Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice

Abstract

Background: Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the regulation of the hypothalamic-pituitary gonadal axis in a sex-specific manner. We hypothesized that the differences seen in reproductive functions of males and females are associated with a sexually dimorphic gene expression profile of GnRH neurons. Methods and Results: We compared the transcriptome of GnRH neurons obtained from intact metestrous female and male GnRH-green fluorescent protein transgenic mice. About 1,500 individual GnRH neurons from each sex were sampled with laser capture microdissection followed by whole-transcriptome amplification for gene expression profiling. Under stringent selection criteria (fold change >1.6, adjusted p value 0.01), Affymetrix Mouse Genome 430 PM array analysis identified 543 differentially expressed genes. Sexual dimorphism was most apparent in gene clusters associated with synaptic communication, signal transduction, cell adhesion, vesicular transport and cell metabolism. To validate microarray results, 57 genes were selected, and 91% of their differential expression was confirmed by real-time PCR. Similarly, 88% of microarray results were confirmed with PCR from independent samples obtained by patch pipette harvesting and pooling of 30 GnRH neurons from each sex. We found significant differences in the expression of genes involved in vesicle priming and docking (Syt1, Cplx1), GABAergic (Gabra3, Gabrb3, Gabrg2) and glutamatergic (Gria1, Grin1, Slc17a6) neurotransmission, peptide signaling (Sstr3, Npr2, Cxcr4) and the regulation of intracellular ion homeostasis (Cacna1, Cacnb1, Cacng5, Kcnq2, Kcnc1). Conclusion: The striking sexual dimorphism of the GnRH neuron transcriptome we report here contributes to a better understanding of the differences in cellular mechanisms of GnRH neurons in the two sexes.

Glucagon-Like Peptide-1 Excites Firing and Increases GABAergic Miniature Postsynaptic Currents (mPSCs) in Gonadotropin-Releasing Hormone (GnRH) Neurons of the Male Mice via Activation of Nitric Oxide (NO) and Suppression of Endocannabinoid Signaling Pathways

“…In our present study, the mRNA content of the individual GnRH neurons of male mice used in PCR experiments was also harvested using the patch clamp pipette, as described earlier (Farkas et al, 2013; Vastagh et al, 2015) with slight modification. Patch pipettes were pulled from capillaries sterilized at 180°C for 6 h and filled with autoclaved intracellular pipette solution containing (in mM) HEPES 10, K-gluconate 130, KCl 10, NaCl 10, EGTA 1 and MgCl 2 0.1 (pH 7.3 with KOH, osmolarity adjusted to 300 mOsm with D-sorbitol).…”

“…The resistance of the patch electrodes was 2–3 MΩ. The RNA samples were collected from GnRH-GFP neurons of acute brain slices kept in oxygenated aCSF at 33°C (Farkas et al, 2013; Vastagh et al, 2015) using extra care to avoid any glial RNA contamination with the protocol suggested by Fuzik et al (2016). The collected cytoplasm was reverse transcribed directly in 20 μl reactions using the ViLO SuperScript III cDNA reverse transcription (RT) kit (Thermo Fisher Scientific, Waltham, MA, USA).…”

Altered Expression of Genes Encoding Neurotransmitter Receptors in GnRH Neurons of Proestrous Mice

“…Brain fixation, preparation of sections for the subsequent laser capture microdissection (LCM) and microarray profiling were performed as reported elsewhere (Khodosevich et al, 2007 ; Vastagh et al, 2015 ). Briefly, metestrous ( n = 6) and proestrous female ( n = 6) mice were deeply anesthetized and perfused transcardially with 80 ml 0.5% paraformaldehyde followed by 20% sucrose.…”

Impact of Proestrus on Gene Expression in the Medial Preoptic Area of Mice

“…GABA- and glutamate-driven events of neurotransmission onto GnRH neurons have been the most comprehensively studied regulatory mechanisms of these neurosecretory cells (Christian and Moenter, 2010; Iremonger et al, 2010; Moenter, 2010; Penatti et al, 2010; Liu and Herbison, 2011; Taylor-Burds et al, 2015). A wide repertoire of GABA, glutamate and DA receptors characterizes the GnRH neurons (Todman et al, 2005; Vastagh et al, 2015, 2016), as a prerequisite of their control by neurotransmitters. The upregulation of solute carrier family 17, member 1 gene ( Slc17a6 ) that codes for vesicular glutamate transporter 2 (VGLUT2) indicates the probability of enhanced packaging of glutamate into synaptic vesicles at the level of axon terminals in proestrus.…”