Overall our results indicated the involvement of

Overall, our results indicated the involvement of GABAAR in controlling GnRH/LH release through mechanism which included GABA action on molecular processes leading to GnRH biosynthesis. This is consistent with previous in vivo [14,54] and in vitro [55] studies in ewes suggesting that GABA inhibits GnRH release with participation of GABAA, rather than GABAB, receptor mechanism. In support, the present study was carried out on ewes in the breeding season and the inhibition of LH secretion in sheep during this period is strongly controlled by GABAAR in vivo [13]. A study in rats, in which down-regulation of pulsatile LH release induced by stress was inhibited by bicuculline in vivo [15] further strengthens the importance of GABAAR mechanism in GABA-induced suppression of GnRH/LH secretion. The mode of GABA action via the mechanism including GABAAR effect on molecular processes leading to GnRH biosynthesis is poorly recognized. However, immunohistochemical studies in sheep showing that GABA terminals are in close contact with GnRH neurons in the POA [8] provide an opportunity for a direct action of GABA on the expression of gene encoding GnRH. Subsequently, from studies in mice it appears that muscimol and/or bicuculline could inhibit or activate GnRH gene expression directly through GABAAR located on GnRH Aldicarb [56,57]. The question remains whether GnRH cells in ewes, just as it is in mice, contain GABAAR. Nonetheless, we cannot ignore the possibility of indirect GABA action via this class of receptor molecules which are expressed in other neuronal systems in the hypothalamus having a synaptic contact with the GnRH neurons. From studies in rats and ewes, it appears that such intermediary function may be performed by noradrenergic, dopaminergic and β-endorphinergic neurons [11,22,58]. However, further investigations are needed to clarify this point. In the present study, both activation and blockade of GABAAR had no evident effect on the levels of mRNAs encoding Kiss 1 and Kiss1r in hypothalamus. However, functional interconnections between inhibitory GABAergic and stimulatory kisspeptidergic inputs appears to govern pronounced changes in the pattern of GnRH/LH secretion in other species. Indeed, in the female rhesus monkey, administration of bicuculline during the prepubertal period, but not during the pubertal period, stimulated kisspeptin release, and the bicuculline-induced GnRH secretion was blocked by the kisspeptin antagonist, peptide 234 [29]. Furthermore, an in vitro study in rats indicated that the activation of GABAA R by muscimol can increase Kiss 1 gene expression in kisspeptin-producing cell model [59]. There was also an inverse relationship in mice suggesting that GABAergic cells mediate the effect of kisspeptin on inhibition of GnRH secretion [28]. Although it is premature to exclude GABA action via kisspeptin in sheep, our mRNAs expression data did not confirm the dogma that kisspeptin is a mandatory intermediate for GABA-induced changes in GnRH release. Given that there are many processes between Kiss 1 mRNA and mature peptide, and also Kiss 1 stability is an important factor, we were taking into account that transcription and translation might be far from having a linear and simple relationship. It must be emphasized that due to lack of information on transcript degradation or utilization in biosynthetic processes, it is now impossible to define the relationship between the post-transcriptional and post-translational products of Kiss 1 and Kiss1r genes. Limited data on rats suggested that changes in GABA-ergic neurotransmission affect GnRH gene expression in a different manner, depending on the mode of agonist administration. For example, chronic activation of GABAAR by muscimol decreased GnRH transcript level [32], whereas acute treatment with low, but not high, doses of this drug increased GnRH mRNA [31]. From these conflicting results, it is impossible to clarify the mode of GABA action on GnRH gene expression. A study in mice suggested that the inconsistency between the results might be attributable to the heterogeneity of GnRH neurons in their response to GABA depending on GnRH cells location in hypothalamus [60,61]. Another possible explanation for the contradictory data may be the assumption that GABAAR exist in numerous stimulatory and inhibitory hypothalamic neuronal systems with respect to the control of GnRH expression. Furthermore, the discordance between transcript and protein expression levels should also be considered.