Laser-Captured Single Digoxigenin-Labeled Neurons of Gonadotropin-Releasing Hormone Types Reveal a Novel G Protein-Coupled Receptor (Gpr54) during Maturation in Cichlid Fish

Abstract

GPR54 is a novel G protein-coupled receptor speculated to be essential for sexual development. However, its role in the regulation of GnRH types is unknown. To address this issue, we cloned GPR54 from the brain of a cichlid fish (tilapia Oreochromis niloticus) and determined its expression in immature and mature males using our newly developed technique: laser-captured microdissection of single digoxigenin-labeled GnRH neurons coupled with real-time quantitative PCR. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. Absolute copies of GnRH1 and GnRH3, not GnRH2, mRNAs were significantly high in mature compared with immature males. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P < 0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1, GnRH2, and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain; on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P < 0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence of the structure of a nonmammalian GPR54, which is highly conserved during evolution and is expressed in GnRH1, GnRH2, and GnRH3 neurons. Furthermore, we propose that the expression of GPR54 is a "stop signal" for GnRH1, GnRH2, and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.

Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling

“…If kisspeptin neurons communicate directly with GnRH neurons, one would expect GnRH neurons to express GPR54. Parhar et al (2004) isolated GnRH neurons from the cichlid fish Oreochromis niloticus by laser capture microscopy and identified GPR54 mRNA transcripts in these cells by realtime quantitative RT-PCR. They found that approximately 50% of GnRH neurons in these animals coexpress GPR54.…”

Kisspeptin antagonist prevents RF9-induced reproductive changes in female rats

“…Kisspeptins are termed kisspeptin 10, 13, 14, and K54 according to the number of amino acids in length, and the decapeptide kisspeptin 10, which is shared by all the members of kisspeptin family, is required for the biological activity (Kotani et al 2001). Gonadotropinreleasing hormone (GnRH) neurons have been shown to express GPR54 receptor (Parhar et al 2004), through which kisspeptins activate GnRH secretion (Messager et al 2005). In a recent study (Beale et al 2014), kisspeptin knockdown in the arcuate nucleus (ARC) has been reported to result in a decrease in luteinizing hormone (LH) pulse frequency, which suggests that maintenance of ARC-kisspeptin levels is essential for normal pulsatile LH release and estrous cyclicity.…”

Endocannabinoids and Kisspeptins: Two Modulators in Fight for the Regulation of GnRH Activity

“…In tilapia brain, GPR54 co-localizes with neurons expressing GnRH1, GnRH2 and GnRH3, suggesting an involvement of kisspeptin system in the regulation of GnRH system expression as in mammals (Parhar et al, 2004). Higher levels of GPR54 expression in the brain have been discovered in mature than in immature animals, thus to hypothesize a link between gonadal development and encephalic GPR54 expression (Parhar et al, 2004). Similarly to mammals, kiss1 gene has been identified in two distinct neuronal populations that exhibited a differential response to steroid milieu (Kanda et al, 2008).…”