Indian Journal of Experimental Biology
Vol. 51, March 2013, pp. 218-227
Regulation of luteinizing hormone receptor in hippocampal neurons following
different long-lasting treatments of castrated adult rats
H Abtahi1, M Shabani
2, S B Jameie
3, A H Zarnani
4,5, S Talebi
6, N Lakpour
4, H Heidari-Vala
7, H Edalatkhah
7,
M.A Akhondi7, M Amiri
1, A R Mahmoudi
6 & M R Sadeghi
6,*
1Department of Basic sciences, Faculty of Allied Medicine, Gonabad University of Medical Sciences, Gonabad, Iran
2Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3Department of Basic Sciences, Faculty of Allied Medicine & Cellular and Molecular Research Center,
Tehran University of Medical Sciences, Tehran, Iran
4Nanobiotechnology Research Center, Avicenna Research Institute,
Academic Center for Education Culture and Research (ACECR), Tehran, Iran
5Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
6Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
7Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Received 4 July 2012; revised 27 November 2012
The aim of this study was to investigate the effects of different Luteinizing hormone (LH) and steroid hormones levels
on LH receptor (LHR) expression in the hippocampal cells. Rats (24 males and 24 females) were assigned to four groups:
one control and three experimental [gonadectomy (GDX), gonadectomy + gonadotropin releasing hormone analogue
(GDX+GnRHa) and GDX+GnRHa+estradiol (E2) or testosterone (T)] independently for each gender. All experimental rats
were gonadectomized; then GnRHa was administrated to GDX+GnRHa group, and GnRHa plus steroid hormone to
GDX+GnRHa+E2 or T group in both genders for four-month. LHR mRNA expression and its protein level in hippocampal
cells were measured using QRT-PCR and Western blotting. Quantification of mRNA revealed a decrease in LHR transcripts
level in GDX+GnRHa group of females. A significant change was observed between GDX groups and GDX+GnRHa+E2 or
T versus GDX+GnRHa group in females. High levels of LH decreased significantly the immature isoform of LHR in GDX
group compared to control group in both genders, but low LH concentrations in GDX+GnRHa group induced immature
LHR isoform production only in females. Therefore increased LH concentration induces production of incomplete LHR
transcripts in hippocampal cells and decreases immature LHR at the protein level. This implies that LH decreases the
efficiency of translation through either producing non-functional LHR molecules or preventing their translation.
Keywords: Hippocampus, Immature isoform, LHR, Luteinizing hormone, Regulation, Steroids
Luteinizing hormone receptor (LHR) is a member
of G protein-coupled receptor (GPCR) family. It is
a sialylated glycoprotein with molecular weight of
80-90 kDa in its mature isoform. The contribution of
carbohydrate in its molecular weight is about 15 kDa.
However, the immature isoform contains 674 amino
acids and a molecular weight of 75 kDa1.
LHR has been found on different tissues
of mammalian species, including gonadal and
non-gonadal tissues: testis, ovary, uterus, placenta,
mammary gland, brain, and many other tissues2-5
.
In brain, spinal cord6, hippocampus
7-12 and all
types of human neuronal cells are among tissues
expressing LHR13,14
. Accordingly, the highest density
of LHR belongs to the neurons of hippocampus in
mammalian brain while this density decreases in
the neurons of the hypothalamus, cerebellum, and
cerebral cortex15-17
.
LH is a heterodimeric glycoprotein hormone
secreted by pituitary gland in response to
gonadotropin releasing hormone (GnRH) into blood
stream. It can cross blood brain barrier (BBB) and
reach neurons in some brain areas such as
hippocampus. Even though the effects of LH on
reproductive organs through LHR are well-known,
its effects are still unknown in the case of the brain.
——————
*Correspondent author
Telephone: +98 2122432020
Fax: +98 2122432021
E-mail: [email protected]
ABTAHI et al.: LHR REGULATION IN HIPPOCAMPAL NEURONS
219
The roles of LH in both physiological processes
such as reproductive behaviours, memory, and motor
coordination18
and pathological conditions including
neurodegenerative age-dependent disorders in adult
brain are well-established19
. LH is involved in the
regulation of neuronal development during the normal
development of the brain through synthesis of
neurosteroids19
. Further, it has been shown that high
level of LH or lack of sex steroid hormones during
menopause/andropause could be responsible for
neurodegeneration in certain areas of the brain
including hippocampus, which leads to development
of neurodegenerative diseases such as Alzheimer's13,14
.
LHR like other receptors is regulated at levels
of transcription and post-transcription by different
mechanisms. High levels of LH or human chorionic
gonadotropin (hCG) down-regulate both LHR content
and its associated transcripts20-25
. However, in the case
of hCG, LHR down-regulation was not significant26
.
The LHR receptor and its corresponding mRNA have
been down-regulated in rat ovary during acute phase
of LH surge in estrous cycle. This decrement in
mRNA is not due to the decrease in LHR gene
transcription but it is due to the increase of mRNA
degradation20,21,23,27
. It is postulated that the LH
receptor binding protein 1 (LRBP1), which is
responsible for LHR mRNA stability may be involved
in this process28
.
In addition to LH, sex steroid hormones might be
involved in modulating neuronal expression of LHR.
It has been demonstrated that physiological levels of
estradiol increase the immature isoform of LHR while
treatment with increased levels of estradiol decrease
the expression of immature LHR in a dose-dependent
manner. In contrast to immature isoform, mature
isoform increases at high levels of estradiol. In other
words, high levels of estradiol might induce LHR
maturation14
.
The pathway(s) involved in regulation of LHR by
different levels of LH or other sex steroid hormones
in hippocampus of adult rat is/are not fully
understood. Therefore, the present study has
been designed to investigate the effects of LH and
sex steroid hormones on both transcript and protein
levels of LHR in the hippocampus of adult male
and female rats.
Materials and Methods
Animal treatment—In this study, 48 6-month old
Wistar rats (24 males and 24 females), weighing
386-478 g, (Razi Institute, Tehran, Iran) were
maintained at 12 h light/dark cycle at 21 °C. Standard
rat chow and tap drinking water ad libitum were
provided. The study procedures were approved by
Local Bioethics Committee of Avicenna Research
Institute and Tehran University of Medical Sciences
(TUMS). Correspondingly, both male and female rats
were randomly divided into following four groups of
6 each: control (intact animals, both genders), GDX
(the animals underwent gonadectomy, both genders),
GDX+GnRHa (gonadectomized animals received
subsequently long-acting GnRHa as gonadotropin
releasing hormone analogue [Diphereline S.R. 3.75 mg,
IPSEN, France] to suppress the release of LH, both
genders), and GDX+GnRHa+E2 or T (gonadectomized
rats were subsequently administered long-acting
GnRHa in conjunction with sex steroid hormone
replacement therapy (HRT), estradiol in females and
testosterone in males).
The animals were anesthetized using 100 mg/kg
Ketamine and 10 mg/kg Xylazin, next ovariectomy
was performed via abdominal incision and excision of
ovaries while the fallopian tubes were ligated.
Orchidectomy was also done via two10 mm lateral
incisions on the scrotum, and the testicles were excised
after ligation of the spermatic cord to prevent bleeding.
Hormonal treatment for related groups was
started after a 7-10-day period of recovery and
continued for a four-month period as follows:
Triptorelin acetate (Diphereline) as a GnRHa was
injected intramuscularly 0.5 mg/kg every 21 days. To
assure the suppression of increased LH concentration,
the applied dose was almost 10 times more than the
dose usually used for human. Estradiol valerate
(Estradiol-Depot 10 mg Jenapharm, Germany) as
slow-release estradiol was injected to female rats
in GDX+GnRHa+E2 or T group intramuscularly,
1.76±0.14 mg/kg, for each 14 days to supply the
physiological levels of estradiol29
. To obtain the
physiological level of testosterone, male rats in
GDX+GnRHa+E2 or T group received Testosterone
undecanoate (Nebido 1000 mg, Bayer Schering
Pharma, Germany) as long-acting testosterone,
120.07±1.68 mg/kg, for every 2 months 29
. Female
rats in control groups were sacrificed during their
metestrus phase of their estrous cycles. To achieve
this goal, the estrous cycle was monitored by daily
inspection on vaginal cytology before scarification.
Sample preparation—Rats were sacrificed by rapid
decapitating in the morning and their hippocampi
INDIAN J EXP BIOL, MARCH 2013
220
were dissected on ice according to Paxinos et al30
.
The tissues were immediately transferred into liquid
nitrogen for instantaneous freezing. For long storage,
they were stored at -70 °C. Blood samples were
collected from trunk, and sera were stored at -70 °C
for subsequent determination of LH and sex steroid
hormones concentrations. The hippocampi were
powdered into fine homogenate using liquid nitrogen
to obtain a homogenous sample representing various
parts of tissue. Subsequently, the powder was put into
two separate sterile micro-tubes one of which was
used to extract mRNA and the other to prepare cell
lysate for protein assay and Western blotting.
Hormone assay—Blood levels of testosterone
(DiaSorin Liaison, Italy), estradiol (DiaSorin Liaison,
Italy), and LH (Uscn Life Science Inc. Wuhan, China)
were determined to evaluate the efficiency of various
treatments following gonadectomy in different
groups. Some specifications of the mentioned
commercial kits are as follows: the LH assay kit
(Enzyme-linked immunosorbent assay Kit for Rat
Luteinizing Hormone) has a detection range of
15.6-1000 µIU/mL, the minimum detectable dose of
<3.9 µIU/mL, and no significant cross-reactivity or
interference. Both estradiol and testosterone assay kits
used competitive 2-step chemiluminescence assay
using directly coated magnetic microparticles with
the detection limit of ≤12 pg/mL and ≤ 0.05 ng/mL;
and the measuring ranges of 12-1100 pg/mL and
0.05-16 ng/mL for estradiol and testosterone assay,
respectively.
Primer design—According to Swissprot database,
at present, eleven different isoforms have been
recognized for rat LHR (http://www.uniprot.org/
uniprot/P16235, retrieved 2010/10/29). Accordingly,
two sets of primers which could be attached to LHR
cDNA with accession number of NM_012978.1 at
NCBI were designated (http://www.ncbi.nlm.nih.gov/
nuccore/6981159, retrieved 2010/10/29). The first set,
named LHR1 set primers, recognizes all of the LHR
isoforms and the second one, termed as LHR2,
recognizes cDNAs corresponding to isoforms 1 and
4 in Swissprot; P16235-1 and P16235-4, respectively.
GAPDH cDNA with accession number of
NM_017008.2 at NCBI (http://www.ncbi.nlm.nih.
gov/nuccore/31377487, retrieved 2010/10/30) was
also used as housekeeping gene for normalization.
The primer sequences and their characteristics have
been shown in Table 1.
Reverse transcription - Polymerase chain reaction
(RT-PCR)—Total RNA was extracted using RNAzol
(Cinna/Biotecx, Friendswood, TX, USA). RNA
concentration was determined via UV absorption at
260 nm using Picodrop (Picodrop, Cambridge, UK).
One microgram of the total RNA was reverse-
transcribed into cDNA using 200 U of Molony
Murine Leukemia Virus (RTM-MULV) reverse
transcriptase enzyme (Fermentase, Vilnius, Lithuania),
and 20 pmol of random hexamer primers (Cybergene,
Stockholm, Sweden) in MasterMix containing dNTPs.
Real-time PCR using SYBR-Green I—Real-time
quantitative PCR was carried out in 96-well
polypropylene microplates by ABI Prism 7000
(Applied Biosystems, Foster City, CA, USA) using
SYBR-Green I Real-time PCR Master Mix (Takara,
Japan) according to the manufacturer’s instructions.
The profile of amplification was one cycle at 95 °C
for 10 sec, and 40 cycles each at 95 °C for 5 sec and
60 °C for 34 sec. All PCR reactions were performed
in triplicate wells. Melting curve analysis was
performed to assure of amplification specificity and
PCR product purity. Double distilled water (DDW)
was used as the negative control. The PCR products
were visualized through ethidium bromide staining
following electrophoresis in a 1.5% agarose gel, and
visualized bands were purified using QIAquick Gel
Extraction Kit (QIAGEN, Germantown, MD, USA).
The purified PCR products were used for DNA
sequencing (ABI 3130 genetic analyzer, Applied
Biosystems, Foster City, CA, USA). LinRegPCR
Table 1—The primers sequences and their characteristics
Set Name Primer Sequence Template Length
(bp)
Template Location at cDNA
of Gene (bp)
Sense 5’-CAGTCCCGAGAGCTGTCAG-3’ LHR1
Antisense 5’-TTACGACCTCATTAAGTCCC-3’
169 116- 284
Sense 5’-AATTCACGAGCCTCCTGGTC-3’ LHR2
Antisense 5’-CTGGAGCACATTGGAGTGTC-3’
246 846- 1091
Sense 5’- AAGGTCATCCCAGAGCTGAA-3’ GAPDH
Antisense 5’-ATGTAGGCCATGAGGTCCAC-3’
338 1497- 1835
ABTAHI et al.: LHR REGULATION IN HIPPOCAMPAL NEURONS
221
software was applied for calculation of the relative
amount of RNA using the ∆∆Ct method following
sample's efficiency determination.
Western blot analysis—Tissue lysate was prepared by adding lysis buffer containing 20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet-P40 (NP40), 2 mM EDTA, and protease inhibitor (Mini Protease Inhibitor Cocktail Tablets, Roche Applied Science, Germany) while kept on the ice all the time. Then, it was homogenized immediately and centrifuged at 10000 g for 5 min. The supernatant was transferred into a separate tube for protein determination and Western blotting. Protein concentration in cell lysate was determined using BCA assay kit (Pierce, USA). Exactly 20 µg total proteins of tissue lysate were loaded into each lane. Testis and HeLa cell lysates were used as positive and negative controls for LHR, respectively. Lysate proteins were resolved on 10% SDS-PAGE under denaturing condition at 100 mV. Subsequently, protein transfer was performed on PVDF membrane at 100 mV for 60 min. The membranes were blocked with 5% skim milk in TBST overnight and probed with primary antibodies as follows:
At first, the membrane was treated with rabbit
anti-rat LHR antibody (2 µg/mL) (H-50, Santa Cruz
Biotechnology, USA) for 1.5 h at room temperature.
After washing, it was reprobed with rabbit anti-rat
GAPDH antibody (0.3 µg/mL) (Abcam, Cambridge,
UK) for 1 h at the same temperature.
Treated membranes with primary antibodies were incubated with secondary antibody (0.3 µg/mL) (Sheep HRP-conjugated anti-rabbit antibody, ARI, Iran) for 30 min at room temperature. Finally, the membrane was developed by advanced ECL kit (Amersham, UK) for 5 min. The changes in LHR level were analyzed by densitometry and then quantified using AlphaEase FC software (Alpha Innotech, USA). LHR level was normalized through calculation of relative level of LHR to GAPDH proteins for all the samples. This ratio was considered as the LHR concentration to compare the effects of different treatments on LHR levels in control and treatment groups.
Statistical analysis—Real-time PCR data were analyzed using REST-RG beta software version 3. Statistical analyses for other results were performed using SPSS software V.13.0. All comparisons between groups were carried out by Two-Sample Kolmogorov-Smirnov test, and the groups with normal distribution were compared through Independent-
Sample T test; otherwise, Mann-Whitney test was used. All values are presented as mean±SD. A P <0.05 was considered statistically significant.
Results
Following gonadectomy and HRT, levels of
testosterone in males, estradiol in females, and LH
levels in both genders were determined for different
groups. Due to the lack of inhibitory effects of
gonadal hormones on the cells of anterior pituitary,
LH levels increased following gonadectomy in GDX
groups of female and male rats (P = 0.002 and 0.003,
respectively). Administrating GnRHa continuously,
which suppresses LH secretion, decreased LH levels
in GDX+GnRHa groups and GDX+GnRHa+E2 or
T of both sexes. GnRHa administration to
GDX+GnRHa group of female rats decreased LH to
the levels which have no significant difference
compared with the female control group (P = 0.063).
Despite marked decreases of LH levels in male rats of
GDX+GnRHa group due to administrating GnRHa,
the difference in LH levels was significant in
comparison with corresponding control group
(P = 0.002). Due to the co-administration of GnRHa
and sex steroid hormones in GDX+GnRHa+E2 or T
group of both genders, LH levels decreased to the
values in accordance with control groups (P = 0.719
for females and 0.85 for males) (Fig. 1). Decreased
testosterone and estradiol levels in gonadectomized
groups compared with those of control groups were
related to the absence of functional gonads to produce
these steroid hormones. There were significant
decreases in sex steroid hormone concentrations in
GDX and GDX+GnRHa groups of both genders of
rats compared with the corresponding control groups
(P = 0.05). However, HRT compensated the lack
of steroid synthesis in gonadectomized rats in
GDX+GnRHa+E2 or T group of males and females. Quantification of LHR transcripts by real-time
PCR using the LHR1 and LHR2 primer sets (the
LHR1 primers recognize all LHR transcripts in
contrast to LHR2 primers which amplify the most
complete mRNA variant of LHR) indicated that the
only significant change was the decreased LHR
mRNA in GDX+GnRHa group (P = 0.012 and 0.003,
respectively) while the difference in expression
ratio of LHR in the other groups was not significant
for both sexes. However, there were significant
differences between LHR expression using both
LHR1 and LHR2 primers between GDX and
GDX+GnRHa groups of female rats (P = 0.007
INDIAN J EXP BIOL, MARCH 2013
222
and 0.002, respectively). But comparison of
LHR expression between GDX+GnRHa and
GDX+GnRHa+E2 or T groups showed a significant
increase only with the LHR2 primer in female rats
(P = 0.005) (Fig. 2). By using LHR1 and LHR2
primer sets, the LHR has been expressed more in the
hippocampus of female rats than that of male ones,
but this difference was not significant (P = 0.45 and
0.084, respectively).
Relative expression of LHR using LHR1/LHR2 in
different groups of both genders is shown in Fig. 3.
This ratio was different in various groups of this
study, however the differences were not significant
compared to the corresponding controls. For example,
despite the fact that this ratio increased to more than
one in GDX group of females and GDX+GnRHa
group of male rats, it decreased markedly in
GDX+GnRHa+E2 or T group of both sexes.
It is noteworthy that the expression ratios of LHR
using LHR1 and LHR2 primers in hippocampus were
about 20000 and 10000 times less than those in rat
testis, respectively, while the concentration of LHR in
hippocampus was 20 ± 2 folds less than that in testis
at the protein level.
The immature protein isoform of LHR in GDX rats
had a significant decrease (Fig. 4) in comparison to
the control groups of both genders (P = 0.001 and
0.001). The mature isoform of LHR has increased in
the gonadectomized groups in both sexes, however
these increases were not significant (P = 0.439 for
females and 0.138 for male rats). The immature
isoform of LHR has decreased considerably
(P = 0.016) in GDX+GnRHa group of male rats,
but in female rats of the same group there was no
Fig. 1—Hormonal assays in control and experimental groups of
both male and female rats.[Plot A shows the serum levels of LH
and estradiol in female rats while plot B shows the LH and
testosterone levels in serum of male rats. The * represents a
significant difference between an experimental group and its
corresponding control group].
Fig. 2—Expression ratio of LHR using LHR1 and LHR2 primers
in different groups of female and male rats.[The * shows a
significant difference between an experimental group and its
corresponding control group. The number of rat in each group was
six. This figure shows that increased LH in GDX rats has
decreased LHR mRNA, especially complete transcript and this
decrease has been reinforced by decreasing LH in GnRHa treated
rats. HRT increased the LHR mRNA].
Fig. 3—Comparison of relative expression of LHR isoforms
using LHR1/LHR2 in different groups of female and male
rats.[Speaking specifically, ratio of LHR1/LHR2 expression ratios
less than one indicates that non-functional LHR transcripts have
been suppressed. However, with the emergence of these kinds of
LHR mRNA isoforms, the above-mentioned ratio has increased.
The * represent a significant difference between an experimental
group and its corresponding control group].
ABTAHI et al.: LHR REGULATION IN HIPPOCAMPAL NEURONS
223
significant increase in LHR level in hippocampus
compared to the corresponding control groups
(P = 0.07). However, there were insignificant
decreases in mature LHR concentration in
GDX+GnRHa group of both female and male rats
(P = 0.14 and 0.478, respectively). Finally, in
GDX+GnRHa+E2 or T group, immature isoform of
LHR decreased significantly in males (P = 0.023)
and insignificantly in females (P = 0.771) compared
to corresponding control groups. Nevertheless, the
mature LHR has increased in female and male rats of
GDX+GnRHa+E2 or T group (P = 0.158 and 0.053,
respectively); these increases were not significant in
comparison to the corresponding control groups.
The ratios of mean values of immature/mature
LHR for female and male groups were 39.1±17.5 and
25.8±10.1, respectively. Whereas the pattern of this
ratio was not the same in different groups of both
genders, this ratio decreased in GDX group of both
sexes (P = 0.44 and 0.017 for female and male rats
respectively) and increased in GDX+GnRHa group
(P = 0.122 for females and 0.856 for male rats). This
ratio increased in GDX+GnRHa+E2 or T group of
females (P = 0.173) while decreased significantly in
male GDX+GnRHa+E2 or T group compared to the
corresponding control group (P = 0.023) (Fig. 5).
Due to significant difference in concentration of
mature and immature isoforms of LHR in rat
hippocampus (1:25), the mature isoform of LHR
was not detectable for some samples in blotted lanes
(Fig. 6). Therefore, three samples were located in
Fig. 4—Comparison between immature and mature LHR levels
in different groups of female and male rats.[The * shows a
significant difference between an experimental group and its
corresponding control group. Increased LH in GDX rats has
decreased level of immature LHR while it has increased the
mature LHR level. Conversely, decreasing LH in GDX+GnRHa
rats increased immature LHR while it decreased level of mature
LHR. HRT also acted in the same way as gonadectomy did].
Fig. 5—Ratio of immature LHR/mature LHR isoforms in
different groups of both genders.[This ratio was achieved by
dividing the values of the immature LHR content to the
corresponding values of mature LHR content in each group of
rats. * : a significant difference between an experimental group
and its corresponding control group. This figure shows maturation
process of LHR in different groups. Increase of LH increases
maturation and vice versa. Sex hormone replacement therapy
modulates this process].
Fig. 6—Western blot analysis of hippocampus and testis
tissue lysates using anti-rat LHR and anti-rat GAPDH
antibodies.[Lanes 1, 2, and 3 with 20 µg protein of rat
hippocampus lysate and lane 4 with 0.8 µg protein of rat
testis lysate were loaded. No GAPDH band could be observed in
lane 4. This could be due to the amount of protein which has been
loaded. Technically, this amount of protein lacks enough GAPDH
protein to be detected. The presence of GAPDH band in nearby
lanes verifies that lack of GAPDH band in lane related to testis
lysate is correct. Lane 5 represents the molecular weight marker
(Prestained Protein Molecular Weight Marker, Fermentas life
sciences, Lithuania)].
INDIAN J EXP BIOL, MARCH 2013
224
each group instead of six samples for immature
isoform of LHR and analyses were carried out on, at
least, three samples in each group with detectable
bands in Western blotted lanes.
Discussion
Nowadays, it is almost well-known that menopause
and, at a lesser extent, andropause accompany many
unwanted changes in different tissues and organs such
as bones, muscles, cardiovascular system, and brain31
.
The menopause/andropause complications are major
consequences of severe variations of gonadotropins
and sex steroid hormones following diminished
gonadal functions. It is clear that the hippocampus, as
an area of the brain involved in memory and
cognition, is much more affected than other areas of
the brain following menopause/andropause onset32
.
Neurodegenerative diseases such as Alzheimer are the
cases whose incidence increase following onset of
menopause/andropause13,14
. Due to the absence of
follicle stimulating hormone (FSH) receptor in the
brain33
, the effects of gonadotropins on the brain
may be limited to the LH functions that mediate
through LHR33
.
LHR is distributed on different areas of the brain34
,
whose structure and function may be affected
following menopause/andropause onset. Therefore,
this study was designed with the aim of recognizing
LHR variations at molecular level in experimental
models of menopause/andropause and the effects of
HRT on this receptor. Based on the difference in the
female and male reproductive physiology, using the
same intervention for both genders do not yield
similar outcomes. According to the results of
hormonal assay, gonadectomy did not increase the
levels of LH in male as much as those in female rats.
Despite lower level of LH in gonadectomized male
rats, following GnRHa administration, LH level did
not decrease in the males as much as that in the
females. Therefore, this distinctive pattern might
explain different outcomes and overlaps of results in
the male and female rats.
The doses of estradiol and testosterone for HRT
were chosen according to the doses applied by Banu,
et al29
. However, in the case of estradiol, this dose
verified by other studies was injected to provide the
physiological level of estrogen in female rats35-37
.
It was difficult to select a unique dose due to
different results on physiological levels of estradiol at
various phases of estrous cycle29,35,36,38-40
. Although
the applied dose was estimated to be at the
pharmacological level of estradiol (based on
hormonal assay results), it might not have affected
the present results drastically.
Based on real-time PCR and Western blotting
results, there are significant changes of LHR mRNA
and protein levels associated with the changes of LH
concentration.
The present observations showed that although
LHR expression was not significantly different
between control and GDX groups, there was a
significant decrease of LHR expression in
GDX+GnRHa group compared to the control group
due to the low level of LH (Fig. 2). Moreover, at the
protein level, high concentration of LH might be an
explanation not only for decreased immature LHR
but also for increased mature LHR isoforms in
GDX group while the low concentration of LH
had the opposite effects in GDX+GnRHa group.
In brief, high level of LH decreases LHR
expression, decreases immature isoform and increases
mature isoform of LHR. Nonetheless, discrepancy of
the present results with previous studies, in which LH
has decreased significantly LHR mRNA might be
explained to some extent by considering the
LHR1/LHR2 ratio as an indicator for expression
of incomplete LHR mRNA. Hence, it might be
concluded that high level of LH in GDX groups led to
an increase in this ratio (Fig. 3). Increased
LHR1/LHR2 ratio means that, LH has probably
induced post-transcriptional modifications, such as
alternative splicing. Furthermore, lower concentration
of LH in male GDX rats compared to corresponding
female GDX raised complete transcript more than
incomplete isoforms of LHR.
Despite the lack of significant decreases of LHR
expression in GDX groups, several reasons might be
considered for decreasing immature LHR protein in
mentioned group. The first one is down-regulation of
LHR by degrading its mRNA. It has been known that
LRBP1 degrades the LHR mRNA following the LH
level increasing8,41,42
. If degradation of mRNA is the
only cause of LHR down-regulation in hippocampus,
low level of LHR transcripts must be detected
following LH increasing. This assumption is in
contrast with the present findings. Moreover, this
mechanism has been shown not to be involved in the
regulation of LHR expression in the brain33
. The
second reason is the desensitization of hormone-
receptor complex. It might be the mechanism for
ABTAHI et al.: LHR REGULATION IN HIPPOCAMPAL NEURONS
225
explaining down-regulation of LHR following
increased LH level. Prolonged stimulation of LHR
leads to its desensitization so that the LH surge at the
proestrus phase of estrous cycle causes transient
receptor desensitization. The mechanisms of down-
regulating LHR expression and temporariness of
LHR uncoupling are the causes for none-responding
of the follicles to LH changes20-23,27
. Therefore,
desensitization is the reason for unresponsiveness of
primary granulosa cell cultures to hCG treatment43,44
.
If the above mechanism is true, the mature isoform of LHR should decrease on the cell surface; however, the present findings are contrary to this assumption. The unrecognized LHR isoforms other than those detected by the commercial antibody might be the third possible reason for detection of low level
for LHR protein. The forth reason may be the inconsistent expression of various LHR isoforms at different tissues. Apaja et al
45 showed that fetal rodent
gonads consist of just immature isoform; while the mature gonads, adrenal glands and also the kidneys of pregnant rat express both the immature and the
mature isoforms of LHR. Moreover, there were not significant changes in mature isoform of LHR in frontal cortex of rats which were chronically treated with high doses of LH. Although expression, translation, and modification of LHR are time and tissue dependent, the effect of LH on efficiency of
translation could be the last probable reason for the LHR down-regulation. Despite the low levels of LHR mRNA in GDX+GnRHa group, the increments of immature LHR isoform could be a strong reason for high efficiency of translation system in synthesizing LHR protein, whereas its efficiency would decrease
following increase in LH concentrations in GDX group. In addition, LH can influence post-translation modifications so that at high levels of LH, the glycosylated isoform of LHR (mature) increased and its levels decreased following LH suppression in GDX group. Regarding the above-mentioned reasons,
the hippocampus can regulate the LHR expression through different mechanisms.
Despite the presence of the FSH receptor in ovary
and its permissive role for LH function20
. The absence
of FSH receptor in brain abolishes its effect on
developing the LHR in hippocampus. Therefore, FSH
might not be the regulator of LHR expression in brain
of GDX rats46
. This may be, in part, a reason for low
LHR expression in hippocampus compared to the
testis or ovary. Unlike FSH, the estradiol receptor
is found in all of the brain areas47
. Regarding
the up-regulatory function of estradiol on LHR
expression in the ovary during pre-ovulatory phase, it
can be rationally concluded that estradiol could apply
similar function on LHR expression in other tissues
such as hippocampus. However due to the absence of
estradiol receptor, treatment of primary hippocampal
cell culture with high doses of estradiol did not show
significant changes in its LHR concentration33
. In the
present study, an increase in mRNA level of complete
isoform of LHR was found in GDX+GnRHa groups
and, especially, GDX+GnRHa+E2 or T of female rats
with different levels of estradiol and a limited
decrease at levels of immature isoform of LHR
protein concurrent with increased mature isoform of
LHR in GDX+GnRHa+E2 or T group. This finding
is similar to the effect of estradiol on increasing LHR
in the ovary at pre-ovulatory phase.
In the present study, male rats received the same
treatments as their female counterparts; therefore, similar patterns of LHR transcripts and protein could
be expected. Lower increases of LH in the male
GDX in comparison with their female counterparts and inability of diphereline® to decline LH levels in
male GDX+GnRHa group might be the cause of some overlapping effects of LH and sex steroid
hormone on LHR expression. Regarding the different half-life and activities of various glycoforms of
gonadotropins such as LH48-50
, the impact of these
glycoforms on their receptor expression might be different, especially their glycosylation changes
during menstrual cycle, menopause, ovariectomy and HRT. These are the questions which need to be
elucidated in future.
In conclusion, LH decreases, at least, its cognate receptor transcripts in hippocampus, but it decreases
the level of immature LHR protein probably by two mechanisms: (i) increasing incomplete LHR transcripts
instead of complete one using post-transcriptional
modifications, and (ii) decreasing translation of LHR transcripts to protein by reducing the efficiency of
translation process. Further, LH increases mature protein of LHR through post-translation modifications.
Last but not least, sex steroid hormones modulate severe fluctuations of LHR mRNA and protein
isoforms probably through regulating efficiency of
transcription and translation following changes in LH concentration.
Acknowledgment
This study was supported by Tehran University of
Medical Sciences and Avicenna Research Institute
INDIAN J EXP BIOL, MARCH 2013
226
by grant NO: 8708. The authors would like to thank
Dr. K. Kamali for constructive comments and
Dr. M. Kerdari for editing the manuscript.
References 1 Dufau M L, The luteinizing hormone receptor, Annu Rev
Physiol, 60 (1998) 461.
2 Rao C V, Multiple novel roles of luteinizing hormone, Fertil
Steril, 76 (2001) 1097.
3 Zhang M, Shi H, Segaloff D & Van Voohis, Expression and
localization of luteinizing hormone receptor in the female
mouse reproductive tract, Biol Reprod, 64 (2001) 179.
4 Fields M J & Shemesh M, Extragonadal luteinizing hormone
receptors in the reproductive tract of domestic animals, Biol
Reprod, 71 (2004) 1412.
5 Shemesh M, Actions of gonadotrophins on the uterus,
Reproduction, 121 (2001) 835.
6 Rao, S C, Li X, Rao Ch V & Magnuson D S K, Human
chorionic gonadotropin/luteinizing hormone receptor
expression in the adult rat spinal cord, Neurosci Lett, 336
(2003) 135.
7 Lei Z M, Toth P, Rao C V & Pridham D, Novel coexpression
of human chorionic gonadotropin (hCG)/human luteinizing
hormone receptors and their ligand hCG in human fallopian
tubes, J Clin Endocrinol Metab, 77 (1993) 863.
8 Nair A K, Kash J C, Peegel H & Menon K M, Post-
transcriptional regulation of luteinizing hormone receptor
mRNA in the ovary by a novel mRNA-binding protein, J
Biol Chem, 277 (2002) 21468.
9 Reiter E, McNamara M, Closset J & Hennen G, Expression
and functionality of luteinizing hormone/chorionic
gonadotropin receptor in the rat prostate, Endocrinology, 136
(1995) 917.
10 Reshef E, Lei Z M, Rao C V, Pridham D D, Chegini N &
Luborsky J L, The presence of gonadotropin receptors in
nonpregnant human uterus, human placenta, fetal membranes,
and deciduas, J Clin Endocrinol Metab, 70 (1990) 421.
11 Tao Y X, Lei Z M, Woodworth S H & Rao C V, Novel
expression of luteinizing hormone/chorionic gonadotropin
receptor gene in rat prostates, Mol Cell Endocrinol, 111
(1995) R9.
12 Toth P, Li X, Rao C V, Lincoln S R, Sanfilippo J S, Spinnato
J A 2nd & Yussman M A, Expression of functional human
chorionic gonadotropin/human luteinizing hormone receptor
gene in human uterine arteries, J Clin Endocrinol Metab, 79
(1994) 307.
13 Bukovsky Antonin, Indrapichate Korakod, Fujiwara Hiroshi,
Cekanova Maria, Ayala Maria E, Dominguez Roberto,
Caudle Michael R, Wimalsena Jay, Elder Robert F, Copas
Pleas, Foster James S, Fernando Romaine I, Henley Donald
C & Upadhyaya Nirmala B, Multiple luteinizing hormone
receptor (LHR) protein variants, interspecies reactivity of
anti-LHR mAb clone 3B5, subcellular localization of LHR in
human placenta, pelvic floor and brain, and possible role for
LHR in the development of abnormal pregnancy, pelvic floor
disorders and Alzheimer's disease, Reprod Biol Endocrinol,
46 (2003) 1.
14 Bowen R L, Verdile G, Liu T, Parlow A F, Perry G, Smith M
A, Martins R N & Atwood C S, Luteinizing hormone, a
reproductive regulator that modulates the processing of
amyloid-beta precursor protein and amyloid-beta deposition,
J Biol Chem, 279 (2004) 20539.
15 Lei Z M, Rao C V, Kornyei J L, Licht P & Hiatt E S, Novel
expression of human chorionic gonadotropin/luteinizing
hormone receptor gene in brain, Endocrinology, 135 (1993)
2262.
16 Al-Hader A A, Lei Z M, & Rao C V, Neurons from fetal rat
brains contain functional luteinizing hormone/chorionic
gonadotropin receptors, Biol Reprod, 56 (1997) 1071.
17 Al-Hader A A, Tao Y X, Lei Z M & Rao C V, Fetal
rat brains contain luteinizing hormone/human chorionic
gonadotropin receptors, Early Pregnancy, 3 (1997) 323.
18 Pakarainen T, Zang F P, Poutanen M & Huhtaniemi I,
Fertility in luteinizing hormone receptor-knockout mice after
wild-type ovary transplantation demonstrates redundancy of
extragonadal luteinizing hormone action, J Clin Invest, 115
(2005) 1862.
19 Apaja P M, Harju K T, Aatsinki J T, Petäjä-Repo U E &
Rajaniemi H J, Identification and structural characterization
of the neuronal luteinizing hormone receptor associated
with sensory systems, J Biol Chem, 279 (2004) 1899.
20 Segaloff D L, Wang H Y & Richards J S, Hormonal regulation
of luteinizing hormone/chorionic gonadotropin receptor
mRNA in rat ovarian cells during follicular development and
luteinization, Mol Endocrinol, 4 (1990) 1856.
21 Peegel H, randolph J Jr, Midgley A R & Menon K M, In situ
hybridization of luteinizing hormone/human chorionic
gonadotropin receptor messenger ribonucleic acid during
hormone-induced down-regulation and the subsequent
recovery in rat corpus luteum, Endocrinology, 135 (1994)
1044.
22 Hoffman Y M, Peegel H, Sprock M J, Zhang Q Y &
Menon K M, Evidence that human chorionic
gonadotropin/luteinizing hormone receptor down-regulation
involves decreased levels of receptor messenger ribonucleic
acid, Endocrinology, 128 (1991) 388.
23 Lapolt P S, Tilly J L, Aihara T, Nishimori K &Hsueh A J.
Gonadotropin-induced up- and down-regulation of rat
ovarian LH receptor message levels during follicular growth,
ovulation and luteinization, Endocrinology, 126 (1990) 3277.
24 Camp T A, Rahal J O & Mayo K E, Cellular localization
and hormonal regulation of follicle-stimulating hormone
and luteinizing hormone receptor messenger RNAs in the
rat ovary, Mol Endocrinol, 5 (1991) 1405.
25 Hu Z Z, Tsai Morris C H, Buczko E & Dufan M L,
Hormonal regulation of LH receptor mRNA and expression
in the rat ovary, FEBS Lett, 274 (1990) 181.
26 Wang H, Segaloff D L & Ascoli M, Lutropin/
choriogonadotropin down-regulates its receptor by both
receptor-mediated endocytosis and a cAMP-dependent
reduction in receptor mRNA, J Biol Chem, 266 (1991) 780.
27 Lu D L, Peegel H, Mosier S M & Menon K M, Loss of
lutropin/human choriogonadotropin receptor messenger
ribonucleic acid during ligand-induced down-regulation
occurs post transcriptionally, Endocrinology, 132 (1993) 235.
28 Kash J C & Menon K M, Sequence-specific binding of a
hormonally regulated mRNA binding protein to cytidine-rich
sequences in the lutropin receptor open reading frame,
Biochemistry, 38 (1999) 16889.
ABTAHI et al.: LHR REGULATION IN HIPPOCAMPAL NEURONS
227
29 Banu S K, Govindarajulu P & Aruldhas M M, Testosterone
and estradiol up-regulate androgen and estrogen receptors in
immature and adult rat thyroid glands in vivo, Steroids, 67
(2002) 1007.
30 Ken W S & Ashwell G P, Atlas of the developing rat nervous
system, Third ed. (Academic Press, USA) 2008.
31 Jung B H, Jeon M J & Bai S W, Hormone-dependent aging
problems in women, Yonsei Med J, 49 (2008) 345.
32 Lister J P & Barnes C A, Neurobiological changes in the
hippocampus during normative aging, Arch Neurol, 66
(2009) 829.
33 Liu T, Wimalasena J, Bowen R L & Atwood C S, Luteinizing
hormone receptor mediates neuronal pregnenolone production
via up-regulation of steroidogenic acute regulatory protein
expression. J Neurochem, 100 (2007) 1329.
34 Ascoli M, F Fanelli, & Segaloff D L, The
lutropin/choriogonadotropin receptor, a 2002 perspective,
Endocr Rev, 23 (2002) 141.
35 Wang Q, Santizo R, Banghman V L, Pelligrino D A,&
Ladecola C, Estrogen provides neuroprotection in transient
forebrain ischemia through perfusion-independent
mechanisms in rats, Stroke, 30 (1990) 630.
36 Feng, Z. and J.-t. Zhang, Long-term melatonin or 17[beta]-
estradiol supplementation alleviates oxidative stress in
ovariectomized adult rats. Free Radical Biol Med, 39
(2005) 195.
37 Bottner M & Wuttke W, Chronic treatment with
physiological doses of estradiol affects the GH-IGF-1
axis and fat metabolism in young and middle-aged
ovariectomized rats, Biogerontology, 7 (2006) 91.
38 Degano B, Berger P, Molimard M, Pontier S, Rami J &
Escamilla R, Estradiol decreases the acetylcholine-elicited
airway reactivity in ovariectomized rats through
an increase in epithelial acetylcholinesterase activity,
Am J Respir Crit Care Med, 164 (2001) 1849.
39 Wide J K, Hanvally K, Ting J & Galea Lisa AM, High level
estradiol impairs and low level estradiol facilitates non-
spatial working memory, Behav Brain Res, 155 (2004) 45.
40 Varga H, Németh H, Toth T, Kis Z, Farkas T & Toldi J,
Weak if any effect of estrogen on spatial memory in rats,
Acta Biologica Szegediensis, 46 (2002) 13.
41 Nair A K & Menon K M, Regulation of luteinizing hormone
receptor expression: Evidence of translational suppression in
vitro by a hormonally regulated mRNA-binding protein and
its endogenous association with luteinizing hormone receptor
mRNA in the ovary, J Biol Chem, 280 (2005) 42809.
42 Menon K M, Nair A K & Wang L, A novel post-
transcriptional mechanism of regulation of luteinizing
hormone receptor expression by an RNA binding protein
from the ovary, Mol Cell Endocrinol, 246 (2006) 135.
43 Amsterdam A Berkowitz A, Nimrod A & Kohen F,
Aggregation of luteinizing hormone receptors in granulosa
cells: a possible mechanism of desensitization to the
hormone, Proc Natl Acad Sci U S A, 77 (1980) 3440.
44 Amsterdam A Nimrod A, Lamprecht S A, Burstein Y &
Linder H R, Internalization and degradation of receptor-
bound hCG in granulosa cell cultures, Am J Physiol, 236
(1979) E129.
45 Apaja P M, Aatainki J T, Rajaniemi H J & Petäjä-Repo U E,
Expression of the mature luteinizing hormone receptor in
rodent urogenital and adrenal tissues is developmentally
regulated at a posttranslational level, Endocrinology, 146
(2005) 3224.
46 Menon K M Munshi U M, Clouser C L & Nair A K,
Regulation of luteinizing hormone/human chorionic
gonadotropin receptor expression: a perspective, Biol
Reprod, 70 (2004) 861.
47 Rainbow T C, Parson, B, MacLusk N J & McEwen B S,
Estradiol receptor levels in rat hypothalamic and limbic
nuclei, J Neurosci, 2 (1982) 1439.
48 Wide L & Bakos O, More basic forms of both human
follicle-stimulating hormone and luteinizing hormone in
serum at midcycle compared with the follicular or luteal
phase, J Clin Endocrinol Metab, 76 (1993) 885.
49 Dharmesh S M & Baenziger J U, Estrogen modulates
expression of the glycosyltransferases that synthesize
sulfated oligosaccharides on lutropin, Proc Natl Acad Sci U S
A, 90 (1993) 11127.
50 Burgon P G, Stanton P G & Robertson D M, In vivo
bioactivities and clearance patterns of highly purified human
luteinizing hormone isoforms, Endocrinology, 137 (1996)
4827.
All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
Top Related