Circadian Clock Gene Involvement in the Regulation of the Reproductive Neuroendocrine Axis

The key to species survival depends on the ability to reproduce. In mammals ovulation requires a precisely timed luteinizing hormone (LH)-surge on the afternoon of proestrus. It is thought that this LH-surge requires the convergence of two signals one being a hormonal signal (i.e. the positive feedback of estrogen) and the other being a neural signal (i.e. a circadian signal). The circadian clock is known to be involved in regulating numerous biological functions, such as sleep/wake cycles, food intake, body temperature, hormonal regulation, etc. It does this through a series of transcriptional/translational feedback loops involving various clock genes (i.e. Clock, Bmal1, Per1, Per2, Cry1, Cry2, reverbα, etc.). CLOCK and BMAL1 bind to noncanonical E-boxes in the promoters of clock driven genes, which activates their own transcription and subsequent translation. PER and CRY are then translocated back into the nucleus to inhibit their own transcription and that of other E-box driven genes. There have been various published reports on clock mutant animals having impaired fecundity. Mammalian reproduction occurs through the hypothalamic-pituitary-gonadal (HPG) axis where GnRH (gonadotropin-releasing hormone) neurons, located in the hypothalamus, release GnRH in a pulsatile fashion. GnRH acts on the GnRH receptor (GnRHR) on the surface of anterior pituitary cells called gonadotropes. The GnRHR triggers the synthesis and release of LH and FSH, which act on the ovaries to produce estrogen and progesterone. Estrogen then feeds back on the hypothalamus and pituitary gland. In the present studies whole rat pituitaries were used to assess clock gene and protein expression at diestrus a.m./p.m. and proestrus a.m./p.m. It was found that mPer1 increased during the evening of both days, but peaked on the evening of proestrus. It was surprising that PER1 protein was also high during the morning of proestrus, which was not seen in mPer1 mRNA. Since LH is synthesized and secreted only in gonadotropes, which make up 3-5% of the entire pituitary, enriched primary gonadotropes were used to study clock gene expression. Using enriched primary gonadotropes it was seen that mPer1 is indeed expressed in these cells in a rhythmic fashion and also that GnRH induces mPer1 as well as LHβ. Because GnRH induces both mPer1 and LHβ, experiments were performed to determine whether the signaling pathway involved in the GnRH induction of mPer1 correlates to that of the GnRH induction of LHβ. Using various pharmacological approaches it was demonstrated that GnRH induces mPer1 and LHβ through the PKC/MAPK pathways. It has also been shown that the GnRH induction of LHβ requires the early growth response protein 1 (Egr-1). To ascertain the importance of Egr-1 in mPer1 expression, analysis of the mPer1 promoter was performed and an Egr-1 binding site was found. Using various techniques it was determined that Egr-1 is involved in the GnRH induction of mPer1 expression. GnRHR expression fluctuates in accordance with the frequency of GnRH pulses. Little is known about how this change in GnRHR expression occurs. E-boxes are required for circadian regulation of a gene. Through examination of the GnRHR promoter, seven noncanonical E-boxes were found proximal to the transcriptional start site (tss). Analysis of the putative clock gene regulation of the GnRHR was performed and it was found that clock genes are indeed important factors in controlling GnRHR expression. To reach the critical LH-surge, LHβ must be synthesized and released from the gonadotropes. To further the understanding of clock gene involvement in the HPG axis, the LHβ promoter was also examined and six noncanonical E-boxes were found proximal to the tss. Chromatin immunoprecipitation was performed, and it was seen that CLOCK and BMAL1 bind to these E-boxes in the LHβ promoter. To determine whether these elements play a role in the regulation of the LHβ each E-box was mutated to a nonsensical sequence using site-directed mutagenesis. The impact on LHβ expression was analyzed via luciferase reporter activity. Through these techniques it was established that clock genes play an important role in regulating LHβ. These data demonstrate a complex variety of ways by which the circadian clock is involved in the regulation of the reproductive neuroendocrine axis.