The present findings suggest that immortalized GnRH cells exhibit intrinsic daily changes in sensitivity to neurochemicals stimulating their activity (fig. , ). Additionally, these studies have uncovered at least two alternatively spliced isoforms of GPR54 in GT1–7 cells (fig. ), a finding further confirmed in mouse whole-brain cDNA. Other groups have recently reported one isoform of GPR54 in GT1–7 cells [63
]. In contrast to the present work, the primers used in these studies do not cover the alternative splicing region of the gene. Administration of kisspeptin promotes GnRH release in a dose-dependent manner with a saturable concentration in GT1–7 cells (fig. ). Previous reports demonstrated dose-dependent effects of VIP stimulation on GnRH release in this same cell population [40
], and we have replicated and extended these earlier findings, showing daily changes in sensitivity to this peptide. Previous studies in perifused GT1–7 cells suggested that rapid transcriptional and translational regulation is not involved in GnRH pulsatility by applying transcriptional and translational inhibitors to these cells [65
]. In this study, we investigated GnRH production at the transcriptional level, translational level and secretion level. For both kisspeptin and VIP, no significant differences in GnRH mRNA were detected throughout the day (fig. ), implying that rapid GnRH transcriptional regulation does not occur in response to peptide stimulation. In contrast, time-dependent responses to kisspeptin and VIP at the translational/post-translational and secretory levels were observed. Together, these findings indicate that the GT1–7 cells, and perhaps the GnRH system in vivo, exhibit an endogenous time-dependent sensitivity to neurochemical stimulation. However, to confirm a rhythm in sensitivity to GnRH secretagogues, future investigations examining this system across several daily cycles are necessary.
The model system used in the present series of studies allows the investigation of the reproductive neuroendocrine axis uncoupled from the master pacemaker in the SCN. Recent studies suggest that the coordination of subordinate clocks is central for normal physiology [66
]. As described previously, the peripheral time-keeping system is subordinate to the SCN and requires input from the master clock for its maintenance [18
]. Studies using food restriction indicate that peripheral oscillators can become uncoupled from central oscillators when hormonal signals conflict with SCN output [70
]. Under normal circumstances, individual cellular clocks from a given system are synchronized to each other, but this synchrony is lost in peripheral clock cell populations in culture when removed from SCN input [18
]. In vitro, Chappell et al. [58
] found that clock genes in GT1–7 cells oscillate with a circadian period following serum shock, and pulsatile GnRH secretion appears to be regulated by these oscillations. The Period-2 and BMAL-1
mRNA patterns in the present studies are consistent with this original report. Additional evidence for a circadian mechanism in GT1–7 cells comes from studies in which melatonin treatment leads to increases in cellular melatonin receptor expression, but only at distinct circadian periods [72
]. Likewise, the ability of melatonin to inhibit GnRH secretion varies with a circadian period when examined over 48 h in this immortalized population [73
]. Our results demonstrate a daily change in sensitivity of GT1–7 cells to kisspeptin and VIP treatment, suggesting the possibility that the intrinsic circadian time-keeping apparatus in extra-SCN cells provides a mechanism for altering local responsiveness to upstream neuromodulators. Future studies using molecular approaches to disrupt the cellular clock are necessary to determine whether these daily changes in sensitivity are mediated by local oscillators in this cell population.
The SCN neuropeptide VIP is a key regulator of the ovulatory cycle in rodents. Administration of VIP antiserum [34
] or antisense oligodeoxynucleotides [42
] alters the time course and reduces the magnitude of the LH and prolactin surges on the afternoon of proestrus. The present findings indicate that the effects of VIP on the GnRH system depend on the timing of administration. Similar findings have been reported for vasopressinin vivo; vasopressin administration can stimulate the LH surge [74
], and its effects have been shown to depend on timing of administration [75
]. Whether vasopressin acts directly on GnRH cells or via an intermediate system remains unclear. One recent study has demonstrated the innervation of AVPV kisspeptin neurons by vasopressin immunoreactive fibers [76
], suggesting that this kisspeptin pathway may provide an indirect route between the SCN and the HPG axis [47
]. A vasopressinergic pathway from the SCN to AVPV kisspeptin cells would allow circadian control of this potent stimulator of reproductive axis function.
Several lines of evidence support a role for AVPV kisspeptin as an integration point for circadian and steroidal signals necessary for initiation of the LH surge. First, a large proportion of kisspeptin cells express estrogen receptor-α, the receptor subtype important for the positive feedback effects of this sex steroid during the surge [45
]. Additionally, kisspeptin mRNA in the AVPV is increased in a timed manner, with maximum expression on the afternoon of proestrus [50
]. Importantly, this expression pattern is dependent on the presence of high concentrations of estrogen [50
]. Likewise, kisspeptin neurons exhibit an increase in FOS expression concomitant with the LH surge [50
]. Infusion of an anti-kisspeptin antiserum into the pre-optic area of rats blocks the LH surge [83
]. In the ewe, kisspeptin can alter the timing of the LH surge and stimulate ovulation in anestrous animals [84
]. In anestrous female Siberian hamsters housed in short days, kisspeptin treatment does not lead to an increase in LH, indicating an alteration in GnRH cell sensitivity to this peptide as a potential additional mechanism of ovulatory inhibition during winter [85
In rodents whose ovulatory cycle is tied to the circadian system, this mechanism ensures that ovulation is coordinated with female sexual motivation and the time of day during which males are seeking a mate. Additionally, estrogen-dependence of this timing mechanism safeguards against the preovulatory LH surge occurring before the oocyte is fully mature. Together with these previous findings, the present data showing daily changes in GT1–7 cell sensitivity to kisspeptin stimulation suggest that local time-dependent sensitivity of GnRH neurons might be an important additional component of the SCN-HPG and SCN-AVPV-HPG circuits required for proper endocrine timing. Because the present study did not specifically investigate daily changes in the ability of GT1–7 cells to produce GnRH, it is possible that such modifications contribute to observed patterns of expression. Future studies in which GT1–7 cells are stimulated with a neurochemical that consistently causes GnRH release are necessary to examine this possibility.
The present results suggest a mechanism of HPG axis control in which the gating of stimulated GnRH secretion is coordinated locally within the GnRH neuronal network. Whether or not circadian clock genes participate in this rhythm in sensitivity represents an exciting avenue for further investigation. As indicated previously, core circadian clock genes are expressed in neurosecretory cells [87
], the pituitary gland [21
], and other peripheral endocrine glands [23
], suggesting that this mechanism of local temporal control may operate at multiple levels of the HPG axis providing opportunity to explore this novel regulatory system.