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. 2009 Dec 22;4(12):e8400.
doi: 10.1371/journal.pone.0008400.

Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis

Affiliations

Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis

Takayoshi Ubuka et al. PLoS One. .

Abstract

The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH(2)) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH(2) (human RFRP-1) and VPNLPQRF-NH(2) (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca(2+) mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GnIH immunoreactive neurons in the human hypothalamus and GnIH receptor mRNA in the human pituitary.
(A) Coronal section of adult human hypothalamus showing GnIH immunoreactive (-ir) neurons clustered in the dorsomedial region of the hypothalamus with their fibers extending to the infundibulum (INF). III, third ventricle. Bar, 1 mm. (B) Higher magnification of GnIH-ir neuronal cell bodies. Bar, 50 µm. (C) Sagittal section of the median eminence showing dense population of GnIH-ir fibers within the external layer. Bar, 10 µm. (D) GnIH-ir axon terminal like structures (stained in purple, indicated by black arrows) in close proximity to a GnRH-ir neuronal cell body (stained in brown) in the preoptic area. Bar, 50 µm. (E and F) In situ hybridization of human GnIH receptor (GPR147) mRNA in the human anterior pituitary (E) in combination with immunocytochemistry for luteinizing hormone (LH) (F). Red arrows in (E) and white arrows in (F) in equivalent positions show cells which express both GPR147 mRNA and LH. Bars, 30 µm. (G and H) In situ hybridization using sense RNA probe (G) and immunocytochemistry without LH primary antiserum (H) served as controls. Bars, 30 µm.
Figure 2
Figure 2. Identification of human GnIH homologs.
(A) Amino acid sequence of the human GnIH precursor polypeptide. The arrowhead shows the predicted cleavage site of the N-terminal secretory signal peptide . Two peptide sequences which have LPXRF (X = L or Q) sequence at their C-termini with glycine as an amidation signal and arginine as an endoproteolytic basic amino acid are underlined (RFRP-1, RFRP-3). One peptide sequence which has LPLRS sequence at its C-terminus with glycine and arginine is underlined with a broken line (RFRP-2). The basic amino acids which could be proteolytic cleavage sites are shown in red. (B) Chromatogram of MALDI-TOF MS of the native human RFRP-1. (C) Chromatogram of MALDI-TOF MS of the native human RFRP-3.
Figure 3
Figure 3. Gel image of RT-PCR products of human GnIH receptor (GPR147) mRNA.
cDNAs corresponding to 1 µg total RNA extracted from human hypothalamus or pituitary were used for a PCR reaction. Combinations of forward primers (F1, F2 and F3) with a reverse primer (R) produced RT-PCR products of 479, 415 and 325 bp using hypothalamic and pituitary cDNAs as the template. The PCR products were sequenced and confirmed to be derived from GPR147 mRNA (NM_022146). F1/R and F3/R primer combinations produced additional RT-PCR products in the pituitary although the subsequent DNA sequencing identified that they were derived from Homo sapiens RAB11 family interacting protein 1 and NEDD4 binding protein 1 mRNAs, respectively.

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