Original Paper

Neuroendocrinology 2005;81:287–293 Received: January 17, 2005 Accepted after revision: June 21, 2005 DOI: 10.1159/000088170 Published online: September 7, 2005

Identifi cation of Sex Hormone-Binding in the Human Hypothalamus

a b c Zsófi a Herbert Susanne Göthe Jack D. Caldwell e d d Hans-Gert Bernstein Christian Melle Ferdinand von Eggeling f a John Lewis Gustav F. Jirikowski

a b Ludwig-Maximilian-Universität München, Department of Biology 2, Munich , Germany; Friedrich-Schiller- c Universität Jena, Department of Anatomy 2, Jena , Germany; University of Illinois at Rockford, d Department of Biomedical Science, Rockford, Ill., USA; Friedrich-Schiller Universität-Jena, CUCA, e Department of Human Genetics and Anthropology, Jena , Germany, Otto-von-Guericke-Universität Magdeburg, f Department of Psychiatry, Magdeburg , Germany, and Clinical Biochemistry Unit, Canterbury Health Laboratories, Christchurch , New Zealand

Key Words be observed in the posterior pituitary lobe in consecutive Steroid-binding globulin Cerebrospinal fl uid semithin sections. We also found strong immunoreactiv- Paraventricular nucleus Arcuate nucleus Supraoptic ity for SHBG in epithelial cells of the choroid plexus and nucleus Suprachiasmatic nucleus Bed nucleus of the in a portion of the ependymal cells lining the third ven- stria terminalis Clinical neuroendocrinology tricle. Mass spectrometry showed that affi nity-purifi ed SHBG from the hypothalamus and choroid plexus is struc- turally similar to the SHBG identifi ed in the CSF. The mul- Abstract tiple localizations of SHBG suggest neurohypophyseal Gonadal steroids are known to infl uence hypothalamic and neuroendocrine functions. The biochemical data sug- functions through both genomic and non-genomic path- gest that CSF SHBG is of brain rather than blood origin. ways. Sex hormone-binding globulin (SHBG) may act by Copyright © 2005 S. Karger AG, Basel a non-genomic mechanism independent of classical ste- roid receptors. Here we describe the immunocytochemi- cal mapping of SHBG-containing neurons and nerve fi - Introduction bers in the human hypothalamus and infundibulum. Mass spectrometry and Western blot analysis were also used Sex hormone-binding globulin (SHBG) is a glycopro- to characterize the biochemical characteristics of SHBG in tein that has been identifi ed in several tissues of different the hypothalamus and cerebrospinal fl uid (CSF) of hu- mammalian species. Alternate splice variants and glyco- mans. SHBG-immunoreactive neurons were observed in sylation of SHBG have been observed in different tissues. the supraoptic nucleus, the suprachiasmatic nucleus, the The outcome of this is a specifi c molecular weight pattern bed nucleus of the stria terminalis, paraventricular nucle- of the expressed in the different tissues [1–4] . Our us, arcuate nucleus, the perifornical region and the me- laboratory and others have reported an intrinsic expres- dial preoptic area in human brains. There were SHBG-im- sion of SHBG in the hypothalamus of rats [5, 6]. munoreactive axons in the median eminence and the One model of SHBG action suggests that it is a carrier infundibulum. A partial colocalization with oxytocin could protein for steroids [7] while a more recent model suggests

© 2005 S. Karger AG, Basel Zsófi a Herbert 0028–3835/05/0815–0287$22.00/0 LMU-München, Institut für Biologie 2 Fax +41 61 306 12 34 Grosshadernerstrasse 2 E-Mail [email protected] Accessible online at: DE–82152 Planegg-Martinsried (Germany) www.karger.com www.karger.com/nen Tel. +49 89 218074313, Fax +49 89 218074304, E-Mail [email protected] that SHBG may bind to a membrane receptor to induce anol in PBS for 20 min at room temperature (RT), to block endog- a rapid steroid effects through a G-protein-mediated pro- enous peroxidase activity. After washing in PBS for 1 h, PBS con- cess [for review, see 8]. This receptor has been function- taining 2% normal goat serum was applied for 2 h. After another washing step in PBS, sections were incubated in anti-SHBG over- ally identifi ed in a number of tissues including prostate, night at 4 ° C. We used a mouse monoclonal (clone 16D5, testis, breast, liver and brain [8–12], its structural features for production and characterization see Lewis et al. [18] ) at a dilu- however are still unknown. Recent fi ndings show that tion of 1: 250 in PBS containing 2% BSA (PBS-BSA). Sections were SHBG-like secreted from sexually dimorphic then washed in PBS and incubated in biotin-conjugated goat anti- structures in the rodent lacrimal and submandibular mouse IgG (Vector Laboratories, 1: 150 in PBS-BSA, 2 h at RT). After washing, streptavidin-peroxidase-complex was applied (Vec- glands may serve as pheromones [13]. tor Laboratories 1: 150 in PBS-BSA, 2 h at RT). The immunoreac- Little is known about the functional importance of tion was visualized with freshly prepared 3 ,3 -diaminobenzidine SHBG in the brain. It has been shown that SHBG coupled (DAB) and H 2 O 2 in PBS (reagents were obtained from Sigma). Af- to estradiol may play a role in the stimulation of oxytocin ter staining, sections were rinsed in distilled water and then dehy- drated through an ascending series of ethanol and mounted in En- release in the rat hypothalamus [14]. Intraventricular in- tellan ® (Merck). Immunocytochemical controls were performed fusions of SHBG, with and without bound estradiol, fa- with normal goat serum instead of SHBG antibody or with anti- cilitate female sexual receptivity in rats while SHBG cou- SHBG preabsorbed with synthetic SHBG (100 ng/1 ml antiserum pled to dihydrotestosterone blocks this effect [15, 16] , in the fi nal dilution). however the effects have not been tested in humans. Serial 1- m-thick semithin sections of the infundibulum and the ME were incubated in 10% sodium methoxide for 2 min, to The aim of the present study was to assess the distri- remove epoxy resin, prior to immersion in PBS. Consecutive sec- bution of SHBG in the human hypothalamus, choroid tions were stained either for SHBG, as described above, or for oxy- plexus and infundibulum. SELDI-TOF mass spectrom- tocin (OT). Rabbit antiserum to OT (Chemicon AB 911) was used etry was used to characterize the SHBG molecule ex- at a dilution of 1: 1,000 in PBS overnight at 4 ° C. Immunoprecipi- pressed in the hypothalamus and to compare its structure tates were visualized with anti-rabbit IgG 1: 100 for 1 h and rabbit

PAP complex 1: 100 in PBS and stained with DAB (all reagents with that of serum SHBG, which is known to be expressed obtained from Sigma, Munich). Stained slides were coverslipped in liver, and with SHBG from the cerebrospinal fl uid. with Entellan ®. Sections were examined with an Olympus BX50 microscope equipped with a digital camera, Olympus DP10 and DP-Soft 3.0 Materials and Methods software for microphotography. Anatomical locations of the la- beled cells were determined according to the cytoarchitectonic de- Tissue scriptions of Mai et al. [19] . Human hypothalami, both male (n = 4) and female (n = 3) For control purposes, SHBG antibody was preincubated with (mean age 69 8 14 years) with a mean postmortem time !30 h, the antigen before being applied to the tissue sections. To prevent were obtained from the Department of Pathology, FSU-Jena. Ex- the cross-reaction of the second , we omitted the anti- periments were approved by the Ethics Commission, FSU Jena, SHBG step in a further control experiment. protocol No. 1065-03/03. After dissection of the retrochiasmatic portion of the hypothal- Purifi cation of SHBG ami, including the mammillary bodies, tissue samples were fi xed For mass spectrometric analysis, human hypothalamic tissues by immersion in 4% paraformaldehyde for at least 3 weeks, dehy- were homogenized mechanically followed by sonication in lysis buff- drated and embedded in paraffi n. Small blocks containing the hy- er (0.1 M NaPH 4 pH 7.5 containing 2 m M MgCl 2 (Sigma), 0.5% pophysial stalk and the median eminence (ME) were dissected and (v/v), CHAPS (Sigma), 5 m M EDTA (Sigma), 0.02 m M PMSF (Sig- fi xed separately for 1 day in the same fi xative prior to dehydration ma), 0.1 m M leupeptin (Serva), 0.04 l/ml -mercaptoethanol (Sig- in ascending ethanol series and embedding in Epon for semithin ma); 5 ml/hypothalamus) and centrifuged at 10,000 g at 3 ° C for sectioning. Unfi xed samples of hypothalamus and choroid plexus 30 min. CSF and the serum samples were centrifuged the same way.

(n = 3) were dissected and frozen at –80 ° C for biochemical analysis. SHBG was purifi ed from the supernatant of the probes using Cerebrospinal fl uid (CSF) samples were obtained from a depart- IDM affi nity beads (Chipergen Biosystems, Inc.). 10 l protein A mental collection of control samples [ for details, see 17] . Serum (Sigma) were coupled onto 5 l of the beads. Monoclonal SHBG samples were collected from 4 volunteer medical students in the antibody from mouse (clone 16D5) was incubated on the protein laboratory. Both CSF and serum samples were stored at –80 ° C un- A coupled beads overnight at 4 ° C. Binding capacity of protein A til biochemical investigation. beads is 10 mg/ml IgG in 1 ! PBS. We incubated 500 l of each tissue sample with 10 l protein A-anti-SHBG beads overnight at

Immunocytochemistry 4 ° C in an end-over-end mixer. After washing, bound SHBG was Eight- to 10- m thick serial frontal sections were cut on a eluted with 50% (v/v) acetonitrile in 0.5% (v/v) trifl uoroacetic acid Reichert microtome and mounted on glycerol and egg - for mass spectrometric analysis. Alternatively, we used polyclonal

coated glass slides. After removal of the paraffi n with xylene and anti-ABP (653 and 882) raised in rabbit diluted 1: 10 in PBS to elute rehydration, sections were washed in phosphate-buffered saline SHBG from the column. For details of preparation, characteriza- (PBS) for 2 h followed by treatment with 3% H 2 O 2 and 10% meth- tion and specifi city of these antibodies, see Becchis et al. [20] .

288 Neuroendocrinology 2005;81:287–293 Herbert/Göthe /Caldwell /Bernstein /Melle /

von Eggeling/Lewis /Jirikowski Fig. 1. Localization of SHBG-immunoreac- tive structures in the human hypothalamus. A–E Diagrams of frontal hypothalamic sec- tions from anterior to posterior (arc = arcu- ate nucleus; bnst = bed nucleus of the stria terminalis; bstc = central nucleus of the bed nucleus of the stria terminalis; bnst-pm = posteriomedial component of the bed nu- cleus of the stria terminalis; CA = anterior commissure; fo = perifornical region; FX = fornix; inf = infundibulum; pvn = paraven- tricular nucleus; scn = suprachiasmatic nu- cleus; son = supraoptic nucleus; rson = ret- rochiasmatic part of the supraoptic nucleus; ME = median eminence; NO = optic nerve; TO = optic tract; XO = optic chiasm; III = third ventricle). Asterisks represent SHBG- IR cells. Circles represent immunoreactive fi bers. The number of asterisks and circles indicate the relative number of the stained structures in the distinct areas. F Sagittal view of the hypothalamus for the exact lo- calization of the frontal sections. CM = Mammillary bodies.

SELDI-TOF MS Analysis digitizer was operated at 250 MHz. 105 transients were averaged Surface-enhanced laser desorption ionization-time of fl ight over 66% of the target area in a linear sweep to generate each spec- mass spectrometry (SELDI-TOF) analysis was applied to deter- trum. Bovine and chicken conalbumin were used mine the mass of the bound proteins on the beads. 3 l of the elut- for external calibration. A deviation exceeding 0.3% from the pro- ed probes were dried on the hydrophobic surface of a H4 chip array tein standard was considered signifi cant. We used Chipergen Pro- after spots were washed with 27% (v/v) acetonitrile in 0.1 M PBS teinChip Software 3.1 to data analysis and representation. (pH 7.4). We used 2 ! 0.5 l saturated sinapic acid as matrix solu- PBS instead of tissue extract was applied to the protein A beads tion. The chip arrays were analyzed in a ProteinChip Reader (PBS- coupled to anti-SHBG as a control. We also coupled anti-corticoste- II; Chipergen Biosystems, Inc.) according to an automated data roid-binding globulin to the protein A beads to test for the specifi c- collection protocol [21] . The instrument was used in positive ion ity of binding of the beads complex. The eluted samples were exam- mode. Laser intensity was set at 300 (corresponding to approxi- ined in both cases with mass spectrometry as described above. mately 200 J), using a nitrogen laser, emitting at 337 nm. The

Identifi cation of SHBG in Human Neuroendocrinology 2005;81:287–293 289 Hypothalamus Fig. 2. SHBG-labeled structures in the hu- man hypothalamus. A A group of labeled magnocellular neurons in the son. B Higher magnifi cation suggests cytoplasmic staining of SHBG in the neurons of the SON. The neuronal nuclei showed no immunoreactiv- ity. C A population of labeled magnocellu- lar (black arrowheads) and parvocellular (arrow) neurons in the paraventricular nu- cleus. In the neuropil associated with the third ventricle (III) is a specifi c immuno- staining for SHBG. Scattered cells in the ependyma are SHBG positive (empty ar- rowheads). D SHBG-immunoreactive fi ber network with varicosities (arrowheads) in the ME. E SHBG-labeled epithelial cells in the choroid plexus. F No immunoreactivity was detected in the case of omitted primary antibody. Photo was taken in the PVN close to the third ventricle. Scale bars: 10 m for B, D, E; 20 m for A, F , and 40 m for C.

Western Blot Analysis and in nerve fi bers. No labeling in the nucleus could be Further samples of eluted protein were reconstituted in 20 l observed. SHBG-immunoreactive (SHBG-IR) cells and SDS-gel loading buffer (Novagen, Inc.) and boiled for 5 min. Elec- fi bers could be found in the magnocellular portions of bed trophoresis was performed in 4–10% (10 v/w) polyacrylamide gra- dient gel. Following SDS-PAGE, protein bands were blotted at nucleus of the stria terminalis (bnst) ( fi g. 1A–D), supra-

400 Vh, 20 ° C onto PVDF membranes (Amersham) in blotting buff- optic nucleus (son) (fi g. 1A–F, fi g. 2A,B), paraventricular er (6.35 g Tris base, 3.62 g glycine, 50 ml methanol in 250 ml dis- nucleus (pvn) (fi g. 1D,E, fi g. 2C, arrowhead) and the per- tilled water). SHBG was visualized with the ABC method as de- ifornical region (fo) (fi g. 1E). Single stained cells were scribed above. Rabbit protein-weight marker mix (Novagen) was found distributed in the medial preoptic area (mpoa) (fi g. used as molecular weight references. 1C). SHBG-IR cells were also detected rostral to the pvn in the posterior part of the hypothalamus (fi g. 1 E), an area that does not correspond to any previously described hy- Results pothalamic nucleus. Scattered parvocellular neurons were labeled in the arcuate nucleus (arc) (fi g. 1E) and in Localization of SHBG in the Human Hypothalamus the pvn (fi g. 2C, arrows). An overview of the hypothalamic SHBG distribution Several immunoreactive fi bers were seen in the infun- is given in fi gure 1. SHBG immunoreactivity could be dibulum (fi g. 1D,E, fi g. 3A). Strong arborization of stained detected in the perikarya of neurons and ependymal cells fi bers was seen in the ME (fi g. 1D, fi g. 2 D) and in the su-

290 Neuroendocrinology 2005;81:287–293 Herbert/Göthe /Caldwell /Bernstein /Melle /

von Eggeling/Lewis /Jirikowski Fig. 3. Consecutive semithin section of in- fundibulum immunostained for SHBG shows specifi c reaction product in single ax- onal varicosities ( A). The subsequent sec- tion reveals coexistence with OT in some of these structures ( B). Different arrowheads show examples of axons immunoreactive for both OT and SHBG. Scale bar: 5 m.

prachiasmatic nucleus (SCN), while stained perikarya were only seen in the anterior part of this nucleus (fi g. 47,500 50,000 52,500 55,000 1D). SHBG-IR cells were also found in a small portion of 1.0 49,597 + H the ependymal cells, lining the third ventricle (fi g. 2 C, empty arrowheads) and in most of the epithelial cells of 0.5 PC the choroid plexus (fi g. 2E, arrowheads). Diffuse SHBG- 0 IR occurred in the neuropil surrounding the third ven- 2.0 49,609 + H tricle (fi g. 2C); this staining was particularly abundant in 1.5 the mpoa. Immunostaining was completely absent in the 1.0 HT immunocytochemical controls (fi g. 2F), indicating that 0.5 neuronal staining was specifi c for SHBG-IR. 0 In semithin sections it was possible to identify distinct 49,608 + H axonal varicosities in the ME and the infundibulum. Ex- 3 2 amination of consecutive semithin sections revealed co- CSF existence of SHBG (fi g. 3A) with OT (fi g. 3B) in identical 1 axons. 0 53,909 + H

Intensity 15 Mass Analysis of the SHBG Protein SELDI-TOF MS analysis identifi ed two protein peaks 10 SER 49,608 + H with different molecular weights which were specifi cally 5 bound to the monoclonal SHBG antibody (fi g. 4). A 0 1.0 49,605 8 6 Da protein peak was identifi ed in the human 48,367 + H hypothalamus (HT), CSF, serum and choroid plexus. A 0.5 C1 53,909-Da protein was found only in the serum. The con- trol experiments showed only a 48,367-Da protein signal, 0 which is under-represented in the tissue probes. This peak represents an immunoglobulin heavy chain molecule 0.2 originating from the eluted antibody. The second control C2 experiment proving the specifi city of the protocol showed 0.1 no protein peaks. 0 47,500 50,000 52,500 55,000 Mass + charge

Fig. 4. SELDI-TOF analysis of the affi nity beads purifi ed human was found only in the blood plasma. C1: The fi rst control experi- tissue probes. PC: Plexus choroideus; HT: hypothalamus; CSF: ce- ment showed a 48,367-Da protein, which resembles eluted anti- rebrospinal fl uid; SER: serum. A 49,605 8 6 Da protein could be body. C2: Second control experiment shows spectrum of a hypo- identifi ed in all the studied tissue probes. The 53,909-Da protein thalamus sample eluted from unspecifi c antibody coupled beads.

Identifi cation of SHBG in Human Neuroendocrinology 2005;81:287–293 291 Hypothalamus reproductive behaviors [15, 16]. The observation that es- tradiol levels control brain SHBG expression [24] also suggests that SHBG is involved in neuroendocrine con- trol. Serum SHBG levels have been shown to increase in patients with Alzheimer’s disease [25, 26] . Further stud- Fig. 5. Western blot analysis of the affi nity beads purifi ed human tissue probes. Blot shows proteins eluted with polyclonal SHBG ies need to be done to demonstrate whether changes in antibody from the beads. Two distinct SHBG-immunoreactive central SHBG exist in various neurodegenerative ail- proteins with different molecular weights are present in the exam- ments. ined tissues: a smaller one in the hypothalamus, choroid plexus, The distribution of the labeled structures overlaps serum and in the CSF. In serum an additional larger protein was with the known location of hypothalamic neuropeptide observed. HT: hypothalamus; p: plexus choroideus; scf: spinal cord fl uid; s: serum; C: control; SHBG antibody. OT. In semithin sections we found coexistence of OT and SHBG in neurohypophysial projections, indicating that both are packaged for transport out of the cell bodies, pos- sibly into the posterior pituitary. We have previously Western Blot Analysis demonstrated the colocalization of SHBG and OT in the Western blot analysis of the purifi ed samples showed rat hypothalamic nuclei and posterior pituitary [6, 27]. bands at two different molecular weights (fi g. 5). A Our SELDI-TOF MS and Western blot studies show 49-kDa protein band could be found in all tissue probes, two SHBG molecules with different molecular weights. while a higher mass protein (around 53 kDa) was only We confi rmed evidence that there is a 53-kDa SHBG pro- seen in the serum sample. During this process the anti- tein in blood serum, which is known to originate from body was not detected in the mass interval presented. hepatocytes [28]. This 53-kDa moiety did not appear in the brain. However, a 49-kDa protein was identical in the hypothalamus and CSF that was also seen in blood serum. Discussion Alternatively, spliced and glycosylated variants of SHBG have been demonstrated in various species and in differ- Our immunocytochemical fi ndings describe for the fi rst ent tissues. The 49-kDa molecular weight could be identi- time cellular localization and distribution of SHBG in the fi ed as the SHBG variant, ABP (testosterone-estrogen- human hypothalamus. We found that neuronal perikarya binding protein; nomenclature from Swiss-Prot Protein and fi bers were detected in the magnocellular supraoptic knowledgebase. Englebienne and Doyen [29] showed a nucleus, bed nucleus of the stria terminalis, paraventricu- distinct SHBG in the serum, with a molecular weight sim- lar nucleus, perifornical nucleus, arcuate nucleus and in ilar to the one we found. They interpreted this as originat- the parvocellular suprachiasmatic nucleus. SHBG-IR fi - ing from the testis. bers were detected throughout the hypothalamic nuclei, in We suggest that SHBG in the CSF is rather of brain the ME and in the infundibulum. The staining of fi bers in origin than from blood. A portion of the hypothalamic hypothalamus, ME and infundibulum indicates that neuroendocrine cells in humans is likely to express SHBG. SHBG is most likely subject to axonal transport to the pos- Wang et al. [5] demonstrated SHBG production in rat terior pituitary lobe. It seems possible that posterior pitu- brain using Northern blots while we used RT-PCR to itary SHBG is liberated into the systemic circulation along identify production of SHBG in the rat brain [24]. There- with OT. This is also supported by previous immunoelec- fore, it is likely that the SHBG immunoreactivity identi- tron microscopical studies, which show SHBG localized in fi ed in this study in humans is also produced in the posterior pituitary secretory vesicles in the rat where it is brain. colocalized with OT [22] . Further, evidence that SHBG in SHBG, liberated from liquor contacting neurons [30] the hypothalamus shows the same characteristics as SHBG in the PVN, may enter the CSF, which would account for from secretory organs such as the testis and mouse lacrimal the observed diffuse immunostaining of the periventricu- glands [13, 23] may indicate that it is packaged in synaptic lar neuropil. Staining of ependymal and plexus epithelial vesicles for extracellular release. cells could be due to expression or uptake of the steroid- The presence of immunoreactive SHBG in human hy- binding globulin by those cells. It is known that ependy- pothalamus suggests its involvement in human neuro- mal cells are connected by gap junctions to the periven- physiology and possibly neuropathology. Our behavioral tricular neurons, thus allowing proteins from CSF to enter data in rats suggest that SHBG is important in control of the surrounding neuropil.

292 Neuroendocrinology 2005;81:287–293 Herbert/Göthe /Caldwell /Bernstein /Melle /

von Eggeling/Lewis /Jirikowski The CSF is known to contain signifi cant levels of bines with neurosteroids to control the modulation of SHBG [31] , which shows alteration at different physio- mood and behavior. logical and pathological stages such as in Alzheimer’s dis- ease [26]. The extensive distribution of both SHBG-IR neurons Acknowledgment and processes suggests multiple functional properties. SHBG may be synthesized and released into blood along Supported by IZKF, FSU-Jena, Project 1.2 to G. Jirikowski; NBL-2 to H.G. Bernstein; LUBOM Thüringen Award to Z. Her- with the neurohypophysial hormones. Intrahypothalamic bert, and MH069810 (J.D.C.). SHBG may be released within the brain where it com-

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Identifi cation of SHBG in Human Neuroendocrinology 2005;81:287–293 293 Hypothalamus