A role for endocannabinoids in acute stress-induced suppression of the hypothalamic-pituitary-gonadal axis in male rats

Abstract

Objective: Stress is known to be an inhibitor of the reproductive hypothalamic-pituitary-gonadal (HPG) axis. However, the neural and molecular connections between stress and reproduction are not yet understood. It is well established that in both humans and rodents, kisspeptin (encoded by the kiss1 gene) is a strong stimulator of the HPG axis. In the present study we hypothesized that endocannabinoids, an important neuromodulatory system in the brain, can act on the HPG axis at the level of kiss1 expression to inhibit reproductive function under stress.

Methods: Adult male Wistar rats were unilaterally implanted with an intracerebroventricular cannula. Afterwards, the animals were exposed to immobilization stress, with or without the presence of the cannabinoid CB1 receptor antagonist AM251 (1 µg/rat). Blood samples were collected through a retro-orbital plexus puncture before and after stress. Five hours after the stress, brain tissue was collected for reverse transcriptase-quantitative polymerase chain reaction measurements of kiss1 mRNA.

Results: Immobilization stress (1 hour) resulted in a decrease in the serum luteinizing hormone concentration. Additionally, kiss1 gene expression was decreased in key hypothalamic nuclei that regulate gonadotrophin secretion, the medial preoptic area (mPOA), and to some extent the arcuate nucleus (ARC). A single central administration of AM251 was effective in blocking these inhibitory responses.

Conclusion: These findings suggest that endocannabinoids mediate, at least in part, immobilization stress-induced inhibition of the reproductive system. Our data suggest that the connection between immobilization stress and the HPG axis is kiss1 expression in the mPOA rather than the ARC.

Keywords: Cannabinoids; Immobilization; Kisspeptins; Rats; Reproduction.

Figure 1

The serum corticosterone level in the control (without stress group) rats and in those after (post) 1 hour of immobilization stress. Each value represents the mean±SE of 6 animals. ^aIndicates p<0.05 vs. the control group.

Figure 2

The serum LH concentrations across experimental groups, before and after the stress. (A) Experimental time line. (B) Effects of intracerebroventricular (ICV) administration of AM251 (1 and 10 µg/rat) 30 minutes before the onset of stress on serum LH concentrations. Each value represents the mean±SE of 6 animals. ^a,b,cp<0.05.

Figure 3

The effect of acute immobilization stress and AM251 (1 µg/rat, intracerebroventricular) 30 minutes before the onset of stress on the serum testosterone level and compared with the stress group. Each value represents the mean±SE of 6 animals. There were no significant differences between pre and post stress in each group (p>0.05).

Figure 4

The effect of acute immobilization stress and intracerebroventricular administration of AM251 (1 µg/rat) 30 minutes before the onset of stress on kiss1 mRNA expression in the medial preoptic area (mPOA) and arcuate (ARC) nuclei. Relative mRNA expression is represented as a log 2 relative expression ratio (mean±SE). The copy number of the kiss1 transcript was normalized as the ratio to the copy number of the β-actin transcript for each sample. The data are expressed as relative to kiss1 mRNA levels in the control group (non-stressed group). The comparison between the groups was performed by using REST 2009 software; (n=6). ^aIndicates a significant difference versus the stress group in the mPOA nucleus (p<0.05).