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Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 422, Institut Fédératif de Recherche 114, Unité de Neuroendocrinologie et de Physiopathologie Neuronale, 59045 Lille Cedex, France
Address all correspondence and requests for reprints to: Vincent Prevot, Ph.D., Institut National de la Santé et de la Recherche Médicale Unité 422, place de Verdun, 59045 Lille Cedex, France. E-mail: prevot{at}lille.inserm.fr.
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Introduction |
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and neuregulins, are key components of this astrocyte-GnRH communication system in vivo, acting in an autocrine and/or paracrine fashion on astrocytes through receptor tyrosine kinase signaling pathways, which then alter the secretory activity of GnRH neurons by releasing prostaglandin E2 (PGE2) (4, 5, 6, 7). More recently, TGFß1, a second glial growth factor, has also been reported to influence the regulation of GnRH secretion (8). In vitro studies performed on astrocytes cultured together with GnRH-secreting cell lines suggest that astrocyte-derived TGFß1 acts directly on GnRH neurons to modulate both GnRH release and GnRH gene expression (9, 10, 11, 12). However, the morphological and functional basis for this glia-to-neuron signaling mechanism has never been clearly established in vivo. GnRH neurons are located in the basal forebrain and in the rat, GnRH perikarya are diffusely distributed throughout the preoptic region (POA). Those GnRH neurons involved in reproductive function send their axons to the median eminence (ME), where they release the GnRH decapeptide into the pituitary portal blood vessels. One of the most striking morphological characteristics of hypothalamic GnRH neurons is their strong association with glial cells. Astrocytes abundantly appose the plasma membrane of GnRH neuronal cell bodies (13, 14); and tanycytes, the specialized ependymoglial cells of the ME, are intimately associated with the axons of GnRH cells (for review see Ref. 15). Because both astrocytes and tanycytes have been shown to produce TGFß1 in vitro (9, 12, 16), it can be hypothesized that they modulate the secretory activity of GnRH neurons via juxtacrine interactions involving TGFß1 signaling. This glia-to-neuron communication pathway requires both the expression of TGFß1 in the glial cells that appose GnRH neurons and the presence of TGFß receptors in hypothalamic GnRH neurons.
TGFß1 regulates cellular processes by acting on transmembrane receptors with serine/threonine kinase activity (17). The signaling pathway is initiated by binding of the ligand to a TGFß type II receptor (TGFß-RII) homodimer, and this complex then interacts with a type I TGFß receptor (TGFß-RI) homodimer. The subsequent formation of a heterotetrameric complex results in the activation of TGFß-RI through the phosphorylation of its serine/threonine residues by TGFß-RII. Consequently, TßRI phosphorylates the receptor-associated proteins, Smad 2 and/or Smad 3, which are then released from the receptor site to form a new heteromer with Smad 4. This complex then translocates into the nucleus to interact directly or indirectly with TGFß-responsive elements in the genome, thereby regulating transcriptional events (for review see Ref. 18). Recently, TGFß1 mRNA has been shown to be expressed within the hypothalamus (19) and to be involved in the control of proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of the hypothalamus (ARH) (20). However, the anatomical distribution of TGFß1-expressing cells, their phenotypes, and their relationship with GnRH neurons remain largely unexplored, and it is not known whether GnRH neurons have the ability to respond to TGFß1 stimulation in vivo. To address these questions, we examined the expression pattern of TGFß1 mRNA in the adult male rat, focusing on the POA and the ME, which contain the majority of GnRH cell bodies and processes, respectively. We also investigated the phenotypes of TGFß1-expressing cells and whether TGFß1 can directly interact with GnRH neurons to modify their secretion.
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Materials and Methods |
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Ex vivo experiments
The protocols for ex vivo experiments were modified from previously reported methods (20, 21). Animals (n = 5 per group) were killed by decapitation and, after rapid removal of the brain, a block of neural tissue containing the POA was isolated by microdissection under a binocular magnifying glass. The POA explant was delineated by the anterior and posterior borders of the optic chiasm and by the ventral and lateral borders of the anterior commissure. After dissection, explants were washed twice in Krebs-Ringer bicarbonate (KRB)/glucose buffer (KRB, pH 7.4) containing bacitracine (23 µM; Sigma Chemical Co., St. Louis, MO) and incubated in KRB at 35 C in an atmosphere of 95% O2-5% CO2. Explants were then exposed to various concentrations (0, 0.01, 0.1, and 0.4 nM) of TGFß1 (Sigma Chemical) for an additional 120-min incubation period. To determine the time course of TGFß1 activity, a separate set of explants was exposed to 0.4 nM TGFß1 at 0, 30, 60, or 120 min before the end of the incubation period. The specificity of the effects of TGFß1 was assessed in an additional set of explants by adding the soluble form of TGFß-RII (60 nM) to the medium during the 120-min incubation (sRII; Calbiochem, Meudon, France), in the presence or absence of 0.4 nM TGFß1. Throughout all experiments, media were changed every 30 min.
At the end of each experiment, POA explants were fixed by immersion in 4% paraformaldehyde/0.1 M phosphate buffer for 18 h at 4 C, cryoprotected for 6 h in 0.05 M Coons veronal buffer (pH 7.4) containing 20% sucrose, embedded in OCT (Miles Laboratories, Naperville, CA), and frozen in liquid nitrogen. Frozen 16-µm coronal sections were collected throughout the entire explant, mounted onto gelatin-coated slides, and stored at -80 C until processing. To evaluate GnRH mRNA levels, a single-label in situ hybridization was performed on tissue collected from all groups, as described below.
Histochemical procedures
Tissue preparation.
Each rat (n = 4 per experiment) was anesthetized and perfused transcardially with normal saline (2 min) followed by ice-cold 4% paraformaldehyde solution (20 min). The brains were quickly removed, post-fixed for 4 h in the same fixative, and placed in 20% sucrose in 0.05 M Coons veronal buffer (pH 7.4) for 12 h for cryoprotection. Brains were then embedded in OCT (Miles Laboratories), frozen in isopentane (-55 C), and stored at -80 C until sectioning. Using a cryostat, 20-µm serial coronal sections were cut and collected into six series of tissue.
Cloning of the rat TGFß-RII in situ hybridization probe.
Rat TGFß-RII cDNA was obtained by RT-PCR on a random-primed template from rat hypothalami using the primers for rat TGFß-RII sense 5'-AAG TCT TGC ATG AGC AAC TGC-3' and rat TGFß-RII antisense 5'-GAT GTC AGA GAA GAT GTC C-3'. Primers were designed from the NM_031132 Rattus norvegicus TGFß-RII mRNA sequence (22). The PCR was run through 40 amplification cycles at an annealing temperature of 55 C. Elongation of the primers was catalyzed by an AmpliTaq Gold DNA polymerase (Applied Biosystems, Roche, Meylan, France). The final product was cloned into a pCRII-TOPO vector (TOPO TA Cloning, Invitrogen, Cergy Pontoise, France) and sequenced by the IFR of Lille DNA Sequencing Core (Abiprism 377XL). The 698-bp PCR product was identical with bp 444-1142 of the published homolog (NM_031132) (22).
Riboprobe syntheses.
Sequences for cDNA encoding TGFß1, TGFß-RI, GnRH, and GFAP (glial fibrillary acidic protein) are described elsewhere (23, 24, 25). The in vitro transcription was carried out using the Promega kit (Promega, Charbonieres, France), and probes were labeled with digoxigenin-1-d-UTP for the GFAP probe (Roche Diagnostics, Meylan, France), or 35S-UTP for the TGFß1, TGFß-RI, TGFß-RII, and GnRH probes (Amersham, Les Ulis, France).
In situ hybridization.
Single-label in situ hybridizations were performed following a protocol described in detail previously (20). Briefly, after a 30-min proteinase K digestion and acetylation, sections were dehydrated in ascending alcohols and dried for 2 h. Sections were then hybridized overnight at 55 C with a riboprobe-hybridization buffer mix that contained the 35S-labeled cRNA probe (30,000 cpm/µl). The hybridization was followed by ribonuclease treatment and a series of stringent washes, including a high-stringency wash in 0.1x saline sodium citrate at 60 C. After dehydration in ascending alcohols, sections were dipped in NTB2 liquid emulsion (Integra, Biosciences, Saint Oven L’Aumone, France). Slides were developed after 20 d of exposure.
Double-label in situ hybridization was performed following a protocol described in detail previously (20). Prehybridization, hybridization, and posthybridization procedures were similar to those described above, except that the sections were not dehydrated and dried after the last 0.1x saline sodium citrate rinse, but were further processed for localization of the digoxigenin-labeled hybrids. Briefly, sections were incubated in sheep antidigoxigenin primary antiserum conjugated to alkaline phosphatase (Boehringer-Roche Diagnostics, Meylan, France), and incubated in chromogen solution (tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate) (Boehringer-Roche Diagnostics) for 12 h. Sections were then quickly dehydrated in ethanol, dried, and dipped in K5 emulsion (Ilford, Saint-Priest, France).
Immunohistochemical and in situ hybridization labeling.
Sections were incubated for 48 h at 4 C with a rabbit polyclonal anti-TGFß-RII primary antibody (1:250; sc-220; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a mouse monoclonal anti-GnRH primary antibody (1:1000; generously provided by Dr. H. Urbanski, Oregon National Primate Research Center, Beaverton, OR) (26) diluted in Tris-buffered saline (TBS) containing 0.3% Triton-X. For the TGFß-RII immunostaining, the slides were citrate/microwave-treated before incubation with the primary antibody, as described previously (20). Antigen-antibody binding sites were visualized using biotinylated goat antirabbit IgG (1:200 for 90 min; Caltag Laboratories, Inc., Burlingame, CA) or biotinylated-linked goat antimouse IgG (1:200 for 90 min; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) secondary antibodies, standard peroxidase ABC amplification methods (for 90 min; Vector Laboratories, Inc., Burlingame, CA) and DAB chromagen. Sections were rinsed in TBS and then processed for single in situ hybridization labeling (see above) for detection of TGFß-RI or GnRH mRNA expression in TGFß-RII immunopositive cells, or TGFß1 mRNA-expressing cells in close apposition to GnRH immunopositive neurons.
Double-label immunohistochemistry.
After citrate/microwave-treatment, slides were incubated for 48 h at 4 C with a rabbit polyclonal anti-TGFß-RII primary antibody combined with either a mouse monoclonal anti-GnRH primary antibody or a mouse monoclonal antivimentin primary antibody (1:500; clone V9, Dako, Trappe, France). All antibodies were diluted in TBS containing 0.3% Triton. Immunoreactive cells were detected by incubating the sections with a cocktail containing Alexa 568-conjugated antimouse IgG (1:200) and Alexa 488-conjugated antirabbit IgG (1:200; Molecular Probes, Eugene, OR) secondary antibodies for 90 min. The sections were then coverslipped with glycerol-PBS (3:1, vol/vol) containing 0.1% p-phenylenediamine to minimize fading, and examined using a conventional Leica fluorescent microscope (Leica, Rueil Malmaison, France).
In addition, one series of sections was incubated with goat polyclonal anti-Smad2/3 (1:100; Santa Cruz Biotechnology, Inc.) and mouse monoclonal anti-GnRH (1:1000) primary antibodies. Immunopositive cells were detected by incubating the sections with a cocktail containing Alexa 568-conjugated antimouse IgG (1:200) and Alexa 488-conjugated antigoat IgG (1:200) secondary antibodies for 90 min. The sections were then stained for 5 min with the nuclear stain 4'-6-diamidino-2-phenylindole dilactate (DAPI; 1:400 in TBS). Coverslipped sections were analyzed. Sections were mounted and coverslipped as above and were then examined using a confocal laser scanning microscope (Leica).
Control experiments.
Specificity of in situ hybridizations was confirmed by incubation of the sections with 35S- and digoxigenin-labeled sense probes, pretreatment with ribonuclease, and coincubation with a 100-fold excess of unlabeled antisense probe. Verification of specificity of TGFß-RII immunostaining included incubation of sections with the labeled secondary antiserum alone, and blocking the immunohistochemical reaction by coincubating the tissue with both the antibody and the peptide used for immunization (sc-220 P, Santa Cruz Biotechnology, Inc.). No labeling was observed on sections incubated with the secondary antiserum alone, and the immunohistochemical reaction was totally blocked by incubating the brain sections with 0.8 µg rabbit polyclonal anti-TGFß-RII antibody (1:250 final dilution in TBS) together with 8 µg of the immunizing peptide (data not shown).
Quantitative analysis
TGFß-RII protein expression in GnRH neurons.
For each animal, four POA sections were analyzed. In these sections, each GnRH mRNA-expressing neuron was examined for the presence of TGFß-RII immunoreactivity. The numbers of singly or doubly labeled neurons were then counted and averaged. The data are presented as percentage of GnRH neurons containing TGFß-RII immunoreactivity.
Relative GnRH mRNA levels in the POA.
For each POA explant, the density of silver grains per cell was calculated from at least 60 GnRH neurons. Grain density was correlated to relative GnRH mRNA levels as previously published (20, 27). Briefly, sections were viewed using a Zeiss Axioscope microscope (Gottingen, Germany). The density of silver grains was quantified using the DensiRag program of Biocom (Les Ulis, France) under a x40 epi-illumination dark-field objective. Neurons were identified as labeled with GnRH probe if the number of silver grains over the perikaryon was at least three times higher than the background. Differences between groups were analyzed by ANOVA, followed by a post hoc Bonferroni’s t test. Differences between groups and brain areas were regarded as significant at P 
0.05.
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), TGFß1 mRNA transcripts were clearly detected in cells in the median preoptic nucleus (MEPO) and the anteroventral periventricular nucleus (AVPV). Scant hybridization was also seen in the medial preoptic area (MPO). Within the mediobasal hypothalamus, dense hybridization was observed in the ARH, but the highest TGFß1 mRNA expression levels were seen in the ME (Fig. 1B), a region that serves as a final common pathway for neurosecretory nerve terminals that release their products into the pituitary portal vasculature. In the ME, TGFß1 mRNA was preferentially expressed in the external layer, where the vascular endothelial cells of the pituitary portal plexus reside, but hybridization was also observed in cells located in the intermediate layer of the ME (Fig. 1B). TGFß1 hybridization signal was also concentrated in a subset of tanycytes (Fig. 1B).
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). In contrast, GnRH-IR neurons themselves were never found to express TGFß1 mRNA.
Expression of TGFß1 mRNA in hypothalamic astrocytes
To determine whether TGFß1 was expressed in hypothalamic astrocytes in vivo, we performed double in situ hybridization experiments using GFAP as an astrocytic marker (25). The expression pattern of TGFß1 mRNA clearly overlapped the distribution of GFAP mRNA transcripts in the POA. As was seen for TGFß1 mRNA, high levels of GFAP mRNA expression were detected in the MEPO, where most GnRH neuron cell bodies are located. Observations performed at high magnification revealed that many cells of the POA colocalized GFAP and TGFß1 mRNAs (Fig. 1D).
TGFß-RII mRNA expression in the POA and mediobasal hypothalamus
In the POA of adult male rats, TGFß-RII mRNA was primarily seen in the vascular organ of the lamina terminalis (OVLT) (Fig. 2A). High expression levels were also observed in cells of the MEPO and the AVPV (Fig. 2A). Lower levels of TGFß-RII mRNA hybridization signal were seen in the MPO (data not shown). In the mediobasal hypothalamus, TGFß-RII mRNA was predominantly expressed in the ME, specifically within the external zone (Fig. 2B). Numerous parenchymal cells also expressed TGFß-RII mRNA in both the ME and in the ARH (Fig. 2B).
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). Quantitative analysis revealed that in the POA, 38 ± 2% of the neurons that expressed GnRH mRNA were also immunopositive for TGFß-RII. No preferential distribution was noticed for these dual-labeled neurons within the POA. We recently reported that a subset of hypothalamic GnRH neurons also expressed TGFß-RI (23), a type I serine/threonine kinase receptor involved in transducing activin signals and capable of transducing TGFß signals after dimerization with ligand-activated TGFß-RII (28, 29). To determine whether TGFß-RII-expressing cells of the POA also colocalize TGFß-RI mRNA, we combined in situ hybridization and immunocytochemistry methodologies. As illustrated in Fig. 2D, the majority of TGFß-RII-immunoreactive cells coexpressed TGFß-RI mRNA, and vice versa.
We then performed double-immunocytochemical studies in the ME to determine whether TGFß-RII proteins were also expressed in GnRH-containing nerve terminals, as had been seen in the soma. None of the GnRH-IR fibers in the ME exhibited TGFß-RII immunoreactivity (Fig. 2, E–G). Rather, TGFß-RII immunostaining was contained in processes that were immunoreactive for vimentin (arrows, Fig. 2, H–J), an intermediary filament expressed by tanycytes, and in the external zone of the ME where tanycytic "endfeet" specializations and capillaries reside (empty arrows; Fig. 2, H–J).
To determine whether TGFß1 signaling in GnRH neurons could directly be coupled to the regulation of gene transcription, we investigated whether Smad downstream signaling molecules were expressed in GnRH hypothalamic neurons. Double-immunocytochemical experiments showed that Smad2 and/or Smad3 were expressed in GnRH cell bodies and dendrites (Fig. 3). Most of the Smad2/3-immunostaining was contained in the cytoplasm of GnRH neurons (Fig. 3B).
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, A and B), whereas TGFß1 had no effect on GnRH mRNA levels when administered at doses of 0.01 nM or 0.1 nM (Fig. 4B). The inhibitory effect of 0.4 nM TGFß1 on GnRH gene expression was prevented by the addition of 60 nM soluble TGFß-RII to the incubation medium (Fig. 4C). Examination of the temporal characteristics of TGFß activity revealed that an incubation of at least 60 min was necessary for TGFß1 to decrease GnRH mRNA expression in POA explants (Fig. 4D).
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Discussion |
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Whereas previous studies have described the production of TGFß1 by hypothalamic astrocytes in culture (10, 11, 12), those looking at its in vivo expression in the hypothalamus have been limited. Only one recent study using radioprotection assay analysis of the female rat hypothalamus has revealed TGFß1 mRNA expression in vivo (19). In the present morphological study, we show that in adult males, the distribution of TGFß1 mRNA-expressing cells is restricted to discrete areas of the POA and the mediobasal hypothalamus. TGFß1 mRNA hybridization signal is mainly contained in the AVPV, the MEPO, the ARH, and the ME, all of which are hypothalamic areas that are critically involved in the control of GnRH secretion (15, 30, 31) and that are known to express the TGFß-RI (20, 23, 32). These data, together with our results showing that TGFß-RII mRNA is also expressed in these regions, strongly support the possibility of cell-cell communication processes involving TGFß1 signaling within the neuroendocrine brain. The role of TGFß1 signaling in the AVPV, a nucleus that provides direct projections to GnRH neurons (33), remains to be determined. However, TGFß1 has been shown to modulate the expression of POMC mRNA in ARH neurons known to project to the POA (20). In the MEPO, TGFß1 mRNA-expressing cells appear to directly interact with the subpopulation of GnRH neurons that is contained in this hypothalamic nucleus. Interestingly, the highest TGFß1 mRNA hybridization signal in the hypothalamus was detected in the ME, which is the primary target of GnRH neuronal projections. In this area, TGFß1 mRNA was expressed in glial cells and in the endothelium of the pituitary portal vessels. Because all of the cell types that were previously shown to express TGFß-RI mRNA (23) also express TGFß-RII mRNA, TGFß1 may be an important signaling molecule involved in the regulation of ME function through local glial-endothelial interactions. In support of this, recent in vitro experiments from our laboratory (16) indicate that TGFß1 may regulate cellular plasticity in the ME, alternately allowing or preventing direct access of GnRH nerve terminals to the portal vasculature during the rat estrous cycle (15).
The finding that TGFß1 mRNA-expressing cells that are in close apposition to GnRH-containing perikarya colocalize GFAP mRNA suggests that astrocyte-to-GnRH neuron communication involving TGFß1 signaling may occur in vivo. The data showing that a subpopulation of GnRH mRNA-expressing cells exhibit immunoreactivity for TGFß-RII, in addition to TGFß-RI (23), further support this hypothesis. In the present studies, most of the TGFß-RII-immunoreactive cells in the POA were found to also express TGFß-RI mRNA, raising the likelihood that a population of GnRH neurons may coexpress both TGFß-RII and TGFß-RI. Thus, as shown for GnRH-secreting cell lines (11, 12), a significant subset of hypothalamic GnRH neurons may be fully capable of directly transducing astrocyte-derived TGFß1 signals in vivo. The expression of Smad2/3 in GnRH perikarya further supports the hypothesis that astrocyte-derived TGFß1 may directly modulate gene transcription in these hypothalamic neurons. Moreover, consistent with earlier in vitro studies that found that the expression of GnRH mRNA is regulated by treatment with TGFß1 in GnRH-secreting cell lines (10), the present studies demonstrate that TGFß1 specifically down-regulates the expression of GnRH mRNA in individual neurons in ex vivo POA explants. Under our experimental conditions, TGFß1 induced a significant decrease in GnRH mRNA levels in single cells within 1 h of treatment. This rapid decay of GnRH mRNA is fully compatible with the inherently high rate of GnRH mRNA turnover (half-life < 15 min) in hypothalamic explants (34). However, it remains to be determined whether Smad proteins can directly bind to the GnRH promoter to modulate its activity, and whether TGFß1 can activate this signaling pathway to influence GnRH gene transcription in vivo or in POA explants.
Interestingly, although TGFß-RII protein was clearly expressed in cell bodies of GnRH neurons, no TGFß-RII immunoreactivity was detected in GnRH axons and nerve terminals within the ME. These results suggest that TGFß1, which has high mRNA expression levels in the ME, does not act directly on GnRH nerve terminals to modulate the release of their neurohormone. This interpretation is consistent with studies showing that unlike TGF, TGFß1 is ineffective in stimulating GnRH release from ME explants (35). Although previous studies demonstrate that TGFß1 stimulates GnRH release from GnRH secreting cell lines (9, 12), our results suggest that any direct in vivo stimulatory effect of TGFß1 on GnRH release is through the selective actions of TGFß1 on GnRH neuronal cell bodies.
Previous studies have demonstrated that TGF and neuregulins activate astrocytic erbB-1 and erbB-4 signaling pathways, respectively, to increase the production of PGE2, which in turn, directly modulates GnRH release from axon terminals located in the ME (6, 35, 36). In contrast, the present results underscore the absence of direct actions of TGFß1 on the neuroendocrine terminals of GnRH neurons. Rather, the glial-to-GnRH neuron communication system involving TGFß1 appears to modulate GnRH secretion by activating signaling events that occur in GnRH neuronal cell bodies. However, the physiological significance of TGFß1 signaling in GnRH neurons needs to be further explored. For example, we have recently demonstrated in vitro (16) that cross-talk between TGF and TGFß1 signaling occurs in glial cells of the hypothalamus, suggesting that the actions of these two growth factors may be complementary. Because TGF expression increases before that of TGFß1 (4, 19) during the preovulatory surge of gonadotropins in females, it is likely that a similar relationship is involved in the in vivo regulation of cyclic gonadotropin release.
Taken together, the present results and our previous findings (16, 23) provide strong evidence that, in addition to TGF and neuregulins, TGFß1 may contribute physiologically to the processes by which glial cells control the function of GnRH neurons in the hypothalamus.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARH, Arcuate nucleus of the hypothalamus; AVPV, anteroventral periventricular nucleus; GFAP, glial fibrillary acidic protein; KRB, Krebs-Ringer bicarbonate; ME, median eminence; MEPO, median preoptic nucleus; MPO, medial preoptic area; OVLT, vascular organ of the lamina terminalis; PGE2, prostaglandin E2; POA, preoptic region; POMC, proopiomelanocortin; RI, type I receptor(s); RII, type II receptor(s); TBS, Tris-buffered saline.
Received October 30, 2003.
Accepted for publication December 5, 2003.
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gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation. Neuron 9:657–670[CrossRef][Medline]
in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus. Proc Natl Acad Sci USA 87:9698–9702This article has been cited by other articles:
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  N. Fielenbach and A. Antebi C. elegans dauer formation and the molecular basis of plasticity Genes & Dev., August 15, 2008; 22(16): 2149 - 2165. [Abstract] [Full Text] [PDF]
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