The frequency and amplitude of GnRH pulses are tightly regulated for the maintenance of reproductive cycles. Pulsatile GnRH release was shown to be an intrinsic property of GT1 GnRH neurons and primate, placodal GnRH neurons. Based on findings in GT1 cells, we hypothesize that the cAMP signaling pathway participates in the stimulation of GnRH secretion and the timing of GnRH pulses. Our findings suggest that increases in cAMP stimulate GnRH secretion by opening cAMP-gated cation (CNG) channels leading to increased excitability and depolarization of the neuron. We will study the role of the CNG channels in GT1-1 neurons and in GnRH neurons in transgenic rats by expressing a dominant/negative mutant of the CNG channel (mCNG) that will be developed by site directed mutagenesis of the cAMP binding domains. Expression of the mCNG channel should block the stimulation of GnRH by elevations in cAMP, and result in decreased amplitude and frequency of GnRH pulses. In stably transfected GT1-1 cells the effect of expression of mCNG channel will be studied on production of cAMP, and the frequency and amplitude of GnRH pulses. The physiological role of the cAMP pathway will be tested in transgenic rats. Expression of the mCNG channel will be targeted to GnRH neurons in transgenic rats using promoter/enhancer regions of the GnRH gene. Effects on the ovulatory surge and pulsatile LH release in castrated rats will be determined. Understanding the molecular basis of the timing mechanisms for the pulsatile release of GnRH should give us new insights into the control of fertility. Potentially new pharmacological strategies can be formulated to both increase and decrease fertility by regulating the biological clock timing the pulsatile release of GnRH.
