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: sadeghi@avicenna.ac.ir

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.

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