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

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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.

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