Narcotics and opioid peptides are known to inhibit
the activity of the hypothalamic-pituitarygonadal
(HPG) axis in rodents, ruminants and humans.
Central or peripheral injections of morphine
or β-endorphin significantly decrease mean plasma
luteinizing hormone (LH) and circulating gonadal
steroids mainly via binding to opioid μ-type receptors
Kisspeptin-54 is the product of the
In the present experimental study, male Wistar rats (n=60) that weighed 230-250 g were obtained from the Center of Neuroscience Research at Shahid Beheshti University (Iran) and housed individually in cages under controlled temperature (22 ± 2˚C) and light (12 hour light/dark cycle, lights on at 0700 hours). Animals had continual free access to food and water. All procedures for the maintenance and the use of the experimental animals received the approval of the Ethical Committee of the Neuroscience Research Center at Shahid Beheshti University of Medical Sciences (Tehran, Iran).
Animal surgery procedures and handling were carried out as previously described (
After a one-week recovery period, the 60 rats in 12 groups of n=5 per group received either saline (3 μl)/ saline (200 μl), kisspeptin (1 nmol/3 μl)/ saline (200 μl), saline (3 μl)/ morphine (5 mg/kg, 200 μl), saline (3 μl)/ morphine (10 mg/kg, 200 μl), saline (3 μl)/ naloxone (2 mg/kg, 200 μl), saline (3 μl)/ naloxone (2 mg/kg, 100 μl) + morphine (5 mg/kg, 100 μl), saline (3 μl)/ naloxone (2 mg/kg, 100 μl) + morphine (10 mg/kg, 100 μl), kisspeotin (1 nmol/3 μl)/ morphine (5 mg/kg, 200 μl), kisspeptin (1 nmol/3 μl)/ morphine (10 mg/kg, 200 μl), kisspeotin (1 nmol/3 μl)/ naloxone (2 mg/kg, 200 μl), kisspeotin (1 nmol/3 μl)/ naloxone (2 mg/kg, 100 μl) + morphine (5 mg/kg, 100 μl), kisspeotin (1 nmol/3 μl)/ naloxone (2 mg/kg, 100 μl) + morphine (10 mg/kg, 100 μl). kisspeptin was injected via third cerebral ventricle and morphine or naloxone were injected subcutaneously.
For ICV injection, kisspeptin-10 (Ana Spec Co., USA) was dissolved in DMSO and injected with a 27-gauge stainless steel injector that protruded 0.5 mm beyond the cannula, and was connected to a Hamilton microsyringe by PE-20 tubing at 9-9:30 AM. For subcutaneous injection, morphine sulfate (Temad Co., Iran) and naloxone hydrochloride (Tolid Daru Co., Iran) were dissolved in distilled water and injected by an insulin syringe at 9-9:30 AM. In co-administrated groups, either morphine or naloxone injections were administered 15 minutes before the kisspeptin-10 injections.
We collected 0.5 cc of blood at 60 minutes following the injections via the tail vein (
The results showed that injection of kisspeptin/saline significantly increased the mean plasma testosterone concentration compared to saline/saline group. Injection of saline/morphine (5 or 10 mg/kg) significantly decreased the mean plasma testosterone concentration compared to the saline/saline group. A significant difference was not observed between the effects of 5 mg/kg and 10 mg/kg of morphine on mean testosterone concentration . Mean plasma testosterone concentration significantly increased following saline/naloxone injection compared to the saline/saline or saline/morphine (5 or 10 mg/kg) groups.
The results also revealed that in the naloxone pretreated groups, morphine did not significantly decrease testosterone concentration compared to the saline/morphine (5 or 10 mg/kg) groups. We did not observe a significant difference between the effects of morphine on testosterone concentration in the naloxone pretreatment groups. The mean plasma testosterone concentration significantly decreased following co-administration of morphine (5 or 10 mg/kg)/kisspeptin compared to the kisspeptin/saline group (
The results also showed that the mean plasma testosterone concentration significantly increased after co-administration of kisspeptin/naloxone + morphine (5 or 10 mg/kg) compared to kisspeptin/morphine. However a significant difference was not observed between the effects of co-administration of kisspeptin/naloxone + 5 mg/kg morphine and kisspeptin/naloxone + 10 mg/kg morphine on testosterone concentration. A significant difference was not observed between the effects of co-administration of kisspeptin/naloxone + morphine (5 or 10 mg/kg) on testosterone concentration compared to kisspeptin/saline (
Effects of kisspeptin, morphine, naloxone or co-administration of kisspeptin, morphine and naloxone on mean plasma testosterone concentration in male Wistar rats. Significant differences are indicated by letters. a; Compared to saline/saline group, b; Compared to saline/5 mg/kg morphine group, c; Compared to saline/10 mg/kg morphine group, d; Compared to kisspeptin/saline group, e; Compared to saline/naloxone group, f; Compared to kisspeptin/5 mg/kg morphine group, g; Compared to kisspeptin/10 mg/kg morphine group (data presented as mean ± SEM; p<0.05; n=5 per group), NAL; Naloxone and MOR; Morphine.
Effects of kisspeptin, morphine, naloxone or co- administration of kisspeptin, morphine and naloxone on mean plasma testosterone concentration (data presented mean ± SEM, P<0.05, n=5 per groups).
|5.81 ± 0.28|
|2.33 ± 0.26|
|1.85 ± 0.3|
In the present study, we chose the kisspeptin, naloxone and morphine doses based on previous studies which reported their respective stimulatory or inhibitory effects on the HPG axis (
However previous studies have suggested that opioid peptides exert their inhibitory effects on the HPG axis via different interneurons and there is an interaction between kisspeptin and dynorphin in controlling the HPG axis but there is no report about the interaction of morphine and the kisspeptin/GPR54 signaling system on the HPG axis thus far. In the present study, we have investigated the effects of a central injection of kisspeptin on mean plasma testosterone concentration following morphine or naloxone injections. The results showed that morphine significantly attenuated testosterone response to the kisspeptin injection compared to the kisspeptin alone group while a stimulatory additive effect was observed in the kisspeptin/naloxone group compared to only naloxone. In the present study, for the first time, the effect of an interaction between morphine and kisspeptin has been investigated on the reproductive axis. No previous studies exist to compare these results. However morphine may play a partial role in the rate of kisspeptin/GPR54 signaling system activity. Recently it has been established that some important factors involving the control of sexual function that include steroid hormones, fasting or ghrelin exert their inhibitory effects on the HPG axis via down regulation of the kisspeptin/GPR54 signaling system. Ghrelin injection results in a significant decrease in
The results of the present study showed that central injection of 1 nmol kisspeptin or subcutaneous injection of 2 mg/kg naloxone significantly increased mean plasma testosterone concentrations compared to saline while subcutaneous injection of different doses of morphine (5 or 10 mg/kg) significantly decreased mean plasma testosterone compared to saline. Morphine significantly attenuated testosterone secretion after kisspeptin injection compared to the kisspeptin only group while a stimulatory additive effect was observed in the kisspeptin/naloxone group compared to only naloxone or kisspeptin. Thus the morphine and kisspeptin systems might interact with one another to control the HPG axis.