The objective of this present study was to seek insights into the genetic basis of the variability of response to stress in chickens. Corticosteronemia following ACTH injection varied between animals. Individual variations in adrenal responses have already been described in previous study [35
]. This variation can, in part, be related to time course responses to ACTH injection. According to Noirault et al. 1999 [35
], a maximum response is generally achieved 15 min post-injection and lasts for a period of 5 to 20 min, then drops gradually to reach the basal level. In our study, the blood was taken one hour post treatment, therefore animals with the highest level of CORT can be considered either having a high adrenal response or a prolonged response, and the opposite (low adrenal response or short delay response) for those with low level of CORT.
Following microarray analysis, changes in the expression of many genes encoding proteins involved in the response of adrenal glands to ACTH stimulation were observed. To our knowledge, this is the first study investigating gene expression in adrenal tissues following ACTH treatment in birds. A total of 134 transcripts in adrenal glands revealed to be affected by acute ACTH treatment. As expected, the expression level of several genes encoding steroidogenic enzymes and biogenic amines metabolism were significantly affected by ACTH treatment. However, genes commonly described to respond to ACTH treatment with mammals [36
], such as genes belonging to the P450 family (P450 11A1, P450 11B1, P450 11B2, P450 c17 and P450 c21) or the 3β hydroxysteroid dehydrogenase (3β HSD) family, were not found differentially expressed in response to ACTH treatment in the present study with birds. Our results suggest that another member of cytochrome P450 family (CYP51), and another member of the HSDs family (17 β HSD type 7) appeared to be activated in response to ACTH treatment in the chicken. CYP51 or the lanosterol 14alpha-demethylase, is known to be the only cytochrome P450 (P450) that is widely conserved from prokaryotes to eukaryotes, and is believed to represent the ancestral P450 [39
]. In eukaryotes, CYP51 catalyzes the 14α-demethylation of 14α-methylsterol, which is an essential process in cholesterol biosynthesis [40
]. Up to eight (8) isoforms of 17 β HSDs have been identified, but their roles in steroid metabolism remain unclear [41
]. The regulation of all HSDs is controlled by trophic hormones and the signaling mechanisms involve cAMP-dependent protein kinases and protein kinase C, but the exact mechanisms by which this leads to altered gene transcription have not been clearly elucidated.
Data on key regulatory genes involved in the steroidogenic pathway are lacking in chickens. Most of data available are from studies carried out with mammals. It is hypothesized that gene expression steroidogenic enzymes following ACTH treatment is only significantly modulated during long-term stimulation. There seems to be only limited effect on adrenal P450s and steroidogenic enzymes gene expression in response to acute ACTH treatment [11
]. Based on previous work from our laboratory [35
], the dose of ACTH used in the present study should maximally activate corticosterone production as early as 15 min following ACTH treatment and this high response can last up to one hour post-treatment. Therefore, it is not clear whether the lack of modulation of expression of genes belonging to P450 family (P450 11A1, P450 11B1, P450 11B2, P450 c17 and P450 c21) or the 3β hydroxysteroid dehydrogenase (3β HSD) in the present study is due to the nature of stimulation (acute with long duration) or related to the fact that different genes (such as CYP51 and 17 β HSD type 7) may be responsive in chickens compared to mammals.
In the present study, IPKG was activated by ACTH treatment. IPKG is known to be a potent competitive inhibitor of cAMP-dependent protein kinase activity [44
]. It is generally recognized that in mammals, cAMP dependent transcription is the most important level of regulation of steroidogenic genes [45
]. As such, IPKG could be an important regulator of steroid hormones production in animals under stressful conditions.
Up-regulation of StAR (steroidogenic acute regulatory protein) in treated animals compared to controls was observed in the present study. StAR is involved in cholesterol transport into mitochondria [46
]. The up-regulation of StAR in the present study supports previous studies which indicated that a rapid increase in StAR mRNA levels, and steroid hormone production, is observable in the steroidogenic cells of the adrenal cortex in response to acute ACTH/cAMP stimulation [47
]. An up-regulation of ICER (inducible cAMP early repressor) in response to ACTH treatment was also observed in the present study. ICER is involved in the regulation of the cAMP-dependent transcription of StAR [49
]. However, both StAR and ICER were not found to be differentially expressed between the higher and lower responders to ACTH treatment groups in Experiment 2. However, results from microarray analysis revealed an up-regulation of PRAX-1 (peripheral-type benzodiazepine receptor associated protein 1) in animals with higher adrenal response compared to the lower responders to ACTH treatment. This finding failed to be confirmed by RT-PCR analysis probably because the fold-change was not high enough to be detected. However an up-regulation of PRAX-1 in the treated groups (either with high or low response) compared to the control was observed following RT-PCR analysis. PRAX-1 is involved in steroid hormone synthesis by assuring the transport of cholesterol into the mitochondria [50
]. This suggests that enhanced cholesterol transport into mitochondria might contribute to the higher corticosteroid biosynthesis found in animals with a higher response to ACTH treatment. This finding is consistent with what was observed in pigs in a previous study [11
Several other genes were found in the present work to be affected by ACTH treatment. Among those, Sqle (squalene epoxidase) which is involved in endogenous cellular cholesterol synthesis [51
] or Cry (delta 2 crystallin) and Bmal (Aryl hydrocarbon receptor nuclear translocator-like protein) which are known as peripheral clock genes. Both Cry and Bma1 regulate a large number of genes involved in general cellular processes as well as in pathways related to major organ-specific function, such as, corticosteroid biosynthesis. These genes may be important regulators of adrenal sensitivity to ACTH [52
]. In the case of Sqle and Cry, both were up-regulated but Bmal was down-regulated by ACTH. A large number of other genes encoding for protein kinases, protein phosphatases and trancriptional factors were also found to be differentially expressed in the present study. Their involvements in adrenal steroidogenesis have not been characterized and deserve some attention, in particular C-TSK whose expression level was positively influenced by ACTH treatment even in animals from the group with the low adrenal response to ACTH.