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. 2003 Dec 3;23(35):11202-13.
doi: 10.1523/JNEUROSCI.23-35-11202.2003.

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line

Patrick E Chappell  1 , Rachel S White, Pamela L Mellon
Affiliations

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line

Patrick E Chappell et al. J Neurosci. .

Abstract

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

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Figures

Figure 1.
Figure 1.
Multiple core circadian clock genes are expressed in cultured GT1-7 cells. RT-PCR bands (arrows) corresponding to murine circadian clock genes Bmal1, Clock, mCry1, mCry2, mPer1, mPer2, mPer3, and CKIϵ are present in tissue preparations of pituitary and total brain, as well as in cultured GT1-7 cells. No RT (far right lane) served as a negative control. A 100 bp ladder (Promega, Madison, WI) is shown in the far left lane.
Figure 2.
Figure 2.
Multiple stimuli induce transient increases in expression and oscillation of circadian clock gene expression in cultured GT1-7 cells, as measured by RNase protection assay. A, Circadian gene expression of mPer1 and mPer2 oscillates in GT1-7 cells after a shift to SF media. A, B, Fluctuations in mRNA band intensity are presented as percentage change from 0 hr mPer1/cyc or mPer2/cyc optical density (OD) ratios. Traces indicate the mean of three RNA collections per time point. B, mPer1 and mPer2 expression oscillates after a 15 min stimulation by 10.0 μm FSK, followed by a shift to SF for 60 hr. GT1-7 cells remaining in 10% FBS media (no perturbation) demonstrated no significant mRNA oscillations. C, Representative RNase protection assay of mPer1 and cyclophilin mRNA level after 1 hr of exposure of GT1-7 cells to media containing 10.0 μm FSK (15 min exposure; lane 2), 50% horse serum (lane 3), 1.0 μm Ionomycin (lane 4), 100.0 nm PMA (lane 5), and 100.0 μm SNP (lane 6). The bar graph on the right shows mean changes in mPer1 expression at 1 hr after indicated treatments. Each bar represents the mean of four experiments (*p < 0.01, increase above 0 hr level; #p < 0.05, decrease below 0 hr level).
Figure 3.
Figure 3.
Per1 and Bmal1 proteins exhibit circadian oscillations in cultured GT1-7 cells. A, Protein levels of Bmal1 oscillate in GT1-7 cells after a serum shock. A representative Western blot (top) reveals an ∼60 kDa band present in GT1-7 nuclear extracts exhibiting peak levels at 24 and 48 hr. The corresponding graph shows mean protein levels at each time point (n = 3). B, Oscillation of mPer1 protein over 48 hr exhibits a peak at 12-18 hr after serum shock, 6-12 hr out of phase with the Bmal1 protein rhythm (*p < 0.05; significantly different from nadir as determined by ANOVA).
Figure 4.
Figure 4.
The circadian clock maintains transcriptional regulatory relationships in the GT1-7 cell line. A, B, Oscillation of mPer1-luciferase activity in GT1-7 cells under both SF (A) and serum-shock (B) conditions. Filled circles indicate cells cotransfected with control pcDNA3.1 plasmid. Open circles represent cells cotransfected with a Clock-Δ19 expression plasmid. Filled squares represent cells cotransfected with an mCry1 expression plasmid. Values are presented as percentage changes from 0 hr normalized luciferase/β-galactosidase ratios of pcDNA3.1-transfected cells. Two hr serum-shock in B is indicated by the gray area.
Figure 5.
Figure 5.
GFP fluorescence reveals robust transgene expression in GT1-7 cells grown on beads after 48 hr of perifusion. A, Bright-field micrograph of a cluster of GT1-7 cells on Cytodex beads after 24 hr static incubation in DMEM/10% FCS followed by 48 hr of perifusion with KRB. Clusters of multiple cells are visible, adhering multiple beads together. B, Flourescence imaging reveals robust GFP expression in the same cell cluster 72 hr after transfection and perifusion. Scale bar, 100 μm. Ranges of transfection efficiencies for rat GnRH-eGFP and CMV-eGFP are ∼18-36 and ∼25-60%, respectively.
Figure 6.
Figure 6.
Perturbation of the circadian clock by expression of Clock-Δ19 disrupts patterns of GnRH pulse release from perifused GT1-7 cells. A-C, Representative GnRH pulse release profiles from perifused GT1-7 cells that were untransfected (A), transfected with the control vector pcDNA3.1 (B), or transfected with Clock-Δ19 (C). Significant pulses are determined by CLUSTER and PeakFit pulse analysis programs and are indicated by asterisks. Depolarization is indicated by the gray bars.
Figure 7.
Figure 7.
Expression of Clock-Δ19 results in alteration of GnRH pulsatile secretory patterns. A, Representative 5 min GnRH pulse profiles of perifused pcDNA3.1-transfected (top) and Clock-Δ19-transfected (bottom) GT1-7 cells. B, Mean pulse frequency of all sample periods was significantly (*p < 0.01) reduced in perifused Clock-Δ19-transfected cells. C, Representative 1 min GnRH pulse profiles of pcDNA3.1-transfected cells and Clock-Δ19-transfected GT1-7 cells. D, Mean pulse amplitude of Clock-Δ19-transfected cells, although highly variable, was not significantly different from control-transfected cells. Mean pulse amplitude variability, calculated as mean variation from normalized mean pulse amplitude within each 10 hr sampling period, is shown in E and was significantly (*p < 0.01) different between treatment groups. The bar graph in F indicates cumulative GnRH released over the 10 hr sampling period. Although control cells exhibited a stable baseline of release (top), Clock-Δ19-transfected cells displayed high-amplitude bursts of secretion, followed by a return to baseline. Bar graphs in B, D, E, and F represent mean pulse frequency, pulse amplitude, amplitude variability, and cumulative GnRH secretion, respectively, with n = 8 in each experimental and control group, and were derived from both 5 and 1 min sampling data.
Figure 8.
Figure 8.
Overexpression of mCry1 in GT1-7 cells results in a significant increase in mean GnRH pulse amplitude. A, Representative GnRH pulse profiles of perifused GT1-7 cells transiently tranfected with a pcDNA3.1 (top) or a Cry1 expression vector (bottom). B, Mean pulse amplitude of Cry1-transfected GT1-7 cells is significantly (*p < 0.01; n = 6) greater than that of control (pcDNA3.1)- transfected cells (left), whereas pulse frequency is not different between Cry1-transfected or pcDNA-transfected cells (right). Gray bars indicate depolarization.
Figure 9.
Figure 9.
Clock/clock mutant mice exhibit prolonged estrous cycles. A, Representative estrous cycles as measured by vaginal cytology from 2- to 5-month-old female wild-type (top), clock/+ (middle), and clock/clock (bottom) mice. D1, Diestrus-I phase; D2, diestrus-II; P, proestrus; E, estrus. B, Time spent in each phase of the estrous cycle by genotype, represented as percentage of total time. Clock/clock mice spend a significantly greater (*p < 0.05; one-way ANOVA) amount of time in estrus than do the other genotypes. Data shown represent mice housed in 12 hr LD conditions (n = 6 per genotype).
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