Hypothalamic KiSS1/GPR54 Gene Expressions and Luteinizing
Hormone Plasma Secretion in Morphine Treated Male Rats
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/
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
Morphine significantly decreased mean plasma LH concentration and mean relative
Down regulation of
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/
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.
|1||Saline (3 µl, ICV)/saline (200 µl, SC)|
|2||Kisspeptin (1 nmol/3 µl, ICV)/saline (200 µl, SC)|
|3||P234 (1 nmol/3 µl, ICV)/saline (200 µl, SC)|
|4||Kisspeptin (1 nmol/1.5 µl, ICV)+P234 (1 nmol/1.5 µl, ICV)/saline (200 µl, SC)|
|5||Saline (3 µl, ICV)/morphine (5 mg/kg, 200 µl, SC)|
|6||Saline (3 µl, ICV)/naloxone (2 mg/kg, 200 µl, SC)|
|7||Saline (3 µl, ICV)/naloxone(2 mg/kg, 100µl, SC)+morphine (5 mg/kg, 100 µl, SC)|
|8||Kisspeotin (1 nmol/3 µl, ICV)/morphine (5 mg/kg, 200 µl, SC)|
|9||Kisspeptin (1 nmol/3 µl, ICV)/naloxone (2 mg/kg, 200 µl, SC)|
|10||P234 (1 nmol/3 µl, ICV)/morphine (5 mg/kg, 200 µl, SC)|
|11||P234 (1 nmol/3 µl, ICV)/morphine (5 mg/kg, 200 µl, SC)|
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).
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
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 (
R: 5'-AGGCTTGCTCTCTGCATACC-3' (18)
R: 5'-AGTGGCACATGTGGCTTG-3' (18)
R: 5'-CGAAGGTGGAAGAGTGGGAGTTG-3' (19).
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.
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,).
In addition, results showed that morphine induced a
significant decrease in
In animals receiving naloxone+morphine, the mean
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
The results showed that morphine significantly down-
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
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
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/
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
This study was financially supported by a grant from the Iranian National Science Foundation (INSF grant number 92044248). There is no conflict of interest in this article.
H.K., F.M.; Participated in study design, data evaluation, conducted molecular experiments, and statistical analysis. M. J.; Contributed to conception ad study design. All authors read and approved the final manuscript.