noma) cells. The importance of TSH has been documented in women
where its level correlates with the clinical pregnancy outcomes [69].
In the meantime, expression on VDR in the stroma was higher under
P than under E2 influence. It was shown previously that in cow uteri,
VDR expression was high at luteal (P dominant) phase [61], supporting
our findings that this could be due to high expression in the stromal
compartment. It was recently reported that the concentrations of cal-
citriol, an active form of vitamin D in endometrial tissues was higher
during early pregnancy which could affect expression of several genes
related to implantation, vitamin D metabolism, calcium ion regulation,
PG metabolism, and calcium-binding proteins in the endometrium [70].
Thus, high VDR expression, in particular in the stroma could play a role
in mediating vitamin D effect in preparation for pregnancy. Vitamin D
also play a pivotal role in normal decidual immune function via pro-
moting the innate responses to infection [71] which could explain the
reason for the increased VDR expression in the stroma under P influ-
ence. Vitamin D treatment was found to decrease inflammation-induced
cytokines and contractile-associated factors in uterine myometrial
smooth muscles [25].
Besides the increased in stromal TSHR and VDR, expression of RAR
in stroma also increased under P influence. Our findings were consistent
with the report that RAR plays important role in uterine stromal de-
velopment from Mullerian duct in mouse embryo [36]. Ozaki et al.,
[72] also shows that RAR and the retinoic acid pathway are involve in
decuadualization in human endometrial stromal cells, an event which
occur under P influence. In the meantime, our findings that RAR ex-
pression in uterine epithelium was low under P influence was consistent
with a report by Fukunaka et al., [66] who observed that RAR ex-
pression was drastically reduced in epithelial nuclei during the secre-
tory phase in association with reduced in serum E2 level.
In this study, we observed that TRα1, TRβ1, VDR and RAR were
distributed intracellularly as well as at or near the plasma membrane.
Thyroid hormone can mediate both genomic and non-genomic effects.
Among the non-genomic effects are a set of actions initiated at the cell
surface receptor that are relevant to intracellular trafficking of proteins,
serine phosphorylation and acetylation, assembly within the nucleus
and transcription of specific genes [73]. In the meantime, genomic
139
Biomedicine & Pharmacotherapy 100 (2018) 132–141
effects of thyroid hormone involve thyroid hormone response elements
(TRE) on specific genes, complexes of nuclear thyroid hormone re-
ceptors (TRs) and 3,5,3′-triiodo-L-thyronine (T(3)), coactivator or cor-
epressor nucleoproteins, and histone acetylases or deacetylases [74].
Additionally, it was reported that TR especially TRβ1 isoform may be
found in the cytoplasm complexed with other proteins, such as mi-
togen-activated protein kinase (MAPK). Formation of such complexes
may facilitate nuclear import of TR [75]. Furthermore, nuclear reten-
tion of TR occur only following binding to thyroid hormone [75]. It is
likely that the receptors found in the cytoplasm or at the plasma
membrane are TRs that are not complex to thyroid hormone.
In the meantime, our findings that VDR is located at the plasma
membrane is also consistent with a report in the neuron where VDR was
localized to neuronal plasma membrane which involve in the non-
genomic effect of vitamin D [76]. Similarly, RAR has been reported to
be retained in the cytoplasm and moves towards the nucleus only when
retinoic acid is present [77] and this could support our observation for
the cytoplasmic distribution of RAR, rather than its distribution in the
nucleus of the uterine epithelial cells.
In this study, it was found that the level of E2 in ovariectomized rats
was higher than intact rats either at estrus or diestrus phases of the
cycle. The likely reason was that following ovariectomy, there could be
a compensatory production of E2 from the adrenal gland [78] and this
would contribute towards the high E2 levels. Progesterone treatment
did not have any effect on serum E2 levels. In this study, it was also
found that the levels of expression of TRβ1 and RAR in uterus at Es
phase was lower than following E2 treatment, while the opposite was
observed in the ERK1/2 level where its level was higher at Es phase as
compared to following E2 treatment. However, no difference in TRα1
and VDR levels were observed between Es phase and E2 treatment. The
likely explanation was that higher serum E2 levels could induce tran-
scription of TRβ1 and RAR while higher ERK1/2 level in intact as
compared to ovariectomized, E2-treated rats suggests that in addition
to E2, other factors/hormones from the ovary could induce ERK1/2
expression in the uterus.
In conclusions, our study has shown the differential effects of E2 and
P on TRα1 and TRβ1, TSHR, VDR, RAR and ERK1/2 expression and
distribution in the uterus which could facilitate the changing functions
of this organ under these different sex-steroid conditions. The changes
in expression of these proteins could also mediate uterine effects of
thyroid hormone that could be different under the influence of E2 and P.
Conflict of interest statement
Authors have nothing to declare
Acknowledgement
This research is funded by Fundamental Research Grant Scheme
(FRGS) grant; FP028-2015A, Ministry of Higher Education, Malaysia.
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