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Endocrinology Vol. 143, No. 11 4385-4388
Copyright © 2002 by The Endocrine Society

ARTICLE

Immunocytochemical Localization of Vasopressin V1a Receptors in the Rat Pituitary Gonadotropes

Hélène Orcel, Vicky A. Tobin, Gérard Alonso and Alain Rabié

Centre National de la Recherche Scientifique-Université Montpellier II, Unité Mixte de Recherche 5101, Biologie des Neurones Endocrines, Centre de Pharmacologie-Endocrinologie, Montpellier F-34094, France

Address all correspondence and requests for reprints to: A. Rabié, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5101, CCIPE, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail: rabie{at}ccipe.montp.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunocytochemical labeling using a specific antibody against vasopressin V1a receptor allowed the localization of this receptor within a subset of cells from male rat anterior pituitary. The presence of transcripts of the corresponding gene in the anterior pituitary was confirmed by RT-PCR. Multiple immunocytochemical labeling combined with confocal microscopy allowed the identification of the V1a-labeled cells as gonadotropes. At the subcellular level, the vasopressin V1a receptor was mainly associated with cytoplasmic vesicles dispersed throughout the cell, which were not the secretory granules storing LH or FSH. In addition to effects exerted by vasopressin via central targets involved in the reproductive pathways, the presence of vasopressin V1a receptors on gonadotropes supports the controversial hypothesis of a local direct action of the neuropeptide on this cell type.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN A RECENT IMMUNOCYTOCHEMICAL study, we have described the presence of V1a vasopressin receptor in hypothalamic regions (the supraoptic, paraventricular, and suprachiasmatic nuclei) in which vasopressin has been long shown to influence neuronal activity and the presence of vasopressin receptors was expected (1). In the course of the experiments performed during this previous study, we also observed an unexpected labeling for V1a vasopressin receptor in cells of the anterior pituitary. In this gland, vasopressin is known to be involved in the control of the corticotropes (2). It binds to receptors of the V1b subtype and potentiates the effect of the CRH on these cells, leading to the release of ACTH (3, 4, 5), which in turn induces secretion of corticosteroids by the adrenals. Vasopressin is also known to influence the secretion of gonadotropins by pituitary (6). Intravenous or intracerebroventricular administration of vasopressin has been shown to depress the preovulatory LH surge in the female rat (7, 8) and to lower the basal LH release in the male rat (9). Despite the considerable amount of data, the cellular mechanisms involved in the effects of vasopressin on LH secretion remains unclear, mainly because of the multiple potential sites of action described for this neuropeptide in the regulatory pathways of this secretion. First, there are central effects, exerted on the neurons that secrete the GnRH, the hormone that triggers in fine the release of LH by the gonadotropes (10). These central effects appear very sensitive to the experimental protocol used for vasopressin administration, which may explain the contradictory effects of vasopressin on LH secretion reported in the literature (11, 12). Second, a direct stimulatory effect of vasopressin on LH secretion by gonadotropes has been reported, although it also remains controversial (13, 14, 15, 16). Using multiple labeling immunocytochemistry, we now report the expression of vasopressin V1a receptors by gonadotropes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Standard chemicals were purchased from Sigma (St. Louis, MO), Roche Molecular Biochemicals (Mannheim, Germany), or Merck & Co., Inc. (Darmstadt, Germany), unless otherwise indicated. The rabbit anti-V1a vasopressin receptor antibody and the corresponding blocking peptide were purchased from Alpha Diagnostic (San Antonio, TX). The antibody, raised using a synthetic peptide whose sequence is located in the extracellular N-terminal domain of the rat receptor, does not cross-react with the V1b and V2 isoforms of the vasopressin receptor. The goat antihuman LH ß-subunit was purchased from Affiland (Ans, Belgium), the mouse monoclonal antihuman FSH ß-subunit from Zymed Laboratories, Inc. Corp. (San Francisco, CA), and the mouse monoclonal anti-ACTH antibody from Novocastra Laboratories Ltd. (Newcastle, UK). The Cy2-labeled donkey antirabbit IgG, Cy3-labeled donkey antigoat IgG, and Cy5-labeled donkey antimouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The oligonucleotides were purchased from Sigma-Genosys (Pampisford, UK).

Adult male Wistar rats (Charles River Laboratories, Inc., L’Arbresle, France) were used in accordance with the European laws governing the care and use of experimental animals. The animals (280–300 g) were perfusion fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The pituitaries were dissected, postfixed for 3 d in the same fixative and rinsed with PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4). Frontal sections (50 µm thick) were cut with a VT1000S vibratome (Leica Corp., Nussloch, Germany) and processed for immunocytochemistry. The sections were incubated simultaneously at 4 C for 65 h with the rabbit vasopressin V1a receptor antibody (1/1000), the goat anti-LH (1/3000), and the mouse monoclonal anti-ACTH (1/200) or anti-FSH (1/50) in PBS plus 2% BSA buffer. The sections were then rinsed three times for 10 min in PBS. The immunoreactions were revealed using a 2-h simultaneous incubation at room temperature with Cy2-labeled donkey antirabbit IgG (1/500), Cy3-labeled donkey antigoat IgG (1/2000), and Cy5-labeled donkey antimouse IgG (1/500) in PBS plus 2% BSA. The sections were rinsed three times for 10 min in PBS, and mounted in Mowiol (Calbiochem, La Jolla, CA). Image acquisition was made using an MRC 1024 confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA).

Detection of the vasopressin V1a receptor mRNA was performed using RT-PCR. Total RNA was extracted from carefully dissected anterior pituitary using the RNeasy kit (QIAGEN, Courtaboeuf, France). Reverse transcription was performed on the RNA extracts previously digested with deoxyribonuclease I (Life Technologies, Inc., Cergy Pontoise, France), using SuperScript II reverse transcriptase (Life Technologies, Inc.), RNasin (Promega Corp., Charbonnières, France) as a RNase inhibitor, and the reverse primer 5'-CTATCGGAGTCATCCTTGGCG-3'. DNA amplification (40 cycles, 30 sec annealing at 62 C, 90 sec elongation at 72 C) was performed using a PTC-150/16 thermal controller (MJ Research, Inc., Watertown, MA), Taq DNA polymerase (Life Technologies, Inc.), the reverse primer previously used for reverse transcription, and the forward primer 5'-CGAGGTGAACAATGGCACTAAAAC-3'. Sense and reverse primers were chosen on different exons. The PCR products were electrophoresed on a 2% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8) containing 0.5 µg/ml ethidium bromide and digitized under UV light. Specificity of DNA amplification was verified by digestion of the DNA band with BanII restriction enzyme (Promega Corp.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In all the sections examined, a strong immunocytochemical signal for vasopressin V1a receptor was found in a subset of cells of the anterior pituitary (Fig. 1AGo). Preabsorption of the antibody with the blocking peptide or omission of the primary antibody eliminated the signal (not shown). Simultaneous labeling with LH and vasopressin V1a receptor antibodies showed that immunoreactivity to both antibodies was always localized in the same cells, therefore allowing the identification of the V1a receptor-labeled cells as gonadotropes (Fig. 1Go, B and D). In addition, the simultaneous labeling of ACTH confirmed that the labeling for the vasopressin V1a receptor was not associated with corticotropes (Fig. 1Go, C and D) and hence that the V1a receptor antibody does not cross-react with the V1b receptor subtype known to be present in these cells (3, 4, 5).



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  		Figure 1. A–D, Triple labeling confocal microscope views of a section of the anterior pituitary of a male adult rat immunostained for vasopressin V1a receptor (A), LH (B), and ACTH (C). In D, the three stainings have been superimposed and show that V1a receptor immunostaining is localized within the LH-immunostained but not ACTH-immunostained cells. E, High-magnification view of gonadotropes simultaneously stained for vasopressin V1a receptor (green), LH (red), and FSH (blue), showing that most V1a receptor immunostaining is localized to cytoplasmic vesicles, distinct from those immunostained for LH and FSH. Images A–D, The mean of four confocal planes (0.8 µm thick); image E is a single confocal plane (0.5 µm thick). Bar, 50 µm in A–D, 5 µm in E. F, RT-PCR amplification of vasopressin V1a receptor transcripts in RNA extracts of male adult rat anterior pituitary. M, Size markers; the arrowhead points the 500-bp marker; lane 1, V1a band; lane 2, specificity control of the V1a band by digestion with BanII.

 
Confocal images taken at high magnification after triple staining for vasopressin V1a receptor, LH, and FSH showed that labeling for the V1a receptor was not predominantly associated with the plasma membrane but rather with cytoplasmic vesicles dispersed throughout the cell cytoplasm (Fig. 1EGo). Within labeled cells, LH and FSH immunoreactivities were also essentially associated with intracytoplasmic vesicles; however, at the resolution achieved with the confocal system used, the vasopressin V1a receptor and LH or FSH appeared to be located in three different populations of vesicles (Fig. 1EGo).

RT-PCR performed on anterior pituitary RNA extracts confirmed the presence of vasopressin V1a receptor mRNA in this region. The DNA band was 576 nucleotides long, as expected (Fig. 1FGo, lane 1). When cut with BanII restriction enzyme, it gave the expected 207- and 369-nucleotide-long fragments (Fig. 1FGo, lane 2).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here, for the first time to our knowledge, evidence of a strong immunocytochemical signal for vasopressin V1a receptor in the rat anterior pituitary, a region to date known for the presence of vasopressin receptors of the V1b subtype. The staining was located in the gonadotropes, as demonstrated by the colocalization with LH and FSH labeling. RT-PCR performed on anterior pituitary extracts further confirmed the presence of vasopressin V1a receptor mRNA. Together, these results are consistent with the previous observation, by in situ hybridization, of transcripts of the V1a gene in cells of the anterior pituitary, which unfortunately were not further identified (17).

Recently, using the same antibody, we reported immunolabeling of extrahypothalamic (hippocampal formation) and hypothalamic (suprachiasmatic, paraventricular, and supraoptic nuclei) regions in which vasopressin is known to act or the presence of vasopressin receptors has been assessed using ligand binding and in situ hybridization (1). Throughout the brain, the cells exhibiting the highest intensity of immunostaining for the vasopressin V1a receptor were the magnocellular vasopressinergic neurons of the paraventricular and supraoptic nuclei. The immunocytochemical labeling described here in the gonadotropes is of the same magnitude to that obtained in these hypothalamic magnocellular neurons.

The presence of vasopressin V1a receptor in gonadotropes could help to answer one question that remains controversial: does vasopressin exert a direct effect on LH release by these cells? The existence of direct effects of vasopressin on gonadotropes has long been suspected (13). Vasopressin terminals are present in the median eminence in juxtaposition to portal vessels (18), and nanomolar concentrations of vasopressin have been detected in portal blood (19, 20, 21). These concentrations have long been known to be efficient in the control of the corticotropes (2). Experiments performed by perifusion of dissected pituitary or on pituitary cells in culture have shown that vasopressin stimulates LH release (13, 16), although in other reports, vasopressin was found inefficient (14, 15). In view of the presence of vasopressin V1a receptors in gonadotropes as shown here, we suggest that the discrepancies among these previous studies arose from differences in physiological and/or experimental conditions. It is possible that activation of the vasopressin V1a receptors could potentiate the effect exerted on gonadotropes by GnRH, the hormone that triggers LH release. The main effect of vasopressin V1a receptor activation described in many cell types is to increase the intracellular concentration of Ca2+ by acting on phospholipase C and eventually mobilizing intracellular calcium stores (22). The local Ca2+ increase could create favorable conditions for exocytosis of the LH-loaded secretory granules (23). Therefore, the mechanisms could be very similar to that previously described in corticotropes, in which vasopressin is thought to potentiate the triggering effect of CRH on ACTH release, this time by acting on vasopressin V1b receptors (3, 4, 5).

An intriguing observation of the present study is that only a small amount of the vasopressin V1a receptor was found to be localized at the plasma membrane of gonadotropes, in which functional receptors are expected to be located to bind the peptide. A similar situation was observed in the magnocellular vasopressinergic neurons of the paraventricular and supraoptic nuclei. In these neurons, some of the vesicles immunostained for V1a receptors were secretory granules (as attested by their presence in the neurohypophysial nerve terminals) and others were suggested to be receptor recycling vesicles (1). Vasopressin V1a receptors are known to undergo rapid endocytosis upon ligand binding (within a few minutes in human embryo kidney 293 cells transfected with vasopressin V1a receptors) and to be slowly recycled to the plasma membrane (within 1 h in the same cells, without significant degradation of the receptor) (24). With such a kinetics, most vasopressin V1a receptors should be in the recycling pathway in cells continuously submitted to high concentrations of vasopressin. This hypothesis, which has been proposed for magnocellular vasopressinergic neurons (1), is also possible for gonadotropes, given the occurrence of high concentrations of vasopressin in anterior pituitary. There is, however, a difference between the two cell types: at the resolution obtained with the confocal system used in the present report, the vasopressin V1a receptor does not appear to be included in the gonadotrope secretory granules that store LH and/or FSH. Thus, the incorporation of vasopressin receptors in secretory granules remains to date peculiar to the magnocellular vasopressinergic neuron and is probably of major importance in the autocontrol exerted by vasopressin on this cell type (1, 25, 26).

Finally, although the presence of vasopressin V1a receptors on gonadotropes is suggestive of a local direct action of the neuropeptide on these cells, it does not preclude more complex effects involving this receptor on different central targets. There is, for example, morphological and physiological evidence of inhibition of the GnRH neurons by vasopressin (11, 27, 28, 29, 30, 31). Indeed, vasopressin has been reported to exert various and somewhat controversial actions on the preovulatory LH surge, according to species, physiological context, and protocol of neuropeptide administration (7, 8, 11, 12, 30, 31, 32). In this context, the present report of the localization of vasopressin V1a receptors in gonadotropes could certainly help our better understanding of the role of vasopressin on the LH-regulating pathways, by providing evidence for a molecular support to the direct action of the neuropeptide on these cells.


   Acknowledgments
 
We are grateful to Françoise Moos for critical review of the manuscript. Confocal microscopy was performed at the Centre Régional d’Imagerie Cellulaire (Montpellier), with the expert assistance of Nicole Lautrédou-Audouy.

Received June 7, 2002.

Accepted for publication July 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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