Gonadal Cycle-Dependent Expression of Genes Encoding Peptide-, Growth Factor-, and Orphan G-Protein-Coupled Receptors in Gonadotropin- Releasing Hormone Neurons of Mice

Abstract

Rising serum estradiol triggers the surge release of gonadotropin-releasing hormone (GnRH) at late proestrus leading to ovulation. We hypothesized that proestrus evokes alterations in peptidergic signaling onto GnRH neurons inducing a differential expression of neuropeptide-, growth factor-, and orphan G-protein-coupled receptor (GPCR) genes. Thus, we analyzed the transcriptome of GnRH neurons collected from intact, proestrous and metestrous GnRH-green fluorescent protein (GnRH-GFP) transgenic mice using Affymetrix microarray technique. Proestrus resulted in a differential expression of genes coding for peptide/neuropeptide receptors including Adipor1, Prokr1, Ednrb, Rtn4r, Nmbr, Acvr2b, Sctr, Npr3, Nmur1, Mc3r, Cckbr, and Amhr2. In this gene cluster, Adipor1 mRNA expression was upregulated and the others were downregulated. Expression of growth factor receptors and their related proteins was also altered showing upregulation of Fgfr1, Igf1r, Grb2, Grb10, and Ngfrap1 and downregulation of Egfr and Tgfbr2 genes. Gpr107, an orphan GPCR, was upregulated during proestrus, while others were significantly downregulated (Gpr1, Gpr87, Gpr18, Gpr62, Gpr125, Gpr183, Gpr4, and Gpr88). Further affected receptors included vomeronasal receptors (Vmn1r172, Vmn2r-ps54, and Vmn1r148) and platelet-activating factor receptor (Ptafr), all with marked downregulation. Patch-clamp recordings from mouse GnRH-GFP neurons carried out at metestrus confirmed that the differentially expressed IGF-1, secretin, and GPR107 receptors were operational, as their activation by specific ligands evoked an increase in the frequency of miniature postsynaptic currents (mPSCs). These findings show the contribution of certain novel peptides, growth factors, and ligands of orphan GPCRs to regulation of GnRH neurons and their preparation for the surge release.

Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently tagged cholinergic neuron populations

“…Fluorescent proteins expressed in transgenic mice are widely used neuronal phenotype markers. Harvesting fluorescent neurons with LCM for downstream RNA applications, such as RT-qPCR, microarray, and RNA-Seq, requires stabilization of the fluorescent signals with the crosslinking fixative formaldehyde ( 18 , 22 , 23 ). As the first step toward an optimized LCM-Seq strategy, we compared the fluorescence stability and signal intensity of formaldehyde-fixed tdTomato and ZsGreen proteins expressed selectively in cholinergic systems.…”

Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently-tagged cholinergic neuron populations

“…Fluorescent proteins expressed in transgenic mice are widely used neuronal phenotype markers. Harvesting fluorescent neurons with LCM for downstream RNA applications, such as RT-qPCR, microarray and RNA-Seq, requires stabilization of the fluorescent signals with the cross-linking fixative formaldehyde (18, 22, 23). As the first step toward an optimized LCM-Seq strategy, we compared the fluorescence stability and signal intensity of formaldehyde-fixed ZsGreen and tdTomato proteins expressed selectively in cholinergic systems ( Fig.…”

“…However, it was tested and reported to perform very well using 5-10 ng, and well using 1-2 ng degraded RNA (26). Our decision to pool ∼300 neurons for the LCM-Seq method relies on practical considerations: i) ∼300 pooled CPU neurons were enough to provide >1 ng total RNA, the minimal amount tested successfully by other investigators (26); ii) Collecting thousands of neurons within a workday with LCM would be technically infeasible; iii) Preparation of libraries from 30 neurons greatly reduced detection sensitivity and precision; iv) Genetically tagged neuron populations with a strictly defined topography often consist of a few hundred microdissectable cells only (18, 23).…”

“…The brains were snap-frozen on pulverized dry ice and stored at -80 °C until sectioned with a cryostat. Twelve-µm-thick sections containing the MS and the CPU (Atlas plates 20-26 of Paxinos; Bregma +1.34 to +0.62 mm) (35) were thaw-mounted onto PEN membrane glass slides (Membrane Slide 1.0 PEN, Carl Zeiss), air-dried in the cryostat chamber and preprocessed for LCM as reported elsewhere (18, 22, 23). In brief, the slides were immersed sequentially in 50% EtOH (20 sec), n-buthanol:EtOH (25:1; 90 sec) and xylene substitute:n-butanol (25:1; 60 sec).…”

Recent advances in acid sensing by G protein coupled receptors

“…GPR4 appears to be involved in hormone release from the anterior pituitary. Proton-mediated GPR4 activation in a pituitary cell line was shown to increased growth hormone and prolactin secretion [ 55 ], whilst a transcriptome analysis of gonadotropin releasing hormone (GnRH) neurons isolated from mice at different stages of their oestrous cycle showed a significant downregulation of GPR4 during the first half of the reproductive cycle [ 86 ]. These findings are intriguing since GnRH and prolactin have opposite effects on the release of luteinizing hormone from the anterior pituitary, which is essential for triggering ovulation in cyclic ovulators such as humans.…”