Background: The inhibitory effects of morphine and the stimulatory influence of kisspeptin signaling have been demonstrated on gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release. Hypothalamic kisspeptin is involved in relaying the environmental and metabolic information to reproductive axis. In the present study, the role of kisspeptin/ GPR54 signaling system was investigated on relaying the inhibitory influences of morphine on LH hormone secretion. Materials and Methods: In this experimental study, 55 wistar male rats weighing 230-250 g were sub-grouped in 11 groups (in each group n=5) receiving saline, kisspeptin (1 nmol), peptide234 (P234, 1 nmol), morphine (5 mg/kg), naloxone (2 mg/kg), kisspeptin/P234, morphine/naloxone, kisspeptin/morphine, kisspeptin/naloxone, P234/morphine or P234/naloxone respectively. Blood samples were collected via tail vein. Mean plasma (LH) concentrations and mean relative KiSS1 or GPR54 mRNA levels were determined by radioimmunoassay (RIA) and real time reverse transcriptase-polymerase chain reaction (RT-PCR), respectivwely. Results: Morphine significantly decreased mean plasma LH concentration and mean relative KiSS1 gene expression compared to saline; while it did not significantly decrease mean relative GPR54 gene expression compared to saline. Naloxone significant increased mean LH level and mean relative KiSS1 gene expression compared to saline; while it did not significantly increase mean relative GPR54 gene expression compared to saline. Injections of kisspeptin plus morphine significantly increased mean LH concentration compared to saline or morphine, while simultaneous infusions of them significantly declined mean plasma LH level compared to kisspeptin. In kisspeptin/naloxone group mean plasma LH level was significantly increased compared to saline or naloxone. Co-administration of P234/morphine significantly decreased mean LH concentration compared to saline. Conclusion: Down regulation of KiSS1 gene expression may be partly involved in the mediating the inhibitory effects of morphine on reproductive axis.
kisspeptin/GPR54 signaling pathway has a therapeutic
potential, as regulator of gonadotropin releasing hormone
(GnRH)/luteinizing hormone (LH) release and gonadal steroid
hormone secretions. G protein-coupled receptor, GPR54,
is expressed in GnRH neurons and normal pubertal development,
while sexual function is also dependent to normal
actions of it (1, 2). Reproductive process is disrupted by the
mutations of GPR54 receptor or kisspeptin genes (3). Kisspeptin
analogues are introduced as endogenous ligand for
GPR54 receptor and four types of kisspeptin (kisspeptins 10,
13, 14 and 54) have similar affinity to this receptor. They
induce puberty and peripheral or central injections of them
increase the GnRH/LH release and plasma gonadal steroid
(1-4). Infusions of peptide 234 (P234) also block the stimulatory
effects of kisspeptin on LH secretion (5).
Opioids suppress the reproductive process, resulting in
hypogonadotropic hypogonadism (HH) dominantly via
inhibiting the hypothalamus-pituitary-gonadal (HPG)
axis rather than direct effects on pituitary or testes (6).
Morphine, as an alkaloid extracted from poppy plant, is
extremely used as drug abuse and drugs for the suppressing pain.
Injections of morphine decrease the secretion
of GnRH and LH via binding to opioid µ-type recep.
tors (6-8). However, Aloisi and her colleague reported
that morphine treatment may play a role in declining the
mean plasma testosterone level by increasing peripheral
testosterone metabolism in testes, liver and hypothalamus
(9). It has also been found that naloxone, acting as the
antagonist of µ-opioid receptor, blocks the influences of
morphine on the HPG axis. In contrast, it induces puberty
and improves the GnRH/LH as well as gonadal hormone
secretions in males and females of different species (10).
Opioids receptors are not directly expressed on GnRH
neurons and they exert their inhibitory influences on the
reproductive axis via different interneurons pathways (11).
In addition, several studies have established that kisspeptin
has a crucial role in relaying the central or peripheral information
to the reproductive axis (12-16). In order to the
significant importance of physiological action of kisspeptin/
GPR54 signaling pathway for controlling GnRH/LH release
and considering the clinical overuse of opioid drugs, the present
study aimed to investigate that if the level of kisspeptin/
GPR54 signaling system activity may be partly involved in
the morphine- induced decline of LH mean plasma levels.
Materials and Methods
In this experimental research, three months old male wistar
rats (n=55), weighing 230-250 g (provided by the Center of
Neuroscience Research of Shahid Beheshti University, Tehran,
Iran), were housed in the cages under controlled temperature
(22 ± 2°C) and light (12 hours light/dark cycle).
Animals had always free access to food and water. All procedures
for the maintenance and use of experimental animals
were executed with the Guide for the Care and Use of Laboratory
Animals (National Institute of Health Publication No.
80-23, revised 1996, Iran) and were approved by the Ethical
Committee of Neuroscience Research Center of Shahid Beheshti
University of Medical Sciences (Tehran, Iran).
Intra cerebral ventricular cannulation and injections
Animals were anesthetized by intraperitoneal (IP) injections
of a mixture of ketamine and xylezine (ketamine
80 mg/kg bodyweight+xylezine 10 mg/kg bodyweight),
a 22-gauge stainless cannulae was implanted in the third
cerebral ventricle according to coordinates of Paxinos
and Watson Atlas [anterior posterior (AP)=-2.3, midline
(ML)=0.0, dorsoventral (DV)=6.5] (17). After one week,
55 rats were divided into 11 groups (5 in each group),
receiving drugs as mentioned in the Table 1.
Received drugs (name and dose) in each groups (n=5)
ICV; Intra cerebral ventricular and SC; Subcutaneously.
Kisspeptin10 (Ana Spec Co., USA) and P234 (Phoenix
Pharmaceuticals Inc., USA) were dissolved in distilled
water and injected intra third cerebral ventricle by using
Hamilton micro syringe at 09:00- 9:30. Morphine sulfate
(Temad Co., Iran) and naloxone hydrochloride (Toliddaru
Co., Iran) were dissolved in distilled water and injected
SC by an insulin syringe at 09:00-9:30. In simultaneous
groups, naloxone was injected 15 minutes before morphine
injections. The time of blood sampling as well as
kisspeptin, naloxone or morphine doses was chosen based
on our laboratory and other previous studies reporting the
stimulatory or inhibitory effects of these drugs on the reproductive
axis, respectively (2, 3, 9, 10).
Hormone assays
Blood samples were collected in a volume of 0.5 cc at
60 minutes following the injections via tail vein. Heparin
was added to the samples to prevent clotting. Blood samples
were immediately centrifuged for 15 minutes at 3000
rpm and the plasma samples were stored at -20°C until
assayed for LH concentration. Mean plasma LH concentration
was measured by using rat LH kit and the method
of the radioimmunoassay (RIA, Institute of Isotopes Co,
LT'D, Hungary). Sensitivity and intraassay of the kit were
0.09 ng/ml and 4.61%, respectively.
Microdissections and total RNA extraction
Four hours after injections, the rats were sacrificed by
decapitation and the brains were immediately autopsied.
The brains were placed ventral side up, anterior coronal
slices were cut from 1 mm anterior to optic chiasm. The
slices were then dissected laterally up to the hypothalamic
sulci and posterior coronal slices were cut posterior to
the mammillary bodies (17). The samples were frozen by
liquid nitrogen and stored at -80°C for determination of
mRNA levels. Total RNA was isolated from individual
frozen samples using the acid guanidinium thiocyanate-
phenol-chloroform extraction method, according to PureZol
manufacturer instruction (Bio RAD, USA). The
quantification of each RNA sample was performed by
measuring absorbance at 260 nm. The GAPDH gene was
used to normalize the values obtained for each sample.
RNA analysis by real-time reverse transcriptase
polymerase chain reaction
Changes in the gene expression levels were determined
by using the Corbett Real-Time PCR detection system
Rotorgene 6000 (Qiagen Ltd, Germany). Total RNA (100
ng) was treated by DNaseI to remove residual genomic
DNA according to manufacturer instruction (Thermo Scientific
Inc., USA). Then, total RNA was further amplified
in triplicate by using SYBR green I as fluorescent dye and
one step quantitative reverse transcriptase RT-qPCR Master
Mix Plus for SYBR Green I kit in a final volume of
25 µl according to manufacturer instruction (Eurogentec
CO, USA). The PCR cycling conditions were as follows:
reverse transcriptase step 48ºC for 30 minutes, 95ºC for
10 minutes, followed by 40 cycles of denaturation at 95ºC
for 15 seconds, annealing at 54ºC (KiSS1), 54ºC (GPR54)
and 58ºC (GAPDH) for 15 seconds and extension at 72ºC
for 40 seconds. Specific oligo nucleotide sequences for
sense and antisense primers were used as following:
F: 5'-AGCTGCTGCTTCTCCTCTGT-3'
R: 5'-AGGCTTGCTCTCTGCATACC-3' (18)
F: 5'-GGTGCTGGGAGACTTCATGT-3'
R: 5'-AGTGGCACATGTGGCTTG-3' (18)
F: 5'-AAGAAGGTGGTGAAGCAGGCATC-3'
R: 5'-CGAAGGTGGAAGAGTGGGAGTTG-3' (19).
KiSS1, GPR54 and GAPDH amplified product lengths
were 151, 72 and 112 base pairs, respectively. To ensure
the specification of RT-qPCR products the melting curve
for fragments were generated by the Rotorgene 6000
program and the PCR products were evaluated in 1.5%
agarose gel electrophoresis. Calculation of the relative
expression levels of targeted cDNAs were conducted
based on the cycle threshold (Ct) method. The Ct for each
sample was calculated using the Corbett Real-Time PCR
detection system software with an automatic fluorescence
threshold (Rn) setting. Accordingly, fold expression of
target mRNAs over the reference values was calculated
by the equation 2-ΔΔCt.
Statistical analysis
The results are presented as mean ± SEM. The data
were analyzed by using SPSS software (version 16) and
the one- way ANOVA followed by post hoc Tukey test. In
all cases, statistical significance was defined by P<0.05.
Results
Kisspeptin increased significantly the mean plasma LH
concentration by 1.71 times compared to saline. P234 decreased
mean plasma LH concentration by 0.12 compared
to saline; however this decrease was not statistically significant.
Simultaneous injection of kisspeptin and P234 increased
the mean plasma LH concentration by 0.29 times
compared to saline, while this increase was not statistically
significant. In addition, injection of P234 solely or
simultaneous injection of kisspeptin and P234 decreased
significantly mean plasma LH concentration respectively
by 0.67 or 0.52 times compared to kisspeptin.
Morphine decreased significantly mean plasma LH concentration
by 0.48 times compared to saline. Mean plasma
LH concentration increased significantly following naloxone
injection by 0.48 times compared to saline. Simultaneous
injection of naloxone and morphine increased mean
plasma LH concentration by 0.17 or 1.24 times compared
to saline or morphine, respectively. This increase was not
statistically significant compared to saline, while it was
statistically significant compared to morphine.
Co-administration of kisspeptin/morphine increased
significantly mean plasma LH concentration by 0.73 or
2.32 times compared to saline or morphine, respectively.
Additionally, co-administration of kisspeptin/morphine
decreased significantly mean plasma LH concentration by
0.37 times compared to kisspeptin. Co-administration of
kisspeptin/naloxone increased significantly mean plasma
LH concentration by 2.12 or 5.04 times compared to saline
or naloxone, respectively.
Moreover, LH concentration was increased in kisspeptin/
naloxone group by 0.16 times compared to kisspeptin
group, although this increase was not statistically significant.
Co-administration of P234/morphine decreased
mean plasma LH concentration by 0.5, 0.1 or 0.4 times
compared to saline, morphine or P234, respectively. This
decrease was statistically significant compared to saline
or P234 (P<0.05, Fig .1), while it was not statistically significant
in comparison with morphine. Co-administration
of P234/naloxone increased mean plasma LH concentration
by 0.18 times compared to saline, but this increase
was not statistically significant. Furthermore, co-administration
of P234/naloxone decreased mean plasma LH
concentration by 0.21 times compared to naloxone, while
this decrease was not statistically significant (Fig .1).
Effects of kisspeptin (1 nmol), P234 (1 nmol), 5 mg/kg morphine
(MOR), 2 mg/kg naloxone (NAL) or co-administration of kisspeptin/morphine,
kisspeptin/naloxone, P234/morphine or P234/naloxone on mean
plasma LH concentration, in comparison with a; Saline, b; Kisspeptin, c;
P234, d; Morphine, and e; Naloxone. Data are presented as mean ± SEM,
P<0.05 and n=5 in each group.
In addition, results showed that morphine induced a
significant decrease in KiSS1 mRNA expression levels in
the hypothalamic samples compared to saline, naloxone
or morphine plus naloxone injected groups. So that morphine
decreased significantly mean relative KiSS1 gene
expression by 0.89, 0.93 or 0.85 times compared to saline,
naloxone or morphine plus naloxone, respectively. Naloxone
increased significantly mean relative KiSS1 gene expression
by 0.68, 14.27 or 1.21 times compared to saline,
morphine or morphine+naloxone respectively.
In animals receiving naloxone+morphine, the mean
relative KiSS1 gene expression was decreased by 0.24 or
0.54 times compared to saline or naloxone, respectively.
This decrease was not statistically significant compared
to saline, while it was statistically significant compared to
naloxone. Additionally, injections of naloxone+morphine
increased significantly the mean relative KiSS1 gene expression
by 5.9 times compared to morphine (P<0.05,
Fig .2). The mean relative GPR54 gene expressions were
not significantly influenced by the injections of morphine,
naloxone or morphine+naloxone compared to saline
group. Moreover, a significant decrease or increase was
not observed on the GPR54 mRNA levels between different
groups (Fig .3).
Effects of morphine (5 mg/kg), naloxone (2 mg/kg) or simultaneous
injections of morphine and naloxone (n=5 in each group) on KiSS1 mRNA
expression in the hypothalamus of male rats. The cDNA amplified from
GAPDH mRNA was used to normalize corresponding KiSS1 results. The
results are presented as mean ± SEM. In all cases P<0.05 was considered
to be statistically significant. a; Compared to saline, b; Compared to morphine,
c; Compared to naloxone, and d; Compared to morphine+naloxone.
Effects of morphine (5 mg/kg), naloxone (2 mg/kg) or simultaneous
injections of morphine and naloxone (n=5 in each group) on GPR54 mRNA
expression in the hypothalamus of male rats. The cDNA amplified from
GAPDH mRNA was used to normalize corresponding GPR54 results. The
results are presented as mean ± SEM. In all cases P<0.05 was considered
to be statistically significant.
Discussion
The results showed that subcutaneous injection of naloxone
or central injection of kisspeptin increased significantly
the mean plasma LH concentration compared to
saline, while subcutaneous injection of morphine significantly
decreased it, in comparison with saline. These results
are consistent with the other researches which established
the stimulatory effects of naloxone (10), kisspeptin
(1-5) or inhibitory effects of morphine on the sexual hormone
secretions (6-9) and introduced them as important
key regulators for controlling the HPG axis in the male
and females of different species.
In our previous studies, we showed that interaction of
morphine/kisspeptin play a role in the regulating of mean
plasma testosterone concentration in male rats (8). In this
work, our results indicated that morphine injection attenuates
the stimulatory effects of kisspeptin on mean plasma
LH concentrations anf injection of kisspeptin+naloxone
exerts an additive stimulatory effect on mean levels of
LH, compared to naloxone. The precise molecular and
central mechanisms underlying the effects of opioids on
the reproduction neuroendocrine axis is not clear yet.
However previous researches demonstrated that endogenous
opioids, exogenous opiates (e.g. morphine) or
their receptor antagonists influence the release of LH and
subsequently gondal steroid hormones via indirect regulation
of the hypothalamic GnRH release (11). However
Kappa opioid receptors have been found on hypothalamic
kisspeptin neurons of arcuate nucleus (ARC) (20), but
mu opioid receptors mediating the physiological effects
of ß-endorphin or morphine (21) are widely expressed in
the brain stem and thalamic nuclei and lower levels expression
of them has been reported in hypothalamus or
GnRH neurons. Different signaling pathways supposed to
be involved in mediating opioids indirect effects on the
hypothalamic GnRH-producing neurons, which we could
point to noradrenergic, dopaminergic or GABAergic neurons
(11).
It is well established that more than 80% GnRH neurons
express GPR54 receptor and hypothalamic KiSS1
has been proposed as key molecular conduit for relaying
a number of peripheral or central signals including steroid
hormones, fasting, ghrelin, leptin or photoperiod into
the GnRH system (12-16). Therefore we examined the effects
of morphine/naloxone injections on KiSS1/GPR54
mRNA levels to investigate that if the opioids and kisspeptin
pathways may interact to each other in controlling
the HPG axis.
The results showed that morphine significantly down-
regulated the KiSS1 mRNA levels and naloxone blocked
the inhibitory effect of morphine on KiSS1 mRNA expression.
But GPR54 mRNA levels were not significantly influenced
by morphine or naloxone injections. For the first
time in reproductive axis, we investigated the effects of
morphine/naloxone on KiSS1/GPR54 mRNA levels and
no study has previously been performed to compare this
point in any species. However morphine may take part
in the regulating of kisspeptin synthesis partly via other
brain interneurons or peptides. It has been revealed that
ghrelin system negatively influences the gonadal axis (22-
24). It has also been reported that co-administration of
naloxone with ghrelin restores mean LH concentration
and pulse frequency in rats (23). Moreover, ghrelin inhibits
and delays the LH response to naloxone in men (24).
Changes in the hypothalamic KiSS-1 system have been
reported in situations of negative energy balance, which
are linked to the suppressed gonadotropin secretion. Studies
reported that intravenous injection of ghrelin or fasting,
accompanying with increased ghrelin levels, results
in a significant decrease in KiSS1 gene mRNA level in
the rat brain (16-18). Because GnRH pulse generator and
kisspeptin neurons are located in the medio basal hypothalamus
in which the ghrelin receptor is also expressed
(25). Our studies have also shown that morphine increases
hypothalamic ghrelin gene expression in male rats (data
not published). Thus, central opioid system may down-
regulate KiSS1 gene expression partly via up-regulating
ghrelin levels.
There is a close relationship between hypothalamus-pituitary-
adrenal (HPA) and HPG axis activities. Corticotrophin-
releasing factor (CRF), synthesized by hypothalamic
neurons, is a potent inhibitor of the GnRH pulse generator.
Central administrations of CRF decrease the GnRH
concentration in hypophyseal portal system and mean
plasma LH/sex steroid concentrations (26-28). While suppression
of LH secretion, by CRF injection, or a variety of
stressful stimuli, increasing the CRF/cortisol secretions,
can be reversed by CRF antagonists (29). The previous
studies have reported that injections of opioid increase
CRF/ACTH release and pretreatment of the animals with
opioid antagonists especially µ-type receptor antagonists
abolish the inhibitory effects of CRF on GnRH/LH release,
suggesting that the CRF-induced inhibition of gonadotropin
secretion is mediated by opioids (27). Recently
Kinsey-Jones et al. (30) showed that CRF or corticosterone
injections as well as both acute and chronic stressors
down-regulate kiSS1/GPR54 mRNA levels in rat hypothalamic
nuclei. So, a possible functional interaction between
the opioid and CRF/corticosterone systems could
be considered in regulating kisspeptin/GPR54 signaling
system. Leptin, the hormone which is mainly secreted by
adipose tissue, may be involved.
Leptin is a stimulatory factor for controlling reproduction
process and it improves secretion of LH hormone via
projecting direct or indirect signals including kisspeptin
neurons to GnRH ones (31). Studies demonstrated that
kisspeptin mRNA levels are extremely lower in leptin
gene knocked-out mice compared to normal ones and infusion
of leptin reverse the results in these animals. They
contributed to the down-regulation of HPG axis activity to
declined arcuate kisspeptin levels (13). Many other studies
confirmed the mediatory role of kisspeptin/GPR54
signaling pathway for exerting leptin effects on GnRH/
LH release (31, 32). There is also an inverse relationship
between plasma ß-endorphine (endogenous ligand for
mu receptor) and leptin level. It has been established that
ß-endorphine contains lipolytic properties and it plays an
important role in decreasing body weight via declining
leptin secretion (33). So, it is proposed that suppressing
leptin signaling might partly be involved in the inhibitory
effects of mormhine on KiSS1 gene expression.
However for first time our results showed that down-
regulation of kisspeptin pathway may have a role in the
inhibitory effects of morphine on HPG axis. To better understand
mechanisms of opioid-induced hypogonadism
via affecting kisspeptin/GPR54 signaling system, in
further studies we could examine the effects of injection
of other opiates including methadone, codeine or endogenous
opioid such as ß-endorphine on hypothalamic
KiSS1/GPR54 mRNA levels. In addition, the interactions
of morphine and effect of inhibitory/stimulatory factors
involved in the regulation of reproduction including leptin,
alpha melanocyte stimulating hormone (aMSH) or
CRF should be investigated on kisspeptin/GPR54 signaling
pathway and HPG axis activity.
Conclusion
Subcutaneous injection of morphine attenuates the stimulatory
effects of third cerebral ventricular injection of kisspeptin
on mean plasma LH levels. Kisspeptin+naloxone
exerts an additive stimulatory effect on mean plasma levels
of LH compared to naloxone. Additionally, morphine
significantly down-regulates the hypothalamic KiSS1 levels
and naloxone blocks the inhibitory effect of morphine
on KiSS1 mRNA expression. The GPR54 mRNA levels
were not significantly influenced by morphine or naloxone
injections. These results suggest that down-regulation
of the kisspeptin signaling pathway might partly be involved
in opioid-induced infertility.
Khazali, H., Mahmoudi, F., & Janahmadi, M. (2018). Hypothalamic KiSS1/GPR54 Gene Expressions and Luteinizing Hormone Plasma Secretion in Morphine Treated Male Rats. International Journal of Fertility and Sterility, 12(3), 223-228. doi: 10.22074/ijfs.2018.5332
MLA
Homayoun Khazali; Fariba Mahmoudi; Mahyar Janahmadi. "Hypothalamic KiSS1/GPR54 Gene Expressions and Luteinizing Hormone Plasma Secretion in Morphine Treated Male Rats". International Journal of Fertility and Sterility, 12, 3, 2018, 223-228. doi: 10.22074/ijfs.2018.5332
HARVARD
Khazali, H., Mahmoudi, F., Janahmadi, M. (2018). 'Hypothalamic KiSS1/GPR54 Gene Expressions and Luteinizing Hormone Plasma Secretion in Morphine Treated Male Rats', International Journal of Fertility and Sterility, 12(3), pp. 223-228. doi: 10.22074/ijfs.2018.5332
VANCOUVER
Khazali, H., Mahmoudi, F., Janahmadi, M. Hypothalamic KiSS1/GPR54 Gene Expressions and Luteinizing Hormone Plasma Secretion in Morphine Treated Male Rats. International Journal of Fertility and Sterility, 2018; 12(3): 223-228. doi: 10.22074/ijfs.2018.5332