Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues

Abstract

Recently, the G protein-coupled receptor GPR30 has been identified as a novel oestrogen receptor (ER). The distribution of the receptor has been thus far mapped only in the rat central nervous system. This study was undertaken to map the distribution of GPR30 in the mouse brain and rodent peripheral tissues. Immunohistochemistry using an antibody against GPR30 revealed high levels of GPR30 immunoreactivity (ir) in the forebrain (e.g. cortex, hypothalamus and hippocampus), specific nuclei of the midbrain (e.g. the pontine nuclei and locus coeruleus) and the trigeminal nuclei and cerebellum Purkinje layer of the hindbrain in the adult mouse brain. In the rat and mouse periphery, GPR30-ir was detected in the anterior, intermediate and neural lobe of the pituitary, adrenal medulla, renal pelvis and ovary. In situ hybridisation histochemistry using GPR30 riboprobes, revealed intense hybridisation signal for GPR30 in the paraventricular nucleus and supraoptic nucleus (SON) of the hypothalamus, anterior and intermediate lobe of the pituitary, adrenal medulla, renal pelvis and ovary of both rat and mouse. Double immunofluorescence revealed GPR30 was present in both oxytocin and vasopressin neurones of the paraventricular nucleus and SON of the rat and mouse brain. The distribution of GPR30 is distinct from the other traditional ERs and offers an additional way in which oestrogen may mediate its effects in numerous brain regions and endocrine systems in the rodent.

Figure 1

Immunoreactivity for GPR30 in the adult male or female mouse brain: labelled cells in the supplementary motor cortex (A), piriform cortex (B), labelled cells and fibres in the SON (C), with moderate labelling of cells in the paraventricular nucleus of the thalamus (D), labelled cells and fibres in the PVH (E) with an absence of labelling in the PVH with a section incubated with IgG in place of GPR30 antiserum (F). M2, supplementary motor cortex; PIR, piriform cortex; SON, supraoptic nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; PVH, paraventricular nucleus of the hypothalamus; OC, optic chiasm; 3V, third ventricle. Scale bars, 100 μm in (A–B and E–F); 50 μm in (C–D). A lower magnification of the PVT is inserted in (D) (Scale bar=100 μm). D3V, dorsal third ventricle.

Figure 2

Immunoreactivity for GPR30 in the adult male or female mouse brain: labelled cells and fibres in the dentate gyrus of the hippocampus (A), zona incerta (B), arcuate nucleus and cell bodies of the ventromedial hypothalamic nucleus (C), GPR30-ir in cells and fibres of the lateral mamillary nucleus of the hypothalamus (D), labelled cells and fibres of the pars compacta of the substantia nigra (E) and labelled cells of the pontine nuclei (F). DG, dentate gyrus of the hippocampus; ZI, zona incerta; Arc, arcuate nucleus of the hypothalamus; VMH, ventromedial hypothalamic nucleus; LM, lateral mamillary nucleus of the hypothalamus; SNC, pars compacta of the substantia nigra; Pn, pontine nuclei; 3V, third ventricle. Scale bars, 100 μm in (A–D); 50 μm in (E–F). In E and F, lower magnifications of the SNC and Pn respectively, are shown (scale bars=200 μm).

Figure 3

Immunoreactivity for GPR30 in the adult male or female mouse brain: labelled cells of the Purkinje cell layer of the cerbellum (A), locus coeruleus (B) and sensory trigeminal nucleus (C) enlarged in (D). pcL, Purkinje cell layer of the cerbellum; lc, locus coeruleus; PrS, sensory trigeminal nucleus; 4V, fourth ventricle. Scale bars, 100 μm in (A–C); 50 μm in (D). In (C), a lower magnification of Pr5 is shown (scale bar=200 μm).

Figure 4

Double label immunofluorescence for GPR30 and AVP in the adult rat PVH (A–C), SON (D–F) and median eminence (G–I), and GPR30 and OXT in the adult mouse PVH (J, K and L). Immunoreactivities against GPR30 (A, D, G and J; red) and AVP (B, E and H; green) or OXT (K; green) were merged in each right panel (C, F, I and L; overlap yellow) respectively. G–L were captured using a laser confocal microscope. 3V, third ventricle; OC, optic chiasm. Scale bars, 100 μm in (A–F); 80 μm in (G–L).

Figure 5

Double label immunofluorescence for GPR30, OXT and AVP in the adult rat SON and mouse PVH. Immunoreactivities against GPR30 (A, D and G; red) and OXT (B; green) or AVP (E and H; green) were merged in each right panel (C, F and I; overlap yellow) respectively. Arrows indicate OXT or AVP immunoreactive cell bodies that also contain GPR30-immunoreactivity. Note that some GPR30-ir cells (arrowhead) do not express OXT-ir or AVP-ir. In addition to staining of cell bodies, there also appears to be co-expression of GPR30-ir and OXT-ir in processes (that may be axons or dendrites), suggesting that the receptor may be axo-dendritically transported, as has been well-established for the neuropeptide (see open arrows). The presence of possible intracellular staining of GPR30 within OXT and AVP neurones which is consistent with the predominantly cytoplasmic distribution of GPR30 that has been observed in various cell types in vitro (see discussion for details). Pictures captured at the same low magnification using a laser confocal microscope. Scale bars, 10 μm in (A–I).

Figure 6

Immunohistochemical analysis of GPR30 in rodent peripheral tissues. (A) GPR30-ir in rat pituitary is prominent in the nerve terminals of the neural (posterior) lobe (NL) and is present in most melanotrophs of the intermediate lobe (IL) (e.g. indicated by arrows). GPR30-ir is also found in scattered (∼50%) cells in the anterior pituitary (AP) lobe. Whether these represent endocrine cells (e.g. prolactotrophs and corticotrophs) was not determined. (B) High levels of GPR30-ir are present in the rat renal pelvis (RP), an extension of the ureter, with projections into the renal inner medulla (IM). (C) In the rat ovary, GPR30-ir is found mainly in the granulosa cells (G), with some staining of theca cells (T) of the developing follicle. Scale bars, 400 μm in (B); 50 μm in (A and C).

Figure 7

In situ hybridisation of GPR30 mRNA in the rodent PVH, SON and pituitary gland: reversed image of emulsion dipped section of the rat PVH the GPR30 hybridisation grains appear white in the picture (A) (but black on the actual slide); the corresponding sense slide is absent of black GPR30 hybridisation grains (B); low-magnification photographs of film autoradiographic images of a slide mounted hypothalamic section hybridised with a GPR30 probe, with signal in the PVH and SON of the rat brain (C), and pituitary sections hybridised with a GPR30 probe in rat (D) and mouse (E) with intense signal in both intermediate lobes; emulsion dipped section of the mouse pituitary, hybridisation signal for GPR30 mRNA appears as black grains (E). 3V, Third ventricle; PVH, paraventricular nucleus; SON, supraoptic nucleus; AP, anterior pituitary; IL, intermediate lobe of the pituitary; NL, neural lobe of the pituitary. Scale bars, 1 mm in (C–E); 100 μm in (A and F); 50 μm in (B).

Figure 8

In situ hybridisation of GPR30 mRNA in rodent peripheral tissues: low-magnification photograph of a film autoradiographic image of a slide mounted, rat adrenal section hybridised with a GPR30 probe (A); emulsion dipped section of the mouse adrenal medulla-hybridisation signal for GPR30 mRNA appears as black grains on toluidine blue counterstained cells (B); low-magnification photograph of a film autoradiographic image of a slide mounted, rat kidney section hybridised with a GPR30 probe (C), with an absence of signal in the sense control (D); low-magnification photograph of a film autoradiographic image of a slide mounted, mouse ovary section hybridised with a GPR30 probe, with signal localised predominantly in the developing follicles (E), with an absence of signal in the sense control (F). AM, adrenal medulla; ZG, zona glomerulosa; RP, renal pelvis; IM, renal medulla; DF, developing follicle. Scale bars, 1 mm in (A and C–F); 25 μm in (B).