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Department of Anatomy and Neurobiology (H.S., K.-i.M., K.H., M.N., M.K.), Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; Department of Physiology, Anatomy, and Genetics (J.F.M.), University of Oxford, Oxford OX1 3QX, United Kingdom; and Department of Cell Biology and Physiology (E.R.P.), University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Address all correspondence and requests for reprints to: Hirotaka Sakamoto, Ph.D., Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. E-mail: hsakamo{at}koto.kpu-m.ac.jp.
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, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently a G protein-coupled receptor (GPR) for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the paraventricular nucleus and supraoptic nucleus, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system. |
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According to traditional paradigm, estradiol-17ß is a steroid, produced mainly by the ovary, which acts genomically on brain tissues through intranuclear receptor-mediated mechanisms to develop and regulate female reproductive behavior (11). In addition to the genomic actions of steroids, there is now increasing scientific interest in rapid nongenomic actions of steroids via membrane-associated receptors. Although the effects of estrogens on magnocellular functions are well documented, the mode of actions of estrogens, genomic or nongenomic, remains controversial. Many studies have demonstrated the expression of the intranuclear receptor for estrogens (ER)-ß (12, 13, 14, 15, 16, 17, 18, 19), but not ER (12, 20), in MNCs of the PVN and SON, suggesting the potential role for the direct regulation of MNCs by estrogens via ERß-mediated mechanisms (13, 14, 15, 16, 17, 21). Expression of ER in the nuclei of MNCs in the PVN and SON is still not clear, but localization of ERß, as well as ER, at extranuclear sites of the hippocampal neuron including perikarya, dendrites, and axon terminals has been demonstrated by immunoelectron microscopic analysis (22, 23, 24, 25). Because extranuclear ER and ERß may have the capacity to activate rapid signal transduction pathways in response to estrogens (22), it is possible that the rapid actions of estrogens in the hypothalamic MNCs are transduced through extranuclear ERß and/or ER. Because it has been considered that the mode of actions of nuclear ERs mediate long-term effects at the genomic level, these nuclear receptors do not explain all aspects of estrogen function in the MNCs, such as neuronal activation of OT neurons or OT release (26).
Involvement of G protein-coupled receptors (GPRs) in the rapid nongenomic actions of estrogens has long been suggested (27, 28, 29, 30, 31). Recently a membrane-bound G protein-coupled receptor, GPR30, has been identified as a putative ER in several cancer cell lines (32, 33). In cells expressing GPR30, stimulation with estradiol led to the activation of heterotrimeric G proteins with subsequent activation of adenylyl cyclase, intracellular calcium mobilization, and accumulation of phosphatidylinositol-3,4,5-trisphosphate in the nucleus. Because expression of GPR30 in the brain has been previously reported (34, 35), the identification of GPR30 in the magnocellular OT neurons would provide new insights into the transduction of estrogen signals in the hypothalamo-neurohypophyseal system.
In the present study, we used light and electron microscopic immunohistochemistry to demonstrate the presence of GPR30 protein as well as in situ hybridization to show the presence of GPR30 mRNA, in OT neurons. We show that, in the PVN and SON, GPR30 is expressed in magnocellular OT neurons but not in vasopressin (VP) neurons and that it is localized mainly in the membrane structures of the Golgi apparatus.
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Tissue preparation
Rats were anesthetized with 50 mg/kg body weight sodium pentobarbital and perfused via the left ventricle with 100 ml of physiological saline followed by 200 ml of 4% formaldehyde generated from paraformaldehyde (for immunohistochemistry, immunofluorescence, and in situ hybridization) or 0.2% glutaraldehyde (for electron microscopic studies) in 0.1 M phosphate buffer (PB; pH 7.4). Brains and neurohypophyses were immediately removed and immersed in 4% paraformaldehyde in 0.1 M PB for 16–24 h (for immunohistochemistry, immunofluorescence, and in situ hybridization,) or 3 h (for electron microscopy) followed by 25% sucrose in 0.1 M PB for 48 h at 4 C. For cryosections, brains including the diencephalic region and neurohypophysis were quickly frozen using powdered dry ice and cut into 20 µm- (for immunohistochemistry and immunofluorescence) or 14 µm- (for in situ hybridization) thick frontal (hypothalamus) or horizontal (neurohypophysis) sections on a cryostat (CM3050 S; Leica, Nussloch, Germany). For electron microscopic observation, the brains and neurohypophyses were sectioned with a microslicer (Dosaka EM, Kyoto, Japan) in the frontal (hypothalamus) or horizontal (neurohypophysis) plane at a thickness of 30 µm and were placed in PBS (pH 7.4). Sections were washed several times with PBS for 5 min each.
Immunohistochemistry and immunofluorescence
For immunohistochemistry, endogenous peroxidase activity was eliminated from the sections by incubation with 1% H2O2 in absolute methanol for 30 min, and the sections were then rinsed with PBS three times for 5 min each. These processes were omitted for immunofluorescence and electron microscopy. After blocking nonspecific binding components with 1% normal goat serum and 1% BSA in PBS containing 0.1% Triton X-100 (0% for electron microscopy) for 1 h at room temperature, the sections were incubated with the primary rabbit antiserum against GPR30 (33) at a dilution of 1:8000 overnight at 4 C. Several concentrations of the antiserum from 1:500 to 1:50,000 were examined preliminarily, and a dilution of 1:8,000 proved most satisfactory for the enzyme antibody technique. Immunoreactive (ir) products were detected with a streptavidin-biotin kit (Nichirei, Tokyo, Japan), followed by diaminobenzidine development according to our previous method (36). Control procedures consisted of substituting the preimmune rabbit serum for the primary antiserum at the same dilution (1:8000). Immunoreactive cells in the rat brain were visualized using an Olympus Optical (Tokyo, Japan) BH-2 microscope.
To determine whether GPR30 was expressed in OT or VP cells and its subcellular localization within GPR30-ir cells, double-immunofluorescence staining of GPR30 (1:5000 dilution) and OT-neurophysin (NP), VP-NP, KDEL (Lys-Asp-Glu-Leu), a marker protein for endoplasmic reticulum (10C3; Calbiochem, Darmstadt, Germany; 1:1000 dilution), or TGN38, a marker protein for Golgi apparatus (2F7.1; Abcam, Cambridge, UK; 1:1000 dilution) was performed. Mouse monoclonal antibodies for OT-NP (PS-60) and VP-NP (PS-41) were used at a 1:100 dilution and have previously shown to be specific for OT and VP neurons, respectively (6, 37). Alexa Fluor 546-linked antimouse IgG (Molecular Probes, Eugene, OR) and Alexa Fluor 488-linked antirabbit IgG (Molecular Probes) were used for detection at a 1:1000 dilution. Immunostained sections were viewed by a confocal laser-scanning microscopy (Fluoview 100; Olympus, Tokyo Japan). Then, by using the double-labeled specimens, OT neurons were semiquantitatively analyzed at a high magnification to determine the percentage of OT neurons expressing GPR30 in the PVN and SON. Digital photomicrographs were processed with Adobe PhotoShop computer software (San Jose, CA) at 300 dots per inch resolution, printed on photo-quality paper, and the number of the immunopositive cells with transected round nuclei clearly visible was counted.
In situ hybridization
Male and female hypothalami (n = 2 in each sex) were lysed in Sepasol (Nakarai Tesque, Kyoto, Japan) as the substrate for PCR. Total RNA was extracted according to the manufacturer’s instructions. Oligo-dT primed cDNA synthesis was carried out with ReverTra Ace (Toyobo, Osaka, Japan). PCR was performed using Taq polymerase (Toyobo) under the following conditions: 95 C for 5 min; 95 C for 60 sec, 55 C for 60 sec, 72 C for 60 sec, 35 cycles. Sequences of primers were 5'-CGAGGTGTTCAACCTGGACGA-3' (forward primer) and 5'-GGCAAAGCAGAAGCAGGCCT-3' (reverse primer). The PCR product was electrophoresed on 2% agarose gel. The PCR product was subcloned into the pGEM-T easy vector (Promega, Madison, WI). Then the plasmid was linearized with NcoI, and T7 polymerase was used to transcribe the digoxigenin-labeled antisense cRNA probe, using the digoxigenin RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). Digoxigenin-labeled GPR30 sense cRNA was transcribed from a linearized SalI template using SP6 polymerase and used as a control to define the background levels on sections adjacent to those probed with the antisense probe. Hybridization histochemistry was performed according to our previous method described elsewhere (38). Briefly, frozen sections (14 µm thick) on glass slides were pretreated with 20 mg/ml proteinase K and 0.25% acetic anhydride for acetylation. The sections were incubated with 100 µl of 500 ng/ml antisense or sense probe in hybridization buffer for 16 h at 55 C. After hybridization, the sections were treated with 12.5 µg/ml RNase A to digest the excess hybridization probe and washed in 2x saline sodium citrate (SSC) (150 mM NaCl, 15 mM sodium citrate), 0.2x SSC, and 0.1x SSC. Subsequently the slides were incubated with antidigoxigenin (Fab) conjugated to alkaline phosphatase (Roche Molecular Biochemicals; working dilution 1:500). Finally, the sections were developed with 75 mg/ml nitro blue tetrazolium and 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt.
Mirror image section
To examine colocalization of GPR30 mRNA and protein in OT neurons, mirror-image sections were obtained from two consecutive sections mounted on glass slides with the upward cut surfaces facing each other so that the adjoining surfaces of the consecutive sections were stained by two different methods. The first section was turned over and mounted on the glass slide for observing the double immunofluorescence of GPR30 protein and OT-NP, whereas the second section was directly mounted on the glass slide for in situ hybridization of GPR30 mRNA. After double-immunofluorescence staining of GPR30 and OT-NP, the image of the section was captured with a confocal laser-scanning microscopy. The image was turned once to be in the same direction as the in situ hybridization image. Individual cells in the mirror-image sections were identified by their position relative to surrounding blood vessels.
Immunocytochemical detection of GPR30 by electron microscopy
After blocking nonspecific binding components with 1% normal goat serum and 1% BSA, the sections were immersed overnight at 4 C with the anti-GPR30 serum at a dilution of 1:8000. Immunoreactive products were detected with a streptavidin-biotin kit followed by diaminobenzidine-nickel intensification. After being washed, the sections were placed for 2 h in 1% OsO4 in 0.1 M PB, dehydrated, and flat embedded in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan) according to our previously described method (36). Alternatively, some sections stained with anti-GPR30 were incubated with 1.4-nm gold particle-conjugated goat antirabbit IgG (Nanoprobes, Stony Brook, NY) at a dilution of 1:200. Sections were postfixed for 20 min with 1% glutaraldehyde in 0.1 M PB at 4 C, washed in distilled water, and then silver developed in the dark with a HQ silver kit (Nanoprobes). After being washed, the sections were placed for 45 min in 1% OsO4 in 0.1 M PB, dehydrated, and flat embedded in epoxy resin as described above. Ultrathin sections (60 nm in thickness) containing GPR30-ir MNCs in the SON or neurohypophysis were collected on grids coated with collodion film, contrasted with uranyl acetate and lead citrate, and viewed using a JEM-1220 electron microscope (JEOL, Tokyo, Japan).
Western immunoblotting
Hypothalami and neurophypophyses (n = 3 rats of each sex) were weighed, immersed in 4 vol PBS, and lysed with 5 volumes of sample buffer containing 200 mM Tris-HCl, 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, and a small amount of bromophenol blue, and then the lysates were boiled for 5 min. The lysates (6 µl) were run on a 7.5% SDS-PAGE. Samples were electroblotted onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) from the gel by a semidry blotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). The blotted membrane was blocked with 5% skimmed milk, 0.05% Tween 20 in Tris-buffered saline (TBST) for 30 min at room temperature and then incubated with primary antibody against GPR30 (1:2000 dilution in TBST) at 4 C overnight. Blots were washed three times with TBST and incubated with alkaline phosphatase-conjugated goat antirabbit IgG second antibody (1:1000 dilution in TBST; Chemicon, Temecula, CA) for 2 h at room temperature. After being washed three times with TBST, blots were visualized by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt staining kit (Nakarai Tesque). Control procedures consisted of preadsorbing the working dilution (1:2000) of the primary antiserum with a saturating concentration of GPR30 antigen peptide (100 µg/ml) overnight at 4 C before use and substituting preimmune rabbit serum for the primary antiserum at the same dilution (1:2000) (data not shown). The membranes were incubated with these control sera, using the same procedure as that for the GPR30 antibody.
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), meaning that GPR30 was expressed in adult male and female hypothalamus (data not shown) at the mRNA level.
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, an intense protein band was observed in the hypothalamus. Judging from its electrophoretic mobility, the molecular mass of this immunoreactive protein band was approximately 38 kDa (Fig. 1B, arrow). Preabsorption of the antiserum with an excess amount of antigen peptide (100 µg/ml) failed to stain the 38-kDa protein band in the hypothalamus (Fig. 1C). No obvious sex difference in the expression of GPR30 protein was observed in the hypothalamus (data not shown). In addition, the GPR30-like 38-kDa band was also detected in the rat neurohypophysis of both sexes (data not shown).
Immunohistochemical detection of oxytocin and vasopressin neurons with GPR30 in the hypothalamus
Immunohistochemical analysis for GPR30 (n = 4 rats of each sex) revealed that GPR30-ir in the hypothalamus was present in somata and proximal dendrites of MNCs in the PVN and SON (Fig. 2). In the PVN, the GPR30-ir cells were observed in the rostrocaudal extent of the medial and periventricular parts of the PVN, which typically contain OT neurons (Fig. 2A). In the SON, the GPR30-ir was preferentially localized in the anterodorsal part of the SON, which also typically contains OT neurons (Fig. 2B). Controls in which preimmune rabbit serum was substituted for the anti-GPR30 serum showed no immunoreactivity in the hypothalamus (Fig. 2, B and D). No obvious sex difference in the distribution of GPR30-ir was observed in the PVN and SON of the hypothalamus (data not shown).
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, B and E; red) cells exhibited GPR30-ir (Fig. 3, A and D; green) in the perikarya (Fig. 3, A–F) and proximal dendrites (Fig. 3, G–I). Dual-labeled somata (Fig. 3, C and F; yellow) appeared along the rostrocaudal extent of these nuclei. Semiquantitative analysis of OT neurons (nine sections from three rats were analyzed) further revealed that 94.7 ± 1.2% of a total of 933 inspected OT-NP-ir neurons in the female PVN, 95.2 ± 1.5% of a total of 615 inspected OT-NP-ir neurons in the female SON, 93.0 ± 1.1% of a total of 675 inspected OT-NP-ir neurons in the male PVN, and 94.6 ± 1.8% of a total of 585 inspected OT-NP-ir neurons in the male SON also expressed GPR30-ir in their perikarya (Fig. 3). In contrast, in both sexes, few or no VP-NP-ir neurons in the PVN or SON exhibited GPR30-ir in the perikarya (Fig. 4, A–F) or proximal dendrites (Fig. 4, G–I). In addition, no VP-NP-ir neurons in the suprachiasmatic nucleus (a nucleus that contains parvocellular VP neurons) exhibited GPR30-ir (data not shown).
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) and SON (Fig. 5C). The distribution of GPR30 mRNA expressing cells in the PVN and SON was similar to that of GPR30 protein. In addition, hybridization specificity was indicated by the absence of any labeling by the sense RNA transcript in the brain (Fig. 5, B and D).
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). In sections incubated with antisense cRNA probe for GPR30, GPR30 mRNA-expressing cells were also observed in the PVN (Fig. 6, A and E) and SON (Fig. 6, I and M). In the adjacent section incubated with anti-GPR30 (Fig. 6, B, F, J, and N; green) and anti-OT-NP (Fig. 6, C, G, K, and O; red) antisera, GPR30/OT-NP dual-immunolabeled cells (Fig. 6, D, H, L, and P; yellow) were distributed in a similar pattern to the GPR30 mRNA-positive cells (Fig. 6, A, E, I, and M). At a higher magnification, in a pair of mirror sections, both the GPR30 mRNA signal and dual GPR30/OT-NP-ir were detected in a number of identical cells in both the PVN (Fig. 6, E–H; arrowheads) and SON (Fig. 6, M–P; arrowheads).
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; red), and GPR30-ir (Fig. 7A; green) was preferentially colocalized with the TGN38 signal (Fig. 7C; yellow). In addition, in the ventral part of the SON, which typically contained many dendrites of OT neurons, small clusters of GPR30-ir were also observed (Fig. 7D) and were preferentially colocalized with TGN38 signal (Fig. 7, E and F; arrowheads). The signal localizing a marker for endoplasmic reticulum, KDEL, was also mainly observed around the nucleus of the neuron (Fig. 8B). Although the GPR30-ir partially overlapped with the KDEL signal, the area in which GPR30-ir was most prominent did not colocalize with the KDEL signal (Fig. 8, A–C). Likewise, in the ventral part of the SON, most KDEL-ir was not colocalized with GPR30-ir (Fig. 8, D–F; arrowheads). These results indicate that GPR30 is localized mainly in the Golgi apparatus of SON neurons. In addition, in the PVN, double-immunofluorescence studies exhibited similar subcellular distribution patterns to those seen in the SON (data not shown).
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, arrows). GPR30-ir was associated with membrane structures mainly of the Golgi apparatus and, to a lesser extent, in the endoplasmic reticulum (Fig. 9, A and B), whereas other cellular structures, such as mitochondria, the nucleus, the nuclear membrane, the plasma membrane, and dense-cored neurosecretory vesicles showed only background labeling (see arrowheads for the site of the plasma membrane in Fig. 9, A, B-i, and B-ii). We also observed GPR30-ir associated with the membrane of small electron-lucent vesicles (Den2), which presumably derive from the Golgi apparatus but not dense-cored vesicles localized in the dendrites (Den1) (Fig. 9, C-i and C-ii).
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). Controls in which preimmune rabbit serum was substituted for the anti-GPR30 serum showed no immunoreactivity in the neurohypophysis (Fig. 10B). No obvious sex difference in the distribution of GPR30-ir was observed in the neurohypophysis (data not shown).
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, C and D). GPR30-ir was associated with membrane structures of some population of dense-cored vesicles in the neurosecretory axonal swellings (Ax marked with asterisk in Fig. 10, C-I and C-ii), whereas other cellular structures, such as mitochondria and the plasma membrane, showed only background labeling. Some dense-core-vesicle-filled neurosecretory axonal swellings showed no immunoreactivities for GPR30 (see Ax without asterisk in Fig. 10, C and D). Silver-enhanced immunogold labeling recapitulated that immunogold particles were associated with the same structure as in C (Fig. 10D). |
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) was localized, even within the membranes of synaptic vesicles, suggesting that secretion is also a parameter that could be modulated by the traditional ER. Although intra- and/or extranuclear ERß are expressed in the MNCs of the PVN and SON (12, 13, 14, 15, 16, 17, 18, 19), the presence of GPR30 strongly suggests that GPR30 signaling may play a role in the rapid estrogen actions within the hypothalamic OT neurons or that a synergistic interaction of ERß and GPR30 might occur in response to estrogens. Furthermore, in the present study, the expression of GPR30 is also detected in the rat neurohypophysis. Thus, taken together, these findings provide new insight into the transduction of estrogen signals in the hypothalamo-neurohypophyseal system. Classically, hypothalamic MNCs were thought to secrete OT and VP only from their axon terminals located mainly in the neurohypophysis into the blood or, in part, in the extrahypothalamic brain areas as a neuromodulator. However, it is now clear that, in the PVN and SON, OT is also released locally from neuronal dendrites or somata and functions as an intrinsic (presumably by both the autocrine and paracrine mechanisms) self-neuromodulator involved, for instance, in the synchronization of the firing of OT neurons during lactation (6, 42, 43, 44, 45). Furthermore, it has been reported that estradiol exerts acute nongenomic actions on the magnocellular neurons to promote intrahypothalamic release of OT from dendrites or somata (6, 26). The present study clearly showed that GPR30 was localized in not only the somata but also the dendrites in which it was associated with electron-lucent vesicles, which presumably derive from the Golgi apparatus. Accordingly, we propose that GPR30 localized in the dendrites may directly affect the rapid release of OT from the dendrites or somata. Future study should be focused on the precise physiological characterizations of GPR30 located in the dendrites.
In the present study, we detected GPR30 in magnocellular OT neurons in not only the female but also the male hypothalamus. It is well known that, in the male brain, testosterone can activate ERs (including ER, ERß, and also GPR30) after its aromatization to estradiol by hypothalamic cytochrome P450 aromatase activity (46, 47). Because MNCs in the PVN of males concentrate estradiol (48), circulating testosterone could potentially influence magnocellular functions through mechanisms that are GPR30 mediated by neuroestrogens in the male hypothalamus. In addition, OT neurons in the PVN have been shown to project to extrahypothalamic brain areas (49) and the lumbosacral spinal cord (50, 51). The PVN contains retrograde-labeled neurons from the sexually dimorphic spinal nucleus of bulbocavernosus located in the fifth and sixth lumbar segments of the spinal cord, innervating the bulbocavernosus muscle and levator ani that attach to the penis, which have an important role in copulatory behavior (50, 52). Additionally, OT is transiently released at the time of ejaculation in men, and induces epididymal contractility, by PVN neurons projecting to the neurohypophysis (53). Thus, it is now considered that the OTergic system plays an important role in the control of male reproductive functions such as penile erection and ejaculation (for reviews, see Refs. 54 and 55). Therefore, the present and previous studies taken together suggest that estradiol derived from circulating testosterone in the male hypothalamus may also have rapid effects on the neuronal activation of OTergic system via GPR30-mediated mechanisms and, consequently, modulate male reproductive functions and sexual behaviors.
Our double immunostaining of GPR30 and OT-NP indicated that GPR30 has a clustered distribution restricted to somata and proximal dendrites of the OT neurons in the PVN and SON. This suggests that GPR30 protein is concentrated in certain subcellular membranes in the OT neurons. The use of specific markers for intracellular organelles revealed that GPR30 is preferentially localized to the Golgi apparatus of the somata and dendrites in the OT neurons. This intracellular pattern was confirmed by electron microscopic analysis. At higher magnification, patches of immunoreactivity were localized primarily on the trans-membrane surface of the Golgi apparatus. In hippocampal CA1–3 pyramidal neurons, GPR30-ir was also detected in the somata and dendrites, in which its distribution was also preferentially in the Golgi apparatus in both in vivo and in vitro studies (our unpublished observations), supporting the present findings. Although we did not detect GPR30 in other cellular structures including the plasma membrane, two other groups have reported plasma membrane localization of GPR30 in cultured cell lines (32, 35). Notwithstanding this discrepancy, the present and previous studies with mammals indicate that GPR30 is a membrane-associated protein. It is, of course, possible that the antiserum used in this study specifically recognized a (precursor) form of the GPR30 molecule localized to the Golgi apparatus. This possibility is, however, unlikely because the antibody used in this study was raised against the same C-terminal epitope as that used in studies reporting GPR30 in the plasma membrane (32, 35). Furthermore, exogenous expression of tagged forms of GPR30, as well as staining with fluorescent estrogen derivatives, show exclusively intracellular localization of GPR30 (33). Because estrogens are lipophilic membrane-permeable molecules, it may diffuse across the plasma membrane and bind to GPR30 localized in intracellular organelles just as it binds to ER localized in the nucleus. In addition, it has been reported that stimulation of GPR30 induces calcium mobilization and regulates MAPK and phosphoinositide 3-kinase-Akt pathways in cultured cell lines (22, 32, 33, 35, 56, 57, 58, 59). Together GPR30 appears to have the capacity to recruit downstream molecules to the membranes of intracellular organelles after estrogen binding. To further our understanding of GPR30 function within the hypothalamic OTergic system, electrophysiological and/or behavioral studies after GPR30 stimulation are needed.
In conclusion, the present studies have localized, for the first time, both protein and mRNA of GPR30 in OT neurons but not VP neurons of the PVN and SON of the rat hypothalamus. These findings provide new insights into the possible transduction mechanisms of estrogen signals in the hypothalamo-neurohypophyseal system.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online September 13, 2007
Abbreviations: ER, Estrogen receptor; GPR, G protein-coupled receptor; ir, immunoreactive; KDEL, Lys-Asp-Glu-Leu; MNC, magnocellular neurosecretory cell; NP, neurophysin; OT, oxytocin; PB, phosphate buffer; PVN, paraventricular nucleus; SON, supraoptic nucleus; SSC, saline sodium citrate; TBST, Tween 20 in Tris-buffered saline; VP, vasopressin.
Received April 4, 2007.
Accepted for publication September 4, 2007.
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and ERß) throughout the rat brain: anatomical evidence of distinct roles of each subtype. J Neurobiol 36:357–378[CrossRef][Medline] and -ß mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline], is expressed in prolactin neurons of the female rat paraventricular and supraoptic nuclei: comparison with other neuropeptides. J Comp Neurol 484:28–42[CrossRef][Medline] and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319 protein in neuronal processes of cultured hippocampal neurons. Neuroendocrinology 71:34–42[CrossRef][Medline] estrogen receptors are located at extranuclear sites. J Comp Neurol 429:355–371[CrossRef][Medline]-immunoreactive vesicles in inhibitory presynaptic boutons in hippocampal CA1. J Neurosci 27:2102–2111This article has been cited by other articles:
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G. G J Hazell, S. T Yao, J. A Roper, E. R Prossnitz, A.-M. O'Carroll, and S. J Lolait Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues J. Endocrinol., August 1, 2009; 202(2): 223 - 236. [Abstract] [Full Text] [PDF]
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R. Sirianni, A. Chimento, C. Ruggiero, A. De Luca, R. Lappano, S. Ando, M. Maggiolini, and V. Pezzi The Novel Estrogen Receptor, G Protein-Coupled Receptor 30, Mediates the Proliferative Effects Induced by 17{beta}-Estradiol on Mouse Spermatogonial GC-1 Cell Line Endocrinology, October 1, 2008; 149(10): 5043 - 5051. [Abstract] [Full Text] [PDF]
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C. Wang, E. R. Prossnitz, and S. K. Roy G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary Endocrinology, September 1, 2008; 149(9): 4452 - 4461. [Abstract] [Full Text] [PDF]
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