Expression of Circadian Rhythm Genes in Gonadotropin-Releasing Hormone-Secreting GT1-7 Neurons

Sep 10, 2003Endocrinology

Daily Rhythm Gene Activity in Hormone-Releasing Brain Cells

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Abstract

The GT1-7 cell line expresses circadian rhythm genes with a rhythmic pattern over 54 hours.

Circadian rhythm genes, including clock, BMAL1, timeless, period1, period2, cryptochrome1, and cryptochrome2, are expressed in the GT1-7 cell line.
BMAL1, period1, period2, and GnRH mRNAs exhibit a circadian-like rhythm after synchronization.
CLOCK, BMAL1, and PERIOD1 proteins are present in GT1-7 cells, with BMAL1 showing a rhythmic expression pattern.
Melatonin receptors, mt1 and MT2, also display circadian expression patterns in these cells.
Melatonin treatment down-regulates the expression of mt1 and MT2 receptors.

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The center for circadian rhythms in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus, composed of single cell circadian oscillators driven by a transcriptional/translational feedback loop where clock proteins drive clock gene expression. These genes are expressed in peripheral tissues and several brain areas outside the SCN. It is likely that some peripheral oscillators are synchronized by the SCN. The pineal hormone melatonin plays an important role in the entrainment of circadian rhythms through feedback to the SCN. Melatonin also plays a role in reproduction, including direct effects on GnRH-secreting GT1-7 neurons. The intrinsic rhythmicity of GnRH neurons suggests that these neurons may express the components of the circadian oscillator. Using the GT1-7 cell line, we demonstrate expression of the circadian rhythm genes, clock, BMAL1,timeless (tim), period1,period2, cryptochrome1, andcryptochrome2. Furthermore, semiquantitative RT-PCR demonstrates that BMAL1, period1, andperiod2 as well as GnRH mRNAs are expressed with a circadian-like rhythm after synchronization over 54 h. With available antibodies, we demonstrated CLOCK, BMAL1, and PERIOD1 protein expression in these cells, with BMAL1 protein levels showing a rhythmic expression pattern. In addition, receptors for melatonin, mt1 and MT2, also show a circadian expression pattern in the GT1-7 cells, and their expression is down-regulated by melatonin treatment. These findings suggest that the components of the clock machinery in mammals may play a role in GnRH neuronal function.

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