Insulin-like Growth Factor 1, Growth Hormone, and Anti-Müllerian Hormone Receptors Are Differentially Expressed during GnRH Neuron Development

Abstract

Gonadotropin-releasing hormone (GnRH) neurons are key neuroendocrine cells in the brain as they control reproduction by regulating hypothalamic-pituitary-gonadal axis function. In this context, anti-Müllerian hormone (AMH), growth hormone (GH), and insulin-like growth factor 1 (IGF1) were shown to improve GnRH neuron migration and function in vitro. Whether AMH, GH, and IGF1 signaling pathways participate in the development and function of GnRH neurons in vivo is, however, currently still unknown. To assess the role of AMH, GH, and IGF1 systems in the development of GnRH neuron, we evaluated the expression of AMH receptors (AMHR2), GH (GHR), and IGF1 (IGF1R) on sections of ex vivo mice at different development stages. The expression of AMHR2, GHR, and IGF1R was assessed by immunofluorescence using established protocols and commercial antibodies. The head sections of mice were analyzed at E12.5, E14.5, and E18.5. In particular, at E12.5, we focused on the neurogenic epithelium of the vomeronasal organ (VNO), where GnRH neurons, migratory mass cells, and the pioneering vomeronasal axon give rise. At E14.5, we focused on the VNO and nasal forebrain junction (NFJ), the two regions where GnRH neurons originate and migrate to the hypothalamus, respectively. At E18.5, the median eminence, which is the hypothalamic area where GnRH is released, was analyzed. At E12.5, double staining for the neuronal marker ß-tubulin III and AMHR2, GHR, or IGF1R revealed a signal in the neurogenic niches of the olfactory and VNO during early embryo development. Furthermore, IGF1R and GHR were expressed by VNO-emerging GnRH neurons. At E14.5, a similar expression pattern was found for the neuronal marker ß-tubulin III, while the expression of IGF1R and GHR began to decline, as also observed at E18.5. Of note, hypothalamic GnRH neurons labeled for PLXND1 tested positive for AMHR2 expression. Ex vivo experiments on mouse sections revealed differential protein expression patterns for AMHR2, GHR, and IGF1R at any time point in development between neurogenic areas and hypothalamic compartments. These findings suggest a differential functional role of related systems in the development of GnRH neurons.

Keywords: AMHR2; GHR; GnRH; GnRH secretion; IGF1R; hypogonadotropic hypogonadism; neuron migration.

Figure 1

IGF1R is expressed in olfactory neurogenic niches at E12.5. (A) Coronal sections of E12.5 mouse embryo heads at VNO level were immunolabeled for IGF1R (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. (B) High magnification of squared box area in (A). Solid arrowheads indicate examples of nTUBB3-positive cells/axons exiting the VNO that express IGF1R in the nasal parenchyma. White arrows indicate strong expression of IGF1R in neurogenic epithelia of the VNO and the OE. Single-channel images are shown beside each panel. Abbreviations: IFG1R, insulin-like growth factor 1 receptor; VNO, vomeronasal organ; OE, olfactory epithelium. Scale bars: 250 μm (A) and 50 μm (B).

Figure 2

GHR is expressed by migratory mass at E12.5. (A) Coronal sections of E12.5 mouse embryo heads at the VNO level were immunolabeled for GHR (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. White arrows indicate the expression of GHR in the axon bundles at the CP level. (B) High magnification of squared box area in (A). Solid arrowheads indicate examples of nTUBB3-positive cells/axons exiting the VNO that express GHR in the nasal parenchyma. White arrows indicate the expression of GHR on some cell bodies in neurogenic epithelia of the VNO and the OE. Single-channel images are shown beside each panel. Abbreviations: GHR, growth hormone receptor; VNO, vomeronasal organ; CP, cribriform plate; OE, olfactory epithelium; and FB, forebrain. Scale bars: 250 μm (A) and 50 μm (B).

Figure 3

AMHR2 is expressed in neurogenic niches of VNO, OE, and migratory mass at E12.5. (A) Coronal sections of E12.5 mouse embryo heads at the VNO level were immunolabeled for AMHR2 (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. White arrows indicate the expression of AMHR2 in the parenchyma at the CP level. (B) High magnification of squared box area in (A). Solid arrowheads indicate examples of nTUBB3-positive cells/axons exiting the VNO and cell bodies in neurogenic epithelia of the VNO/OE that express AMHR2. Single-channel images are shown beside each panel. Abbreviations: AMHR2, anti-Müllerian hormone receptor 2; VNO, vomeronasal organ; OE, olfactory epithelium; and CP, cribriform plate. Scale bars: 250 μm (A) and 50 μm (B).

Figure 4

IGF1R and GHR are expressed by GnRH neurons migrating in the nasal parenchyma, but not by GnRH neurons residing in the VNO. (A) Coronal sections of E12.5 mouse embryo heads at the VNO level were immunolabeled for IGF1R (green) and GnRH (red) to reveal GnRH neurons. Solid arrowheads indicate examples of GnRH-positive cells exiting the VNO that express IGF1R. Empty arrowheads indicate examples of GnRH-positive cells within the VNO that do not express IGF1R. (B) Coronal sections of E12.5 mouse embryo heads at the VNO level were immunolabeled for GHR (green) and GnRH (red) to reveal GnRH neurons. Solid arrowheads indicate examples of GnRH-positive cells exiting the VNO that express GHR. Empty arrowheads indicate examples of GnRH-positive cells within the VNO that do not express GHR. Single-channel images are shown beside each panel. Abbreviations: IFG1R, insulin-like growth factor 1 receptor; GHR, growth hormone receptor; GnRH, gonadotropin-releasing hormone; VNO, vomeronasal organ. Scale bars: 50 μm.

Figure 5

IGF1R is expressed by neurons and axons migrating/extending from the VNO. (A,B) Coronal sections of E14.5 mouse embryo heads at the VNO level were immunolabeled for IGF1R (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. White arrows indicate the expression of IGF1R in the VNO/dorsal OE neurogenic epithelia. The high magnification of the squared box area is shown in (B). Solid arrowheads indicate examples of nTUBB3/IGF1R-positive cells/axons exiting the VNO. (C,D) Coronal sections of E14.5 mouse embryo heads at the OB level were immunolabeled for IGF1R (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. The high magnification of the squared box area is shown in (D). Solid arrowheads indicate examples of nTUBB3/IGF1R-positive axons innervating the OB. Single-channel images are shown below each panel. Abbreviations: IFG1R, insulin-like growth factor 1 receptor; VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulb; and NS, nasal septum. Scale bars: 250 μm (A,C) and 50 μm (B,D).

Figure 6

GHR is expressed by neurons and axons leaving/extending from the VNO. (A,B) Coronal sections of E14.5 mouse embryo heads at the VNO level were immunolabeled for GHR (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. White arrows indicate the expression of GHR in the nasal septum and nasal capsule cartilage primordia. The high magnification of the squared box area is shown in (B). Solid arrowheads indicate examples of nTUBB3/GHR-positive cells/axons exiting the VNO. White arrows indicate the expression of GHR in the nasal septum. (C,D) Coronal sections of E14.5 mouse embryo heads at the OB level were immunolabeled for GHR (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. The high magnification of the squared box area is shown in (D). Solid arrowheads indicate examples of nTUBB3/GHR-positive axons innervating the OB. Single-channel images are shown below each panel. Abbreviations: GHR, growth hormone receptor; VNO, vomeronasal organ; OE, olfactory epithelium; NS, nasal septum; OB, olfactory bulb. Scale bars: 250 μm (A,C), 50 μm (B,D).

Figure 7

AMHR2 is expressed by neurons and axons of the vomeronasal/olfactory systems. (A,B) Coronal sections of E14.5 mouse embryo heads at the VNO level were immunolabeled for AMHR2 (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. White arrows indicate the expression of AMHR2 in the VNO/dorsal OE neurogenic epithelia. The high magnification of the squared box area is shown in (B). Solid arrowheads indicate examples of nTUBB3/AMHR2-positive cells/axons exiting the VNO. (C,D) Coronal sections of E14.5 mouse embryo heads at the OB level were immunolabeled for AMHR2 (green) and nTUBB3 (red) to reveal neuron cell bodies and axons. The high magnification of the squared box area is shown in (D). Solid arrowheads indicate examples of nTUBB3/AMHR2-positive axons innervating the OB. Single-channel images are shown below each panel. Abbreviations: AMHR2, anti-Müllerian hormone receptor 2; VNO, vomeronasal organ; OE, olfactory epithelium; NS, nasal septum; and OB, olfactory bulb. Scale bars: 250 μm (A,C), 50 μm (B,D).

Figure 8

IGF1R is not expressed by migrating and hypothalamic GnRH neurons. (A,B) Coronal sections of E14.5 mouse embryo heads at the VNO level were immunolabeled for IGF1R (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (B). Empty arrowheads indicate examples of GnRH-positive cells that are in close contact with IGF1R-positive axons but do not express IGF1R themselves. (C,D) Coronal sections of E14.5 mouse embryo heads at the CP level were immunolabeled for IGF1R (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (D). Empty arrowheads indicate examples of GnRH-positive cells that are in close contact with IGF1R-positive axons but do not express IGF1R themselves. (E–H) Coronal sections of E14.5 (E,F) and E18.5 (G,H) mouse embryo heads at the MPOA level were immunolabeled for IGF1R (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (F). Empty arrowheads indicate examples of GnRH-positive cells that do not express IGF1R. Note the high expression of IGFR in the OVLT area. Single-channel images are shown beside each panel. Abbreviations: VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulb; and MPOA, medial preoptic area. Scale bars: 250 μm (A,C,E,G) and 50 μm (B,D,F,H).

Figure 9

GHR is expressed by migrating but not by hypothalamic GnRH neurons. (A,B) Coronal sections of E14.5 mouse embryo heads at the VNO level were immunolabeled for GHR (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (B). Solid arrowheads indicate examples of GnRH-positive cells that express GHR. (C,D) Coronal sections of E14.5 mouse embryo heads at the OB level were immunolabeled for GHR (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (D). Empty arrowheads indicate examples of GnRH-positive cells that do not express GHR. (E–H) Coronal sections of E14.5 (E,F) and E18.5 (G,H) mouse embryo heads at the MPOA level were immunolabeled for GHR (green) and GnRH (red) to reveal GnRH neurons. The high magnification of the squared box area is shown in (F,H). Empty arrowheads indicate examples of GnRH-positive cells that do not express GHR. Single-channel images are shown beside each panel. Abbreviations: VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulb; and MPOA, medial preoptic area. Scale bar: 250 μm (A,C,E,G,H), 125 μm, and 50 μm (B,D,F).

Figure 10

AMHR2 is expressed by hypothalamic GnRH neurons expressing PLXND1. (A–E) Coronal sections of E14.5 (A,B) and E18.5 (C–E) mouse embryo heads at the MPOA level were immunolabeled for AMHR2 (green) and PLXND1 (red) to reveal GnRH neurons. The high magnifications of the squared box areas are shown in (B–D). Solid arrowheads indicate examples of GnRH-positive cells that express AMHR2. Single-channel images are shown beside each panel. Abbreviations: VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulb; MPOA, medial preoptic area. Scale bar: 250 μm (A,C), 50 μm (B,D,E).