Hy of future exploration as kisspeptin processing enzymes, especially since we
Hy of future exploration as kisspeptin processing enzymes, especially since we show strong evidence of regulation in response to sex steroids. Tmem144 was the only transcript that was consistently up-regulated in the KKO mice as compared with WT mice, independent of testosterone exposure and in all groups of KKO mice. We confirmed this up-regulation by RNA in situ hybridization and western blot analysis of TMEM144 in the hypothalamus of KKO mice as compared to WT mice. Tmem144 encodes an orphan 10-transmembrane family receptor, whose solutes and activities are unknown. This class of transmembrane proteins can transport a wide variety of molecules, ranging from sugars and amino acids to solutes and are generally linked to Ca 2+ mediated activation of gene expression. Activation of non-selective cation channels and inhibition of inwardly rectifying potassium channels have been shown to be necessary for Kp to depolarize GnRH neurons [39,40]. It is possible that TMEM144 is somehow implicated in the regulation of kisspeptin expression, its expression was high in the KKO mice lacking Kiss1 transcripts and low in the GKO micePrentice et al. BMC Genomics 2011, 12:209 http://www.biomedcentral.com/1471-2164/12/Page 10 ofwhere Kiss1 is over-expressed, suggestive of a direct or indirect negative regulator of kisspeptin. Future genetic studies of Tmem144 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 will be required to evaluate this possibility. The transcription factor gene Npas4 was up-regulated in the GKO mice with one of the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28388412 largest fold changes after the Kiss1 transcripts yet was down-regulated by 1.7 fold in castrated mice independent of hormonal feedback. This up-regulation in non-castrated mice was confirmed by IHC showing expression of Npas4 in the hypothalamus of Gpr54-/- mice while Npas4 expression was mostly absent in the wild-type mice. Npas4 has been L-660711 sodium salt site credited with regulating the development of inhibitory synapses in activating neurons in the hippocampus [24]. Interestingly, Npas4 is activated by an influx of calcium [24] and is independent of the MAP kinase pathway [41]. It has been recently reported that Kp-GPR54 signaling in the hypothalamus of rats is also independent of the MAP kinase pathway but can signal with calcium release [39]. Whether the Npas4 regulation is direct or an indirect consequence of sexual immaturity, remains to be seen, but its role in calcium dependent activation taken together with our data suggest it may play a role in the gonadotropic axis. Estrogen receptor alpha (Esr1) expression was higher in both GKO and KKO mice compared to sexually mature wild-type males which probably reflects the absence of negative feedback by testosterone in the mutant mice. Consistent with this, Esr1 expression was decreased by 1.2 fold in the castrated KKO mice that were give testosterone implants. In contrast, expression of the estrogen receptor beta (Esr2) was 1.6 fold lower in the GKO mice independent of testosterone effects. Esr2 function in the hypothalamic-pituitary-gonadal axis is currently unknown as hormonal feedback occurs almost entirely through the estrogen receptor alpha [17,19,22] as supported in our study. This is the first report that Esr2 may be regulated by Kp and GPR54. Previous studies have identified genes that show an upregulation in the hypothalamus during mammalian puberty including Eap1 (enhanced at puberty 1) [42], Oct2 [43] and Ttf1 (thyroid transcription factor 1) [44]. The role of these genes as key regulators of pubertal initiation is not clear.