Abstract Tanycytes are specialized ependymal cells lin-ing the infundibular recess of the third ventricle of thecerebrum. Early and recent investigations involve tany-cytes in the mechanism of gonadotropin-releasing hor-mone (GnRH) release to the portal blood. The present in-vestigation was performed to obtain a specific immuno-logical marker of tanycytes and to identify the com-pound(s) responsible for this labeling. After 30 days oforgan culture, explants of bovine median eminenceformed spherical structures mostly constituted by tany-cytes. These tanycyte spheres were xenotransplanted torats, and the antibodies raised by the host animalsagainst the transplanted living tanycytes were used forimmunochemical studies of the bovine and rat medianeminence. This antiserum immunoreacted with two com-pounds of 60 kDa and 85 kDa present in extracts of bo-vine and rat median eminence. The individual immuno-blotting analysis of rat medial basal hypothalami showeda decrease in the amount of the 85-kDa compound incastrated rats as compared to control rats processed atoestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections through-out the rat central nervous system, immunoreacted spe-cifically with the hypothalamic tanycytes. Within tany-cytes, P-85 immunoreactivity was exclusively present inthe basal processes. It is suggested that the 85-kDa and60-kDa compounds correspond to two novel proteins se-lectively expressed by tanycytes. The possibility that

they are secretory proteins involved in GnRH release isdiscussed. Anti-P85 appears to be the first specific mark-er of hypothalamic tanycytes.

Keywords Median eminence · Tanycytes · Specificmarker · Organ culture · Xenotransplantation · Bovine ·Rat (Sprague Dawley)

Introduction

Tanycytes (Horstmann 1954) are specialized ependymalcells that line, among other parts of the central nervoussystem, the infundibular recess of the third ventricle ofthe cerebrum. They share some morphological and func-tional features with the radial glia present in the develop-ing central nervous system (McQueen 1994). At variancewith radial glia, hypothalamic tanycytes are presentthroughout the life span of all species investigated. Adistinct structural feature of tanycytes is that they pos-sess a single and long basal process. The cell bodies oftanycytes line the infundibular recess, and the basal pro-cesses project to distinct regions of the hypothalamus.Tanycytes do not constitute a homogeneous cell popula-tion. Thus, in the rat, four types of tanycytes have beendistinguished (Akmayev and Fidelina 1976; Rodríguez etal. 1979). Two of these types, beta-1 and beta-2 tany-cytes, are restricted to the median eminence. Beta-1 tan-ycytes are located in the lateral extensions of the infun-dibular recess and project their processes to the lateralregion of the median eminence, where they form a con-tinuous cuff separating neurosecretory fibers, mostlycontaining gonadotropin-releasing hormone (GnRH),from the portal capillaries lying laterally (Rodríguez etal. 1979, 1982; Flament-Durand and Brion 1985; Wittkowski 1998). Beta-2 tanycytes line the median emi-nence, and their processes terminate on the portal capil-laries lying in the medial region of the median eminence(Rodríguez et al. 1979, 1982; Flament-Durand and Brion1985; Kozlowski and Coates 1985). Beta tanycytes, thus,establish an anatomical link between the cerebrospinal

Financial support was provided by grants SA53/99, from Junta deCastilla y León, to J.L.B., and 1000435, from Fondecyt, Chile, toE.M.R.

J.L. Blázquez · F. Pastor · P. AmatDepartamento de Anatomía e Histología Humanas, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain

M. Guerra · B. Peruzzo · E.M. Rodríguez (✉ )Instituto de Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Casilla de Correo (P.O. Box) 567, Valdivia, ChileTel.: +56-63-221207, Fax: +56-63-221604

Cell Tissue Res (2002) 308:241–253DOI 10.1007/s00441-002-0528-9

R E G U L A R A R T I C L E

Juan Luis Blázquez · Montserrat GuerraFrancisco Pastor · Bruno Peruzzo · Pedro AmatEsteban Martín Rodríguez

Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes

Received: 8 October 2001 / Accepted: 16 January 2002 / Published online: 13 April 2002© Springer-Verlag 2002

fluid (CSF) and the portal blood. Furthermore, beta-1and -2 tanycytes play different roles in the barrier systemoperating in the medial basal hypothalamus (Peruzzo etal. 2000).

Although numerous experiments have been carriedout to elucidate the function of the hypothalamic tanycytes, conclusive evidence has not yet been ob-tained (Rodríguez 1976; Akmayev and Fidelina 1981; Flament-Durand and Brion 1985; McQueen 1994; Wittkowski 1998; García-Segura et al. 1999). One of thepossibilities more thoroughly investigated is the proba-ble involvement of tanycytes in the release of hypotha-lamic hormones from their terminals to the portal blood(Rodríguez et al. 1982, 1985; Flament-Durand and Brion1985; Wittkowski 1998; García-Segura et al. 1999). Theinvolvement of tanycytes in the regulation of GnRH re-lease into the portal blood has been proposed by Rodríguez et al. (1982, 1985). This possibility has beenstrongly substantiated by recent investigations. Tany-cytes appear to participate in the release of GnRHthrough two different mechanisms. One of them impliesthe transient and cyclic remodeling of the spatial rela-tionship between the GnRH terminals and the tanycyteprocesses, leading to a retrieval of the tanycyte terminalsduring proestrus, thus facilitating the release of this hor-mone into the portal blood (Hökfelt 1973; King and Rubin 1994; Wittkowski 1998; Prevot et al. 1999). Thepresence of estrogen receptors in tanycytes (Langub andWatson 1992), and the capacity of these cells to incorpo-rate and accumulate insulin growth factor I (Fernández-Galaz et al. 1996) has led to the suggestion that thesetwo compounds are involved in the cyclic plastic chang-es of tanycytes (García-Segura et al. 1999).

The second method of tanycyte-GnRH neuron com-munication, via cell-cell signaling mechanisms, isstrongly supported by the finding that estrogen stimulat-es tanycytes to sequentially synthesize and secrete trans-forming growth factor alpha and then prostaglandin E2,which in turn stimulates GnRH release (Ojeda et al.1990, 1992, 1997; Ojeda and Ma 1998). Furthermore,the use of conditioned medium of cultured hypothalamicastrocytes-tanycytes indicates that these cells secrete astill enigmatic compound that potentiates the stimulatoryeffect of prostaglandin E2 on GnRH release (Ma et al.1997). On the other hand, the ultrastructural characteris-tics of beta-1 tanycytes support the possibility that thesecells secrete peptides and/or proteins (Rodríguez et al.1979). The aim of the present investigation was twofold:to identify a compound that might be exclusively or pref-erentially located in tanycytes, and then to obtain someevidence about its functional significance. For this pur-pose, explants of bovine median eminence organ cul-tured for several weeks were xenotransplanted to rats.Antibodies raised by the host animals against the trans-planted living tanycytes were used for immunochemicalstudies of the bovine and rat median eminence.

Materials and methods

Organ culture of bovine median eminence

The hypophysial neural stalks of cows of either sex were obtainedat the Valdivia (Chile) slaughterhouse, from 9:30 a.m. to 12:00a.m., during the summer. Immediately after dissection, the neuralstalk was opened by inserting one arm of a pair of fine scissorsthrough the infundibular recess, thus exposing the ventricular sur-face. This tissue lamina, constituted by the median eminence andthe pars tuberalis, was immersed in cold (4°C) Hanks’ solution(Sigma, St. Louis, USA), and transported to the tissue culture lab-oratory. The time elapsed between death and immersion in Hanks’solution was about 10–20 min. Under a dissecting microscope, thetissue laminae were divided into several strips to facilitate the sep-aration of the median eminence from the pars tuberalis. In order toavoid damage to the ependymal endings lying close to the pars tu-beralis, a thin layer of pars tuberalis tissue was left attached to themedian eminence. Explants of median eminence of about0.5–1 mm each side were cultured for 7 days, 14 days, and30 days in nutrient mixture F-12 (HAM; Sigma, St. Louis, USA)mixed with Dulbecco’s modified Eagle’s medium (DME; Sigma,St Louis, USA) at a ratio of 1:1, containing an antibiotic/antimy-cotic cocktail. Each group of 20–25 explants was cultured in a 10-cm Petri dish containing 15 ml of medium. The explants wereincubated at 37°C in an atmosphere of 95% air and 5% CO2. Themedium was changed at 24 h, and thereafter every 3 days. After7 days, 14 days, and 30 days of culture, a few explants were col-lected and processed for light and scanning electron microscopy.

Microscopic control of explants

Explants were fixed by immersion in Bouin’s fluid for 24 h, dehy-drated, and embedded in Paraplast. Adjacent 5-µm-thick sectionswere stained with hematoxylin-eosin or processed for the immu-noperoxidase method (Sternberger et al. 1970), using antiseraagainst the following compounds: (1) glial fibrillary acidic protein(GFAP, 1:1,000; Sigma, St Louis. Mo., USA); (2) glucose trans-porter I (Glut I, 1:2,000; kindly provided by Coralia I. Ribas, Me-morial Sloan-Kettering Cancer Center, N.Y., USA); (3) bovineneurophysins (1:1,000; obtained at Instituto de Histología y Pat-ología, Universidad Austral de Chile, Valdivia, Chile); (4) GnRH(1:1,000; Sigma); (5) 200-kDa polypeptide of neurofilaments(1:1,000; obtained from Instituto de Histología y Patología, Uni-versidad Austral de Chile, Valdivia, Chile).

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Fig. 1A Scanning electron microscopy of an explant of bovinemedian eminence cultured for 30 days; the median eminence tis-sue is mostly established by tanycytes (T) forming a globularstructure, whereas the pars tuberalis tissue (PT) keeps its originalpattern.B Paraffin section through a median eminence explant cul-tured for 30 days, immunostained for GFAP; arrows point to bun-dles of immunoreactive tanycyte processes. C Section next to thatshown in B, immunostained for Glut I; arrows point to bundles ofimmunoreactive tanycyte processes. D Paraffin section of a bovinemedian eminence immunostained for GFAP; the basal processesof tanycytes (T) are immunoreactive (III V third ventricle, PV por-tal vessel). E Detailed magnification of B. Arrows point to GFAP-immunoreactive tanycytes. F Detailed magnification of C. Glut Iimmunoreactivity is located in the tanycyte processes (arrows). G Paraffin section through the bovine median eminence (ME). Im-munoreaction using anti-P85. Tanycyte processes are strongly re-active (arrow); their fine distal branches are less reactive (asterisk;IR infundibular recess). H Detailed magnification of G. Immuno-reactivity is missing from the tanycyte cell body (arrowheads) andpresent in the basal processes (arrow). I High magnification oftwo basal processes of tanycytes showing that the P-85 immunore-activity is mostly concentrated in the “plasma membrane region”(arrows). Bars A 100 µm; B C 66 µm; D–F 33 µm; G 50 µm; H 23 µm; I 9 µm

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For scanning electron microscopy, explants were fixed with2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2 h,and then postfixed in 1% osmium tetroxide in the same buffer. Af-ter critical-point drying, the explants were covered with gold andvisualized under a scanning electron microscope.

Xenotransplantation of bovine median eminence explants

After 30 days of culture, the median eminence tissue of each ex-plant formed an ovoid or spherical structure, which remained at-tached to a plate-like structure corresponding to the pars tuberalistissue (Fig. 1A). Under a dissecting microscope, most of the parstuberalis tissue of the 30-day-old explants was removed and thespherical median eminence explants (Fig. 1B, C) were transplant-ed to adult female Sprague-Dawley rats (Holtzman strain). Underether anesthesia, each rat (n=6) was grafted with approximately 30explants distributed under the kidney capsule, peritoneal cavity,and the dorsal skin. Rats used in this investigation were handledaccording to the guidelines of the National Research Council ofChile and to Spanish regulations (R.D. 223/1988).

Collection of antisera

Blood samples were obtained at 8 days, 12 days, 16 days, 20 daysand 24 days after transplantation, under ether anesthesia, by orbit-al puncture. At day 28 the ether-anesthetized rats were bled byheart puncture. Serum obtained from each blood sample was ali-quoted and kept frozen. The presence of antibodies reacting withcompound(s) present in the hypothalamus and/or hypophysis wasinvestigated in all blood samples by immunocytochemistry; thoseblood samples displaying the highest titers of antibodies were alsoused for immunoblotting.

Immunocytochemistry

Twenty male or female Sprague-Dawley rats (Holtzman strain),about 3 months old, were used. In the present investigation, theday of the estrus cycle at which the female rats were killed wasnot established. The central nervous system was fixed by vascularperfusion with Bouin’s fluid. Fixation was with the rats underether anesthesia and was performed around noon. Embedding wasin Paraplast. Some of the brains were oriented to obtain transver-sal sections and others for sagittal sectioning. Frontal serial sec-tions (8 µm thick) through the diencephalon and sagittal sectionsthrough the middle third of the central nervous system were ob-tained. In addition, six bovine pituitary stalks containing the medi-an eminence and the pars tuberalis were collected at the Valdiviaslaughterhouse 10–20 min after death, immersed in Bouin’s fluidfor 2 days and embedded in Paraplast to obtain transversal sec-tions. The tissue sections were processed for the immunoperoxi-dase method (Sternberger et al. 1970). The serum samples ob-tained from the xenotransplanted rats were used as primary anti-body, at 1:250, 1:500, and 1:1,000 dilutions. The sections were se-quentially incubated in (1) the primary antiserum, for 18 h; (2) thesecondary antibody (anti-rat IgG; Sigma, St. Louis, Mo., USA) di-luted 1:15, for 30 min; and (3) rat peroxidase-antiperoxidase(PAP; Dr. H. Folch, Instituto de Immunología, Universidad Aus-tral de Chile, Valdivia, Chile), 1:75 dilution, for 30 min. DAB wasused as electron donor. All antisera and the PAP complex were di-luted in TRIS buffer, pH 7.8, containing 0.7% nongelling seaweedlambda carrageenan (Sigma) and 0.5% Triton X-100 (Sigma). Useof preimmune serum, and omission of incubation in the primaryantiserum in the immunostaining procedure, were used as controltests. All incubations were at room temperature in a moist cham-ber.

Tissue extracts

Bovine median eminences were collected following the same pro-cedure used for the preparation of median eminence explants. Af-

ter dissection, the median eminence tissue obtained from six cowswas pooled and extracted. The brain of 20 female rats was dissect-ed out under ether anesthesia and the medial basal hypothalamusseparated under a dissecting microscope. Two pools of ten rat me-dial basal hypothalami each were prepared. The bovine and rat tis-sue samples were extracted in a medium containing 50 mM am-monium bicarbonate, 0.5 mM phenylmethylsulfonyl fluoride(PMSF) and 1 mM ethylenediaminetetraacetic acid (EDTA). Eachtissue sample was homogenized in 0.5 ml of extraction mediumusing a Potter-Evelhein homogenizer, sonicated for 15 s, andcooled on ice for 30 s. This sonication-cooling cycle was repeatedthree times. The extract was then centrifuged at 12,000g, for45 min, at 4°C. The supernatant was used as a crude extract of bo-vine median eminence or rat medial basal hypothalamus. The pro-tein content was determined by the Bradford’s method. Aliquotswere lyophilized and stored at –20°C.

Immunoblotting

Samples of 90 µg protein of bovine and rat median eminence ex-tracts were subjected to SDS-polyacrylamide gel electrophoresis(Laemmli 1970), using a 5–15% polyacrylamide linear gradient.Gels were electrotransferred to nitrocellulose sheets (Towbin et al.1979) using 25 mM TRIS-HCl, pH 8.3, containing 192 mM gly-cine, 0.2% SDS, and 20% methanol, at 0.1 A; transfer time was8 h. Protein-binding sites were saturated with 5% nonfat milk in0.1 M phosphate-buffered saline (PBS), containing 0.15 mM NaCl.This was followed by immunoperoxidase (PAP) staining, using 4-chloro-1-naphtol (Sigma) as electron donor. One of the antiserathat in immunocytochemistry showed the highest titer of antibodiesreacting with the hypothalamic tanycytes was used as primary anti-body, at a dilution of 1:500 in PBS containing 0.7% lambda carra-geenan (Sigma) and 0.01% thimerosal. The blots were sequentiallyincubated in: (1) the primary antibody, for 18 h; (2) anti-rat IgG,1:50 dilution, for 2 h; (3) rat-PAP, 1:75 dilution, for 1 h. Peroxidasereaction product was visualized by incubating the blots in a solu-tion containing 0.01% 4-chloro-1-naphtol (Sigma) in methanol, and0.01% H2O2 (Merck-Chile, Santiago). Incubations were at roomtemperature, under continuous agitation. Control transfers were in-cubated with preimmune serum or processed for the PAP method,but omitting incubation in the primary antiserum.

Quantitative immunoblotting of median eminence extracts from experimental rats

Three groups of female rats were prepared: (1) at dioestrus (n=4);(2) at oestrus (n=4); (3) castrated for 1 month (n=4). Under etheranesthesia, the medial basal hypothalamus of each rat was dissect-ed out and extracted individually, as described. A 30-µg samplefrom each extract (1/20 medial basal hypothalamus) was used forSDS-PAGE blotting. Trans-blot 0.2-µm nitrocellulose sheets werepurchased from Biorad (Hercules, Calif.,USA). Samples corre-sponding to the three groups of rats were run and processed simul-taneously. Detection of the immunoreactive compound(s) was car-ried out using an enhanced chemiluminescence system (Super Sig-nal, Pierce, Rockford, Ill., USA; Walker et al. 1995). The anti-tan-ycyte antiserum and anti-neurophysin were used as primary anti-bodies. The blots were sequentially incubated in: (1) primary anti-body (anti-tanycyte 1:1,500; anti-neurophysin 1:2,000) dilutedwith 5% nonfat milk in 0.1 M PBS, containing 0.15 mM NaCl, for1.5 h; (2) anti-mouse IgG peroxidase conjugated (Sigma), diluted1:75,000, for 1.5 h; (3) PBS-Tween 20 (0.05%), for 1.5 h; (4) Su-per Signal substrate (1:1 ratio of Luminol/enhancer solution to sta-ble peroxidase solution), for 20 min. The blots were exposed toBiomax MR-1 film (8G; Kodak), for 15–90 s. Development wasin D-72 (Kodak), and fixation in U3 solution (Kodak).

The intensity of the immunoreactive bands from all blots wasquantified by the Un-Scan-It gel automated digitizing system(Asknet, Karlsruhe, Germany). The statistical analysis was per-formed using Student’s t-test, and P<0.005 was considered statisti-cally significant.

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Transmission electron microscopy

The central nervous system of six male or female rats, under sodi-um thiopental (30 mg/kg b.w.) anesthesia, was fixed by vascularperfusion with 5% glutaraldehyde in 0.1 M phosphate buffer, pH.7.4. The brain was dissected out and a block of tissue containingthe medial basal hypothalamus was immersed overnight in thesame fixative. Postfixation was in 1% osmium tetroxide in thesame buffer, for 2 h. Dehydration was in acetone and embeddingin Durcupan. Ultrathin sections were contrasted with uranyl ace-tate-lead citrate and analyzed using a Philips EM 201 electron mi-croscope.

Results

Organ culture of bovine median eminence

The histological and immunocytochemical analyses ofthe median eminence explants at various time intervalsduring the 1st month of culture showed a progressivedisappearance of the nerve fibers immunoreactive forGnRH and neurofilaments; although neurophysin fibersdisplayed progressive regression, many of them still per-sisted in the 30-day-old explants. Tanycytes survivedwell throughout the whole culture period and underwenta progressive spatial rearrangement. They became dense-ly packed in the form of a globular structure (Fig. 1A),

with their basal processes occupying the core of thesphere and their former ventricular pole lining the sur-face of the sphere (Fig. 1B, C). Tanycyte processes werestrongly reactive for GFAP (Fig. 1B, E), whereas theircell body that lines the explant did not show immunore-active GFAP, resembling the situation found in the bo-vine median eminence proper (Fig. 1D). The cell pro-cesses but not the cell body of the in situ and the cul-tured tanycytes reacted with anti-Glut I (Fig. 1C, F). Aspreviously described (Peruzzo et al. 2000), Glut I immu-noreactivity was circumscribed to the plasma membraneregion (Fig. 1F). The free surface of tanycytes wassmooth, lacking microvilli and cilia.

Each explant produced a single sphere that was at-tached to a base-like structure formed by the pars tuber-alis tissue (Fig. 1A). Thus, at variance with the medianeminence tissue, the pars tuberalis present in the explantmaintained its original shape. Before transplantation intorats, most of the pars tuberalis tissue was removed sothat the grafted explants primarily consisted of medianeminence spheres.

Antisera obtained by xenotransplantation of the bovine median eminence reveal two proteins in extracts of the bovine and rat median eminence

The sera collected from two of the rats that receivedgrafts, 24 days and 28 days after transplantation of ex-plants of bovine median eminence, were used to im-munostain blots of extracts of bovine and rat median em-inence. In both types of extract, the antisera revealed twobands, of 60 kDa and 85 kDa, with the latter being morestrongly reactive (Fig. 2A). When the enhanced chemilu-minescence system was used, rat medial basal hypothala-mi extracted individually could be used for immunoblot-ting analysis. In fact, aliquots corresponding to 1/20 ofrat medial basal hypothalamus produced a strong signalin the blot. Under these conditions the two compounds of

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Fig. 2A, B Immunoblotting of median eminence extracts using anantiserum raised in rats xenotransplanted with 30-day-old explantsof bovine median eminence. A Blots of a bovine median eminenceextract processed according to the immunoperoxidase method, in-cubated with antiserum (lane 1) and pre-immune serum (lane 2).Two immunoreactive bands of 60 kDa and 85 kDa are seen. B Blots of medial basal hypothalami of nine individual rats ob-tained at dioestrus (lanes 1–3), oestrus (lanes 4–6), and 30 daysafter castration (lanes 7–9), using the enhanced chemilumines-cence system. The microdensitometric analysis showed that the85-kDa band is about 10 times denser than the 60-kDa band, andthat the density of the reactive bands is greatly reduced in the cas-trated rats

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60 kDa and 85 kDa could be detected in each individualrat; the band of 85 kDa displayed an optical densityabout 10 times higher than the 60-kDa band, as estab-lished by microdensitometry (Fig. 2B).

The individual immunoblotting analysis by microden-sitometry of 12 medial basal hypothalami correspondingto female rats at dioestrus (n=4), oestrus (n=4) and after30 days of castration (n=4), showed a 40% and 64% de-crease (P<0.005) in the amount of the 85-kDa compoundin the castrated rats, as compared to the dioestrus andoestrus groups, respectively (Fig. 2B). The 60-kDa bandwas weakly immunoreactive in the samples from dioes-trus and oestrus rats, and was not detected in the sampleof castrated rats (Fig. 2B). At variance, the blots reactedwith anti-neurophysin, used as an internal control of theprotein content of the samples, showed a 14-kDa immu-noreactive band that was stronger in the castrated ratsthan in the oestrous and dioestrous rats (not shown).

Antisera obtained by xenotransplantation of the bovine median eminence immunostain selectivelythe hypothalamic tanycytes

All blood samples from the six xenotransplanted ratswere used to immunostain sections of rat and bovine me-dian eminence. Four out of the six rats that receivedgrafts raised antibodies that reacted weakly with tany-cytes and with neurophysin fibers. The other two ratsproduced antisera that immunoreacted selectively withtanycytes. The sera collected from these two rats, be-tween 12 and 28 days after xenotransplantation, whenused for immunocytochemistry, immunostain the rat andbovine tanycytes, at dilutions of 1:250 and 1:500. Seraobtained at days 24 and 28 after transplantation alsoshowed immunoreactivity with tanycytes at a 1:1,000 di-lution. According to the method used, only IgG antibod-

ies could be detected. The presence of reactive IgM anti-bodies was not investigated. Since the antibodies react-ing with tanycytes strongly react with a protein of85 kDa, for descriptive purposes this antiserum will belabeled as anti-P85.

In sections through the bovine neural stalk, anti-P85reacted strongly and selectively with tanycytes, especial-ly with their basal processes (Fig. 1G, H, I); the immu-noreaction was stronger in the “plasma membrane re-gion” (Fig. 1I).When anti-P85 was used to immunostainsagittal sections of the rat brain and frontal sections ofthe rat diencephalon, the hypothalamic tanycytes werethe only specifically reactive structure (Fig. 3A), sincethe reaction seen in some ependymal cells of the choroidplexus was also found in control sections immunostainedusing pre-immune serum or omitting the incubation inthe primary antiserum. All subtypes of tanycytes, name-ly, alpha-1 and -2, and beta-1 and -2, were immunoreac-tive with anti-P85 (Fig. 3B–E). At a 1:1,000 dilution ofanti-P85, beta tanycytes were more reactive than alphatanycytes (Figs. 3B, 5A).

The anti-P85 reactive material was not evenly distrib-uted throughout the tanycytes. It was virtually missingfrom the cell body and the ventricular pole (Fig. 3D, E).The basal processes, from their site of origin at the infra-nuclear region, and throughout most of their length, werestrongly reactive (Fig. 3B–E). At high resolution it be-came patent that the immunoreaction was stronger at thesurface (plasma membrane?) than in the core of the basalprocess (Fig. 3E).

The basal processes of beta-1 tanycytes became clear-ly outlined by the immunoreaction. Each beta-1 tanycyteprojected a thick, single process traversing dorsocaudallythe lateral region of the median eminence; when reach-ing the external palisade layer, each process divided intoa few thinner branches that ended at the perivascularspace of the portal capillaries lying in the lateral regionof the median eminence (Figs. 3C, 4A, B). At the rostrallevel, the whole median eminence was lined by beta-1tanycytes (Fig. 3B; cf. Rodríguez et al. 1979). The anti-P85-immunoreactive material was very abundantthroughout most of the length of the basal processes ofbeta-1 tanycytes, but it was scarce in their preterminalportion, and scarce or missing in their terminals contact-ing the perivascular space of the portal vessels (Figs. 3B,4A, B). This “longitudinal” zonation in the distributionof the immunoreactive material throughout tanycytes be-ta-1 became more distinct when anti-P85 was used at1:1,000 dilution (Fig. 5A). A high-magnification analy-sis showed that the immunoreaction was evenly distrib-uted within the cytoplasm of the process (Fig. 4B).

In beta-2 tanycytes, localized in the middle third ofthe median eminence, P85-immunoreactive material wasmissing from the cell body, the proximal third of the bas-al process, and from the terminals contacting the portalvessels (Fig. 4A, C). These tanycytes projected a singlebasal process that, at the external palisade layer of themedian eminence, divided into numerous thin branchesthat ended on the portal capillaries (Fig. 4A, C). A few

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Fig. 3A–E Frontal sections through the medial basal hypothala-mus of female adult rats. Immunoreaction using anti-P85. A Low-power view of the diencephalon at the level of the rostral portionof the median eminence, showing selective immunostaining oftanycytes (thick short arrow); long arrow points to nonreactiveependyma of the third ventricle. B Detailed magnification of theprevious figure showing strong immunostaining of beta tanycytes(large arrow) and a weak reaction of alpha tanycytes (small ar-row). Asterisk points to the weakly reactive tanycyte terminals atthe palisade layer. C Section through the arcuate nucleus (AN)/me-dian eminence region. Beta-1 tanycytes (large arrow) lining thelateral recess (LR) of the infundibular recess are more strongly re-active than alpha tanycytes (small arrow) and beta-2 tanycytes(asterisk; III V third ventricle, LR lateral recess of the infundibularrecess). D Immunoreactive alpha tanycytes project their basal pro-cesses (arrows) into the neuropil of the ventromedial and arcuatenuclei (III V third ventricle). E Detailed magnification of C show-ing immunostaining of the basal processes of beta tanycytes andthe lack of immunoreactivity in their cell body and at their ventric-ular surface (asterisk). Small arrows point to the strong labeling ofthe plasma membrane of a tanycyte process. Large arrow points tothe site of origin of an immunoreactive basal process in the infra-nuclear region (V third ventricle). Bars A 500 µm, B 44 µm, C 40 µm, D 20 µm, E 8 µm

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Fig. 4A–C Frontal sectionthrough the medial basal hypo-thalamus of a female adult rat.Immunostaining using anti-P85 at 1:500 dilution. A Thestrongly reactive beta-1 tany-cytes (large arrow) lining thelateral recess (LR) of the infun-dibular recess project their bas-al processes to the lateral re-gion of the median eminence(LME). Beta-2 tanycytes onlydisplay immunoreactivity in thedistal portion of their basal pro-cesses (small arrows) localizedin the palisade layer of the me-dial region of the median emi-nence (MME). Broken lines in-dicate the approximate borderbetween the medial and lateralregions of the median emi-nence (PT pars tuberalis). B Detailed magnification of be-ta-1 tanycytes; the basal pro-cesses are strongly immunore-active with the exception oftheir terminal portion that ispoorly immunostained (aster-isk; C portal capillary, PT parstuberalis). C Detailed magnifi-cation of beta-2 tanycytesshowing the immunoreactivityof the basal processes and thelack of immunoreactive materi-al in the terminals of these pro-cesses (asterisk). Arrow pointsto an immunoreactive sub-ependymal cell (C portal capil-laries). Bars A 40 µm; B, C 8 µm

Fig. 5A–C Frontal section through the median eminence of a fe-male adult rat. Immunostaining using anti-P85 at a 1:1,000 dilu-tion. A Lateral region of the median eminence. The cell bodies ofbeta-1 tanycytes lack immunoreactivity; the basal processes ofthese tanycytes are strongly immunoreactive (arrow), with the ex-ception of their terminal portion (asterisk). The thin wall of a largevacuole framed by a square, is immunoreactive. Insert: Detailedmagnification of a vacuole to show the immunoreactivity of itsthin wall (arrow). B Lateral region of the median eminence show-

ing a vacuole with an immunoreactive wall (full arrow) and a vac-uole with an immunonegative wall (broken arrow; C capillary, T immunoreactive tanycyte processes). C Transmission electronmicrograph of the lateral region of the rat median eminence show-ing tanycyte processes (TP) displaying vacuoles of different diam-eters (stars) and a large vacuole lined by a thin cytoplasmic ring.Arrows point to caveolar structures suggesting fusion of smallvacuoles to form the large vacuole (L lipid droplets in tanycytes, A axon profiles, C portal capillary). Bars A 20 µm; B 8 µm; C 1 µm

subependymal cells projecting an immunoreactive basalprocess similar to those of beta-2 tanycytes were seen inthe medial region of the median eminence (Fig. 4C).

The use of preimmune serum or the omission of incu-bation in the primary antiserum in the immunostaining ofsections of the rat central nervous system showed somereaction in the ependymal cells of the choroid plexus;however, all cell elements of the hypothalamus, includ-ing tanycytes, were not reactive.

The large vacuoles present in the palisade zone of the lateral median eminence are intracellular formationsof the processes of beta-1 tanycytes

Large vacuoles are consistently found in the lateral re-gion of the median eminence, especially in the vicinityof the tuberoinfundibular sulcus (cf. Brion et al. 1982).Transmission electron-microscopic analysis clearlyshowed that these vacuoles are intracellular structures lo-cated within dilated portions of processes most likelycorresponding to basal processes of tanycytes (Fig. 5C).This was confirmed by the fact that the thin cytoplasmlining the vacuoles was immunoreactive with anti-P85(Fig. 5A, B). Vacuoles whose wall did not react with an-ti-P85 were also found (Fig. 5B). Vacuoles ranging insize between 1 and 60 µm, as well as coalescent vacu-oles, coexisted within the same tanycyte process(Fig. 5C). The content of the vacuoles was electron lu-cent (Fig. 5C).

Discussion

Strategy to raise antibodies specifically marking tanycytes

Antibodies against numerous compounds have been usedfor the immunolabeling of hypothalamic tanycytes. Noneof them has the property to immunolabel tanycytes spe-cifically.

The present strategy, although unconventional, wasdesigned to obtain a specific immunological marker oftanycytes and, if successful, to identify the compound(s)responsible for this labeling. This experimental designincluded several sequential steps: (1) to obtain a prepara-tion of differentiated bovine tanycytes; for this reason or-gan culture was selected instead of cell culture; (2) toobtain a preparation enriched in tanycytes; this wasachieved by a prolonged organ culture of the median em-inence explants that resulted in the degeneration andsubsequent loss of the large mass of nerve fibers formingpart of the median eminence. Under the present cultureconditions, tanycytes remained differentiated and rear-ranged spatially forming an spherical structure; (3) totransplant this preparation of bovine living tanycytes intorats (xenotransplantation) to trigger a host-versus-graftreaction, with the consequent production of antibodiesagainst the grafted cells (Roitt et al. 1998); (4) to search

for anti-tanycyte antibodies at early intervals after trans-plantation.

The xenotransplanted tissue survives for about12 days after grafting; then it starts to become destroyedby the host reaction (E.M. Rodríguez, unpublished ob-servation). Thus, the antibodies detected at day 12 oftransplantation were raised against the living grafted tan-ycytes and, therefore, should be against proteins exposedto the cell exterior. We found that: (1) the immunoreac-tive properties of these early antibodies are not differentfrom those obtained 2 weeks after the graft has been de-stroyed, (2) the titer of the latter is higher, (3) in immu-noblots of fresh median eminence tissue these antiserareact with two bands only, and (4) the material immuno-reacting with these antisera is localized in the cytoplasmand in the plasma membrane of the tanycyte processes.These findings suggest that: (1) the antigen(s) triggeringthe early immune response is (are) also present intracel-lularly; (2) after destruction of the grafted cells, the ex-posure of this intracellular antigen(s) to the immunesystem is equivalent to a reimmunisation; (3) proteinspresent intracellularly but not exposed to the cell exteri-or, do not trigger the production of antibodies.

The 85-kDa and 60-kDa immunoreactive proteins appearto be compounds exclusively present in hypothalamictanycytes

Numerous compounds have been immunocytochemical-ly detected in the hypothalamic tanycytes. Some of themare cytoskeletal proteins, such as GFAP (Redecker et al.1987), vimentin (Leonhardt et al. 1987), and plectin(Errante et al. 1994); others belong to the family ofgrowth factors, namely, the basic fibroblast growth fac-tor (Gibson et al. 2000), the transforming growth factorsalpha (Ojeda et al. 1990, 1992, 1997) and beta (Melcangiet al. 1995; Martini et al. 1997), and the insulin growthfactor I (García-Segura et al. 1991; Dueñas et al. 1994).Several receptor and binding proteins have also been de-tected in tanycytes, i.e., fibroblast growth factor receptor1 (Matsuo et al. 1994), insulin growth factor I receptor(Cardona-Gómez et al. 2000), transforming growth factor alpha receptor (Ojeda and Ma 1998), prolactin re-ceptor (Lerant and Freeman 1998), estrogen receptor (Langub and Watson 1992), glutamate 5–7 kainate re-ceptors (Diano et al. 1998; Eyigor and Jennes 1998), andinsulin growth factor binding protein-2 (Cardona-Gómezet al. 2000). Special attention deserves a dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is present in neurons bearingdopamine D1 receptors (Hökfelt et al. 1988). This pro-tein and its messenger are highly expressed in tanycytesof the medial basal hypothalamus (Everitt et al. 1986;Hökfelt et al. 1988; Meister et al. 1988; Fekete et al.2000) and in pituicytes of the neural lobe (Meister et al.1989).

Considering that: (1) those tanycytic compounds forwhich the molecular mass has been established do not

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correspond to the 85-kDa and 60-kDa compounds de-tected by the anti-tanycyte antisera in the present investi-gation; (2) none of the compounds detected in tanycyteshas been shown to be exclusively present in these cells;(3) the anti-P85 immunoreactive material is exclusivelypresent in the hypothalamic tanycytes, it seems highlyprobable that the 85-kDa and 60-kDa compounds corre-spond to two novel proteins selectively synthesized bytanycytes. Anti-P85 appears to be the first specific mark-er of hypothalamic tanycytes.

Worth considering is the fact that anti-P85, which wasraised against proteins of bovine origin, reveals the 85-kDa and 60-kDa proteins in extracts of bovine and ratmedian eminence, and immunoreacts with the bovineand rat tanycytes, suggesting that the two proteins areconserved in these two mammalian species.

Functional significance of the 85-kDa and 60-kDa compounds present in tanycytes

The reduction in the amount of immunoreactive 85-kDaand 60-kDa compounds in the castrated rats suggests thatsynthesis and/or release of these tanycyte proteins maybe under the influence of ovarian hormones. This possi-bility is supported by the presence of estrogen receptorsin tanycytes (Langub and Watson 1992), and the findingthat estrogen stimulates tanycytes to synthesize and se-crete other compounds such as the transforming growthfactor alpha and prostaglandin E2 (Ojeda et al. 1990,1992, 1997; Ojeda and Ma 1998). More sensitive meth-ods of quantification of the 85-kDa and 60-kDa proteinswill be necessary to evaluate their probable variation inthe estrous cycle.

The intracellular distribution of the anti-P85 immuno-reactivity allows us to make some functional consider-ations. The absence of these proteins from the cell body,bathed by the ventricular cerebrospinal fluid, and fromthe terminals contacting the portal vessels, suggest thatthese compounds are not involved with events occurringat both polar ends of tanycytes. On the other hand, thepresence of these immunoreactive proteins in the basalprocesses of tanycytes would associate them with phe-nomena occurring at this level. Although several types ofpeptidergic and aminergic fibers of the median eminenceappear to establish contacts with tanycytes, the best sub-stantiated neuron-tanycyte relationship is that establishedbetween the basal processes of beta-1 tanycytes and theGnRH axons occurring at the lateral region of the medi-an eminence (Flament-Durand and Brion 1985; King andRubin 1994). The following observations suggest thepossibility that the 85-kDa and 60-kDa immunoreactivecompounds are involved in the tanycyte-GnRH neuroncommunication:

1. The fact that anti-P85 was obtained shortly afterxenotransplantation of living tanycytes, indicates thatthe 85-kDa and 60-kDa compounds are associated tothe plasma membrane and exposed to the cell exterior.

These proteins could correspond to membrane recep-tors or secretory proteins that, somehow, are under theinfluence of ovarian hormones. Worth mentioning isthe fact that the 20-kDa precursor of transforminggrowth factor alpha is synthesized as a transmem-brane protein, with the active processed form of5 kDa resulting from a proteolytic cleavage of the ex-ternal precursor domain (Derynck 1988)

2. Transforming growth factor alpha, a transmembraneprotein, is secreted by hypothalamic astrocytes-tany-cytes to participate in the mechanisms of GnRH re-lease

3. The amount of 85-kDa protein is affected by castra-tion, a condition that also affects the GnRH release.

The large cisternae occurring in the lateral region of the median eminence are located within basal processes of beta tanycytes

These large cisternae were described by Matsui (1966)and Scott and Knigge (1970). They do not incorporatehorseradish peroxidase injected intraventricularly, sug-gesting that they are intracellular structures (Brion et al.1982). Transmission electron-microscopic studies (Brionet al. 1982; Bodoky et al. 1979; Amat et al. 1999) indi-cate that these large cisternae are localized within tany-cytes. The availability of a specific immunological mark-er for tanycytes has allowed us, in the present investiga-tion, to demonstrate that many of these vacuoles are, in-deed, within basal processes of tanycytes labeled withanti-P85. A second look at the ultrastructure of thesevacuoles has shown that the large cisternae are formedby the fusion of smaller vacuoles. On the other hand,there is a remarkably topographical correlation betweenthe distribution of the basal processes of beta-1 tanycytesand, consequently of the large vacuoles they contain, andthat of the GnRH axons and their terminals (Bodoky etal. 1979; Rodríguez et al. 1979). The fact that the contentof the large vacuoles is electron lucent and that it lacksimmunoreactivity with a long series of antibodies againstneuronal and glial markers and neuropeptides (unpub-lished observation by the authors) supports the liquid na-ture of such a content. Since these tanycyte processescontaining the large vacuoles are the ones undergoingspatial plastic changes during the estrous cycle (Kingand Rubin 1994; Wittkowski 1998; Prevot et al. 1999),the possibility that these vacuoles play a role in such aplasticity has to be considered.

Acknowledgements We wish to thank Dr. Sara Rodríguez for hervaluable help in the transplantation experiments, Mr. Genaro Alvi-al for his technical assistance, and Mr. César González for provid-ing his updated monograph on tanycytes.

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