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Single-Cell Real-Time Quantitative Polymerase Chain Reaction of Immunofluorescently Identified Neurons of Gonadotropin-Releasing Hormone Subtypes in Cichlid Fish

Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Ishwar S. Parhar, Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan. E-mail: ishwar{at}nms.ac.jp.

Abstract

The regulatory mechanisms controlling gene expression of GnRH subtypes, particularly of the evolutionarily conserved GnRH2, remain speculative. To address this issue, we have successfully coupled the anatomic specificity of immunofluorescently defined "cell picking" with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of GnRH mRNAs in individual neurons. Here, using RT-Q-RT-PCR, we report change in the levels of transcripts of GnRH subtypes in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. The levels of GnRH1 mRNA per cell and the percentage of neurons expressing GnRH1 transcripts exceeding 0.05 x 10² fg/cell were significantly higher in mature males (44.2%) compared with immature males (4.7%). In contrast, there was no difference in mRNA levels and the percentage of cells expressing GnRH2 and GnRH3 between the two reproductive states. Thus, using a novel approach that enables immunofluorescently labeled single-cell RT-Q-RT-PCR analysis of GnRH neurons, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Furthermore, we speculate that the use of this method will allow the identification and quantification of known and unknown genes in single GnRH neurons, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells of GnRH variants in vertebrate species.

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