Transcriptomic Changes in Broiler Chicken Hypothalamus during Growth and Development

The hypothalamus plays an overarching role that is reflected in the physiological processes observed in the entire organism. The hypothalamus regulates selected metabolic processes and activities of the autonomic nervous system. The avian hypothalamus due to the structural complexity is not well described and has a slightly different function than the mammalian hypothalamus that is the subject of numerous studies. The present study evaluated activities of hypothalamic genes in fast-growing chickens during development (at the 1st day and 3rd and 6th weeks after hatching). The hypothalamic transcriptomes for 3- and 6-week-old cockerels were analysed using an RNA sequencing method in next-generation sequencing (NGS) technology. The differentially expressed gene analysis was conducted using DESeq2 software. In younger 22-day-old cockerels, 389 genes showed higher expression (fold change > 1.5) than that in 45-day-old birds. These genes played a role in several biological processes because they encoded proteins involved in integrin signalling, regulation of hormone levels, camera-type eye development, and blood vessel development. Moreover, surprisingly in the hypothalamus of 3-week-old cockerels, transcripts were identified for proteins involved in both anorexigenic (POMC, NMU) and orexigenic (PMCH, ALDH1A1, LPL, and GHRH) pathways. The RNA-seq results were confirmed by qPCR methods. In summary, the intensive growth of 3-week-old chickens was reflected in hypothalamic activities because the genes associated with the somatotropin axis and regulation of satiety centre showed increased expression.

Identification, Localization, and Function of a Novel Avian Hypothalamic Neuropeptide, 26RFa, and Its Cognate Receptor, G Protein-Coupled Receptor-103

Several neuropeptides with the C-terminal RFamide sequence have been identified in the hypothalamus of a variety of vertebrates. Among the RFamide peptide groups, however, only LPXRFamide peptides, including gonadotropin-inhibitory hormone, have been characterized in the avian brain. In the present study, we sought for the presence of other RFamide peptides in the avian hypothalamus. We identified a cDNA encoding an RFamide peptide orthologous to 26RFa (also referred to as QRFP) in the hypothalamus of the Japanese quail. The deduced quail 26RFa precursor consisted of 120-amino-acid residues, encoding one RFamide peptide with 27 amino acids. This RFamide peptide was flanked at the N terminus by a dibasic amino acid cleavage site and at the C terminus by a glycine amidation signal. Quantitative RT-PCR analysis demonstrated specific expression of quail 26RFa mRNA in the diencephalon including the hypothalamus. Furthermore, mass spectrometry analysis revealed the presence of a peptide exhibiting the mass of mature 26RFa, indicating that the peptide is actually produced from the precursor in the diencephalon. 26RFa-producing cell bodies were localized in the anterior hypothalamic nucleus in the brain. Synthetic 26RFa increased intracellular Ca2+ concentration in HEK293T cells transfected with the chicken G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa in broiler chicks stimulated feeding behavior. These data provide the first evidence for the occurrence of the peptide 26RFa in the avian hypothalamus and indicate that this peptide exerts orexigenic activity.

Cells Expressing RFamide-Related Peptide-1/3, the Mammalian Gonadotropin-Inhibitory Hormone Orthologs, Are Not Hypophysiotropic Neuroendocrine Neurons in the Rat

An RFamide peptide named gonadotropin-inhibitory hormone, which directly inhibits gonadotropin synthesis and secretion from the anterior pituitary gland, has recently been discovered in the avian hypothalamus. It is not known whether the mammalian orthologs of gonadotropin-inhibitory hormone and RFamide-related peptide (RFRP)-1 and -3 act in the same way. We used a newly generated antibody against the rat RFRP precursor combined with retrograde tract tracing to characterize the cell body distribution and fiber projections of RFRP-1 and -3 neurons in rats. RFRP-1/3-immunoreactive cell bodies were found exclusively within the dorsomedial hypothalamus. Immunoreactive fibers were observed in the septal-preoptic area, hypothalamus, midbrain, brainstem, and hippocampus but not in the external zone of the median eminence. Intraperitoneal injection of the retrograde tracer Fluoro-Gold in rats resulted in the labeling of the majority of GnRH neurons but essentially no RFRP-1/3 neurons. In contrast, intracerebral injections of Fluoro-Gold into the rostral preoptic area and CA2/CA3 hippocampus resulted in the labeling of 75 ± 5% and 21 ± 8% of RFRP-1/3 cell bodies, respectively. To assess actions at the pituitary in vivo, RFRP-3 was administered as an iv bolus to ovariectomized rats and plasma LH concentration measured at 0, 2.5, 5, 10, and 30 min. RFRP-3 had no effects on basal secretion, but GnRH-stimulated LH release was reduced by about 25% at 5 min. Together these observations suggest that RFRP-3 is not a hypophysiotropic neuroendocrine hormone in rats. Hypothalamic RFRP-1/3 neurons project to the preoptic area and hippocampus but not the median eminence, suggesting that RFRP-1/3 is not a hypophysiotropic gonadotropin inhibitor in the rat.