Recent studies indicate that almost all cells contain molecular clocks that can be synchronized to master clocks residing in the SCN, such that circadian signals can be effectively distributed to multiple tissues (Balsalobre, 2002
). Our results demonstrate that GnRH neurons are no exception in this regard, and that circadian clock genes and protein products oscillate in cultured GT1–7 cells after exposure to various stimuli, suggesting a functionality that may impact reproductive rhythms. In addition, our data show that the molecular clock may be coupled to the fundamental mechanism of pulsatile GnRH secretion, because perturbation of normal clock oscillation by transfection of Clock
-Δ19 disrupts pulsatile patterns of GnRH secretion.
Because Clock-Δ19 binds in a heterodimeric manner to Bmal1 and will also bind DNA but not activate mPer
transcription (Gekakis et al., 1998
; Jung et al., 2003
), transient overexpression of this dominant-negative protein decreases transcription of Per
, resulting in a subsequent blunting of clock gene oscillations. Results from the above experiments suggest that cycling levels of Per
in GnRH neurons are required for normally observed patterns of GnRH release. Prevention of these oscillations by expression of Clock
-Δ19 leads to an apparent disorganization of GnRH secretion, dramatically slowing pulse frequency and increasing pulse amplitude variability. It is of considerable interest that GnRH pulse patterns were disrupted, although only a percentage of GT1–7 cells expressed Clock
-Δ19, suggesting that synchronous secretion of GnRH relies on homogeneity of gene expression within these neurons. The pattern of circadian gene expression in GnRH neurons in vivo
has not yet been explored; thus, it remains unknown whether stimuli sufficient to reset the clock in these cells is also responsible for maintenance of secretory pulsatility. In fibroblasts and neuronal cell lines, including the GT1–7 cells, a media change is enough to elicit gene expression cycling, suggesting that shifting cultured GT1–7 cells into KRB perifusion buffer may act to not only reset the clock but also to synchronize pulsatile GnRH secretion.
Interestingly, augmentation of the “negative limb” of the clock (i.e., overexpression of mCry
1) significantly increases GnRH pulse amplitude, suggesting that multiple secretory mechanisms may be influenced by intrinsic clock function. The observed increase in GnRH pulse amplitude suggests that the Cry proteins, in addition to their characterized role in the core clock loop, may also interact as promoters of genes required for modulation of neurosecretion. Consistent with this observation, the disruptive effect of Clock
-Δ19 may act by inhibiting transcription of Cry
. Increases in GnRH pulse amplitude resulting from overexpression of mCry
1 suggest that this central clock component is linked to the GnRH secretory machinery in a way that selectively amplifies secreted GnRH. Cry1 and Cry2 are potent inhibitors of Clock/Bmal1-mediated transactivation (Kume et al., 1999
), implying that a constituitive inhibition on GnRH release mediated by the positive limb of the clock may exist. These data also raise the interesting possibility that basal GnRH secretion may be relatively dynamic in vivo
, rising slightly in the afternoon or evening in conjunction with peak Cry1 protein levels, consistent with the observation of diurnal changes in LH secretion in vivo
(Sisk et al., 2001
). The presence of an intrinsic circadian clock within GnRH neurons could thus provide insight into mechanisms mediating the observed circadian window of the LH and precedent GnRH surges. However, at what level the circadian clock exerts control over the secretory process remains unclear, and it is indeed possible that the clock may modulate multiple regulatory pathways at the level of GnRH transcription or by mediating neurosecretion at the cell membrane.
Circadian clock proteins have been shown to regulate transcription of other cycling genes often referred to as “clock-controlled genes” in a circadian manner (Jin et al., 1999
). Previous studies demonstrate that Clock/Bmal1 heterodimers bind to E-box motifs on proximal promoter sites of target genes to activate transcription (Gekakis et al., 1998
; Jung et al., 2003
). Increases in transcription are subsequently inhibited by “negative limb” clock components such as the Period and Cryptochrome proteins that complex with Clock/Bmal1 to prevent transcriptional stimulation. The rat and mouse GnRH proximal promoter regions contain a few consensus E-boxes (CANNTG) but no canonical sites (CACGTGA) found in the promoters of many cycling genes (Panda et al., 2002
). In vivo
studies in both male and female rats have shown that levels of GnRH mRNA, but not primary transcript, exhibit circadian oscillations (Gore, 1998
), suggesting a possible post-transcriptional regulation of GnRH content by the circadian clock. Future studies in GT1–7 cells should provide insight into whether this observed in vivo
rhythm may be mediated by an endogenous circadian oscillator within GnRH neurons.
Alternately, perturbations of clock cycling could affect multiple regulatory pathways affecting secretion, such that GnRH transcription may not be directly influenced. Microarray studies performed on mRNA from clock/clock
mutant mice demonstrate that, whereas most cycling gene expression patterns are disrupted in these animals, many noncycling genes are also either dramatically upregulated or downregulated by this mutation (Panda et al., 2002
). Thus, expression of the dominant-negative Clock
gene may alter GnRH secretion patterns via changes in membrane protein levels or at the level of GnRH transcription or post-transcriptional processing in addition to alterations of the core clock mechanism.
In support of this, GnRH secretion patterns, although undoubtedly influenced by the availability of processed decapeptide, are not likely mediated by transcriptional changes alone. A recent study of perifused GT1–1 cells treated with transcriptional and translational inhibitors suggests that rapid transcriptional and translational cycles do not underlie GnRH pulsatility (Pitts et al., 2001
). However, perifusion after inhibitor treatment by necessity only lasted for 2 hr; thus it remains unknown in this context whether long-term inhibition of the circadian clock transcriptional loop by blockade of protein synthesis would ultimately be reflected in GnRH pulse release. The current study used perifusion periods of up to 24 hr and collection periods of 10 hr, with clock gene overexpression revealing striking differences in GnRH pulse parameters.
Many studies have focused on the unique electrophysiological properties of GnRH neurons that may be required to elicit synchronized release patterns. Recent work investigating activity rhythms of GnRH neurons in slice preparations revealed a complex pattern of rapid conductance oscillations specific to GnRH neurons (Nunemaker et al., 2001
), suggesting that these particular membrane properties may govern episodic release patterns. In a recent study, the same experimenters observed multiple activity rhythms in GnRH neurons spanning milliseconds to minutes and demonstrated that only certain rhythmic parameters may be affected by exogenous signals (Nunemaker et al., 2003
). Thus, there may exist convergent neurosecretory mechanisms that are coupled at various cellular context-dependent strengths to circadian, infradian, and ultradian generators within the same neuron or population of neurons, such that disruption of one of these could lead to arrhythmicity in multiple time domains.
Neuronal firing patterns also play important roles in generating synchronized output rhythms in the SCN. Recent studies in Drosophila
demonstrated that functioning ion channels are required for oscillation of the molecular circadian clock (Nitabach et al., 2002
), suggesting a fundamental interaction between membrane electrical activity and clock oscillation. GnRH neuronal activity and secretion are also intimately linked, because rapid voltage-sensitive Ca2+
oscillations and K+
-mediated action potentials are required for normal pulse release patterns (Charles and Hales, 1995
; Costantin and Charles, 1999
). Primary neuronal SCN cultures from wild-type mice, grown on micro-multielectrode plates, reveal distinct circadian activity patterns, whereas SCN neurons from clock/clock
mice exhibit arrhythmic firing patterns, suggesting that the transcriptional clock is linked to control of neuronal membrane potential (Herzog et al., 1998
). We recreated this mutation effect specifically in GT1–7 cells in culture via Clock
-Δ19 overexpression, raising the possibility that GnRH secretory machinery may similarly be affected by this dominant-negative Clock protein, such that a normal complement of membrane channels, or modification thereof, is absent, thereby perturbing typical release patterns.
The influence of the circadian clock on the reproductive axis can also be observed in mutant mice. Estrous cycle analysis of clock/clock
mutant mice provides insight into their subfertile condition. The presence of persistent estrus smears lasting up to four times longer than typically observed suggests that these animals do not immediately resume cycling after ovulation. The somatic Clock
gene mutation likely affects multiple points within the reproductive axis, including a possible disruption of GnRH secretion. Previous rodent work demonstrated that LH secretion, clearly pulsatile in metestrus and diestrus and surging on proestrus, becomes quiescent on estrus, only to resume pulsatile release again on diestrus (Fox and Smith, 1985
). Observation of clock/clock
mice, in conjunction with the above results from GT1–7 cells, suggests that a functional circadian clock may be required for the resumption of synchronous GnRH secretion after the GnRH surge. Whether this could result from a lack of synchronized activity in the SCN (thus perturbing subsequent output from) or in GnRH neurons themselves is unclear and will require additional studies to determine the extent of the reproductive deficiency of these mice. A previous study revealed a significant decrease in both arginine vasopressin and vasoactive intestinal peptide within the SCN of clock/clock
mice throughout postnatal development (Herzog et al., 2000
). Whether this decrement alters GnRH expression or secretion in vivo
is currently unknown. It is of interest that, in contrast to locomotor activity, reproductive abnormalities in clock/clock
mutant mice persist in both LD and DD conditions, indicating that endogenous circadian clock disruption in GnRH neurons themselves may produce this observed reproductive deficiency.
Although it remains unclear what effect resetting of the circadian clock may have on fibroblasts, the GT1–7 cell line provides a neuroendocrine model that produces a distinctly timed output, a secretory pattern that can be modulated by both neuronal and hormonal signals. Indeed, activity of GnRH neuronal perikarya, not confined to discrete nuclei, may be modulated in a synchronous manner by humoral signals. A recent study using SCN2.2 cells, an immortalized line derived from rat SCN, demonstrates that circadian rhythms of gene expression and glucose utilization in NIH3T3 fibroblasts can be driven by the SCN 2.2 cells in the absence of synaptic contacts (Allen et al., 2001
). These results demonstrate that an uncharacterized humoral factor secreted by the SCN can synchronize other cell types in a paracrine manner. Also, hormonal stimuli shown to modulate the reproductive axis such as retinoic acid (Cho et al., 1998
) and glucocorticoids (De-Franco et al., 1994
) are also capable of exerting effects on the phase and amplitude of the circadian clock (Balsalobre et al., 2000a
; McNamara et al., 2001
). In contrast to fibroblast lines, GT1 cell lines provide a valuable model in which a rhythmic neurosecretory output, albeit ultradian, can be measured after molecular alterations of a circadian transcriptional loop.
In sum, the results of the current study demonstrate that an endogenous circadian clock plays a role in regulating parameters of GnRH pulsatility. Although future studies are required to determine the extent and mode of this regulation, these data provide insight into the fundamental mechanisms underlying GnRH pulsatility, as well as presenting a direct influence of the circadian clock on primary neuronal components of the reproductive axis.