Molecular and Cellular Endocrinology 307 (2009) 68–76 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Expression of the GABA A receptor associated protein Gec1 is circadian and dependent upon the cellular clock machinery in GnRH secreting GnV-3 cells Virginie Mansuy a , Pierre-Yves Risold c , Micheline Glauser a , Annick Fraichard c , Franc ¸ ois P. Pralong a,b,∗ a Services of Endocrinology, Diabetology, and Metabolism of the University Hospital of Lausanne, Switzerland b Services of Endocrinology, Diabetology, and Metabolism of the University Hospital of Geneva, Switzerland c Equipe Estrogenes, Expression Génique et Pathologies du Système Nerveux Central, Universite de Franche-Comte, U.F.R. Sciences et Techniques, Besancon, France article info Article history: Received 3 September 2008 Received in revised form 25 February 2009 Accepted 25 February 2009 Keywords: GnRH Hypothalamus Clock gec1 GABA A receptor abstract The timely regulation of gonadotropin-releasing hormone (GnRH) secretion requires a GABAergic signal. We hypothesized that GEC1, a protein promoting the transport of GABA A receptors, could represent a circadian effector in GnRH neurons. First, we demonstrated that gec1 is co-expressed with the GABA A receptor in hypothalamic rat GnRH neurons. We also confirmed that the clock genes per1, cry1 and bmal1 are expressed and oscillate in GnRH secreting GnV-3 cells. Then we could show that gec1 is expressed in GnV-3 cells, and oscillates in a manner temporally related to the oscillations of the clock transcription factors. Furthermore, we could demonstrate that these oscillations depend upon Per1 expression. Finally, we observed that GABA A receptor levels at the GnV-3 cell membrane are timely modulated following serum shock. Together, these data demonstrate that gec1 expression is dependent upon the circadian clock machinery in GnRH-expressing neurons, and suggest for the first time that the level of GABA A receptor at the cell membrane may be under timely regulation. Overall, they provide a potential mechanism for the circadian regulation of GnRH secretion by GABA, and may also be relevant to the general understanding of circadian rhythms. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The suprachiasmatic nucleus (SCN) is considered as the “clock regulator”. It is directly or indirectly controlling endocrine as well as non-endocrine organs, facilitating the entrainment of physiological systems to the external photoperiod and maintaining synchronic- ity between organs (Schibler, 2006; Reppert and Weaver, 2002). The circadian biological clock modulates the reproductive axis, partic- ularly for the generation of gonadotropin surges, and projections from the SCN to the magnocellular preoptic nucleus provide control over the timing of the release of gonadotrophin-releasing hormone (GnRH) (Boden and Kennaway, 2006; Kennaway, 2005). Pulsatile GnRH secretion is an intrinsic property of GnRH neu- rons: immortalized clones of GnRH (Chappell et al., 2003) as well as explants of embryonic olfactory placode exhibit a pulsatile pattern of GnRH release, demonstrating that GnRH neurons do not require exogenous stimuli to secrete GnRH in pulses (Funabashi et al., 2000; Duittoz and Batailler, 2000). Nevertheless, ablation of the SCN pre- vents the occurrence of luteinizing hormone (LH) surges, resulting in oestrous acyclicity and infertility (Miller et al., 2004; Kalsbeek ∗ Corresponding author at: Service of Endocrinology, BH 10-563, University Hos- pital, 1011 Lausanne, Switzerland. Tel.: +41 21 314 0596; fax: +41 21 314 0597. E-mail address: Francois.Pralong@chuv.ch (F.P. Pralong). et al., 1993). In addition, the LH surge stimulated by 17-estradiol replacement in ovariectomized rodents is temporally confined to the late afternoon and early evening of the following day (Everett and Sawyer, 1950; Legan et al., 1975). These observations suggest that a “pulse generator” synchronizes the activity of GnRH neu- rons to produce a meaningful secretory output, but the mechanisms implicated remain unclear. Because this episodic secretion pattern is species- and cell-specific, a developmentally regulated molecu- lar program is likely responsible for the fundamental machinery of this programmed neuroendocrine pulse generation. In the SCN, rhythmicity is generated by a series of interlocking positive and negative feedback gene transcription and translation loops. The primary positive arm of the rhythmicity is generated by two transcription factors, CLOCK (circadian locomotor output cycle kaput) and BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3). After transcription and translation, the CLOCK and BMAL1 proteins hetero-dimerize to bind to a specific enhancer region (E-Box, CACGTG) in the promoters of the period genes per1 and per2 and of the cryptochrome genes cry1 and cry2 (Reppert and Weaver, 2002). The existence of a functional clock has previously been described in GT1-7 immortalized GnRH neurons (Chappell et al., 2003; Gillespie et al., 2003), a foetal mouse cell line obtained by targeted tumorigenesis, in which constitutive expression of the SV40 onco- gene is driven by the 5 ′ regulatory region of GnRH. Transfection of 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.02.029