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Departments of Physiology (J.M.A.G., B.P.K.C., F.C., D.D.B.), Institute of Medical Science (D.R., D.D.B.), Obstetrics and Gynecology (D.D.B.), and Medicine (D.D.B.), University of Toronto, and Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network (D.D.B.), Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca.
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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