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Endocrinology Vol. 147, No. 6 2670-2674
Copyright © 2006 by The Endocrine Society

BRIEF COMMUNICATION

Sexually Dimorphic Distribution of sst2A Somatostatin Receptors on Growth Hormone-Releasing Hormone Neurons in Mice

Karine Bouyer, Catherine Loudes, Iain C. A. F. Robinson, Jacques Epelbaum and Annie Faivre-Bauman

Unité Mixte de Recherche 549 (K.B., C.L., J.E., A.F.-B.), Institut National de la Santé et de la Recherche, Médicale Faculté de Médecine, Université Paris René Descartes, Institut Fédératif de Recherche Broca Sainte Anne, 75014 Paris, France; and Molecular Neuroendocrinology (I.C.A.F.R.), National Institute of Medical Research, London NW7 1AA, United Kingdom

Address all correspondence and requests for reprints to: Dr. Jacques Epelbaum, Unité Mixte de Recherche 549, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, Université Paris René Descartes, Institut Fédératif de Recherche Broca Sainte Anne, 2ter rue d’Alésia, 75014 Paris, France. E-mail: epelbaum{at}broca.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pulsatile pattern of GH secretion exhibits sexual dimorphism that is likely to depend on somatostatin (SRIH) effects on somatoliberin (GHRH) neurons in the hypothalamus. Using transgenic GHRH-enhanced green fluorescent protein (eGFP) mice, no difference in the total number of GHRH-eGFP neurons or change in somatostatin receptor sst2 and SRIH mRNA levels in ventromedial hypothalamic nucleus-arcuate nucleus and periventricular nucleus regions and GHRH mRNA levels in the ventromedial hypothalamic-arcuate region were observed between male and female mice. However, the percentage of GHRH-eGFP neurons bearing sst2A receptors reached 78% in female vs. 26% in male GHRH-eGFP mice (P < 0.02). This sex difference in sst2A distribution on GHRH neurons may play an important role in the sexually differentiated pattern of GH secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ALL MAMMALIAN species tested so far, GH secretion from pituitary somatotrophs exhibits a typical ultradian pulsatile pattern that is sexually dimorphic. This is particularly evident in the rat. In male rats, GH pulses occur at 3- to 4-h intervals, reaching a peak level of several hundred nanograms per milliliter with undetectable nadir values (1), whereas in females, lower-amplitude pulses are more frequent and nadir values are more elevated (2, 3, 4). Such a secretory pattern of release is established with the onset of the puberty and results mainly from a complex interplay between the two hypothalamic neurohormones, somatoliberin (GHRH) and somatostatin (SRIH). A model has been proposed for generating the sexually dimorphic secretory GH profiles in male and female rats based on different release patterns of the two neurohormones (5). Much less is known about GH secretion in mice, because GH pulsatility studies have been hampered by technical limitations; there are, however, a few reports suggesting a dimorphic pattern of ultradian GH secretion in mice (6, 7).

The mechanisms mediating the sex difference in GH secretory dynamics remain unclear. SRIH and GHRH may interact not only at the pituitary level but also within the hypothalamus, in particular in the arcuate nucleus (Arc), which contains most of the GHRH cell bodies. Several sex-related differences in GHRH neurons have been reported, all pointing to higher activity in male than in female (8, 9, 10, 11, 12). Sexual dimorphism of GH secretion may also be due to sex-related differences in the somatostatinergic inputs on the GHRH neurons or in the pattern of responses to GHRH (13). Indeed, SRIH immunoneutralization induced an increase of GHRH levels in portal blood vessels (14), supporting the notion that SRIH neurons may directly influence GHRH neurons. Moreover, in the adult Rat, somatostatin receptor subtypes sst1 and sst2 are expressed in subsets of GHRH neurons (15, 16, 17). So far, a marked sex-related difference in the Arc was only reported in the case of sst1 expression (18).

In the present work, we took advantage of a GHRH-enhanced green fluorescent protein (eGFP) transgenic mouse model (19) to assess sst2A immunolabeling distribution on GHRH-eGFP neurons, as well as sst2 and neurohormonal (GHRH and SRIH) expression in the hypothalamus. sst2A receptor distribution on GHRH-eGFP neurons was found to differ between male and female mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Heterozygous male and female GHRH-eGFP transgenic mice (19) were produced using transgenic and C57BL/6J breeding pairs. The transgenic offspring were genotyped by PCR amplification of tail DNA. Animals were housed on a 12-h light, 12-h dark cycle at 22 ± 2 C and were given ad libitum access to regular chow and water. Three-month-old mice (body weight for males, 26.4 ± 0.5 g; for females, 21.5 ± 0.4 g) originating from five different litters were used in the neuroanatomical study. For RT-PCR measurements, each animal originated from different litters.

All animal procedures complied with French laws regarding animal experimentation (Decree 87-848 of October 19, 1987 and the Ministerial Decree of April 19, 1988).

Immunohistochemistry
An antimouse GHRH was raised in rabbit against the 25-amino-acid C-terminal part of the mouse GHRH sequence in which the 17-Tyr moiety was replaced by a Cys to allow conjugation to keyhole limpet hemocyanin as carrier. This antiserum (L0851) did not cross-react with rat GHRH, human GHRH, SRIH, TRH, or cortistatin when tested in RIAs. Preincubation with immunogen completely abolished binding and immunocytochemical labeling.

To study colocalization between anti-GHRH antibody and eGFP-labeled neurons, mice were anesthetized with diazepam (100 µg/30 g body weight, ip; Valium; Roche Products, Meylan, France) and slowly injected with colchicine (50 µg in 1 µl 0.9% NaCl; Sigma, Saint Quentin Fallavier, France) intraventricularly (0.007 µl/min). Stereotaxic coordinates for the lateral ventricle were chosen according to the atlas of Franklin and Paxinos (20): 0.07 mm posterior, 0.10 mm lateral, and 0.21 mm deep from bregma. Forty-eight hours later, animals were killed and processed for immunocytochemistry. Briefly, adult male and female mice were anesthetized with ketamine (4 mg/30 g body weight, ip; Sanofi, Libourne, France) and perfused through the aorta with 4% paraformaldehyde in 0.1 M Na/K phosphate buffer (pH 7.4). Brains were cryoprotected by overnight immersion in a 30% sucrose solution, frozen in liquid isopentane (–40 C), and kept at –80 C until use. Sectioning was performed using a cryostat at 30-µm thickness. Slices were then collected in Tris-buffered saline (TBS) and processed for immunocytochemistry on free-floating sections. Incubations were done in TBS (pH 7.4) at room temperature. After blocking in 5% normal goat serum, one of three sections was incubated overnight in TBS containing 0.3% Triton X-100 and 0.5% normal goat serum with the antimouse GHRH (L0851; 1:3000). Sections were further incubated for 1 h in the presence of a goat antirabbit IgG coupled to cyanine 3 (Cy3) (1:2500; Jackson ImmunoResearch, West Grove, PA) in the same buffer, mounted on glass slides, and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA). GHRH-eGFP neurons were counted under a fluorescent microscope (DMRA; Leica, Heidelberg, Germany) at x40 magnification, on consecutive sections, along the rostrocaudal axis. GHRH-eGFP cell surface (and sst2A immunolabeled area, see below) in the Arc were quantified using NIH Image J version 1.32 software (http://rsb.info.nih.gov/ij/) at x40 and x20 magnifications, respectively.

sst2A receptors were immunolocalized in animals not treated with colchicine, using a fully characterized antiserum raised in rabbit against the C-terminal segment 330–369 of the human protein (21) because it was not possible to obtain good sst2 staining in the Arc of colchicine-treated animals (Csaba Z., unpublished results). sst2A immunohistochemistry was performed on 30-µm sections rinsed in TBS, and preincubated for 30 min in 5% normal donkey serum (NDS) in TBS containing 0.3% Triton X-100. Sections were incubated overnight at room temperature in rabbit anti-sst2A receptor antibody (1:2000) in TBS containing 0.5% NDS and 0.3% Triton X-100. Sections were then rinsed in TBS and incubated for 45 min in biotinylated donkey antirabbit IgG (1:500) (Vector Laboratories) in TBS containing 0.5% NDS and 0.3% Triton X-100. Finally, sections were incubated for 30 min in streptavidin-Cy3 (1:4000) (Jackson ImmunoResearch) in TBS to reveal sst2A receptors, rinsed in TBS, and mounted as above.

Sections were examined by confocal microscopy using an SP2 Leica laser scanning microscope equipped with a Leica DMRA microscope and argon/krypton ion laser (488, 543, and 633 nm). GHRH-eGFP cell bodies were counted over a 10-µm depth and checked for sst2A receptor immunoreactivity. Images were acquired at a x40 magnification simultaneously for GFP and Cy3 and processed using the Leica software package.

Real-time PCR
Brains were quickly removed and placed in sterile PBS until dissection. Two hypothalamic fragments containing arcuate and periventricular areas were excised: a mediobasal one containing the ventromedial hypothalamic nucleus (VMH)-Arc region and a more dorsal and anterior segment adjacent to the third ventricle comprising the periventricular nucleus (PeV) region (22). Hypothalamic fragments were immediately placed and disrupted in lysis buffer. RNAs were extracted with a silicium membrane method using an RNeasy Mini extraction kit (Qiagen, Courtaboeuf, France) and quantified by spectrophotometry (Spectronics, Rochester, NY). First-strand cDNA was prepared by reverse transcription using 0.5 µg hypothalamic samples in a 20 µl final reaction mixture containing 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), 0.167 µg random primer (dN6; Promega, Charbonnière, France), 12.5 nmol dNTP (Promega), and 20 U of RNAsin (Promega).

Real-time PCR was performed with an ABI Prism 7000 Sequence Detection System (Applera Corporation, Applied Biosystems, Courtaboeuf, France). Amplification reactions were performed in a 20 µl final volume using a Taqman Universal Master Mix reagent kit for SRIH, GHRH, and sst2 primers (Applied Biosystems). rRNA at 18S was used for standardization.

PCR was initiated after activation of the Amplitaq Gold enzyme (Applied Biosystems) in the reaction mixture by heating for 10 min at 95 C. All genes were amplified by a first denaturation step of 15 s at 95 C, followed by an annealing and amplification step of 1 min at 60 C for 40 cycles. Relative gene expression levels were calculated according to the comparative threshold cycle (CT) method that normalizes the copy number of target genes to that of an endogenous reference gene (23). Based on exponential amplification of the target and reference genes, the amount of amplified molecules at the CT values was given. Normalized target gene expression relative to 18S rRNA is obtained by calculating the difference in CT values, the relative change in target transcripts being computed as 2–{Delta}CT.

Statistical analysis
Results were expressed as mean ± SEM. The data were analyzed by ANOVA, followed by Student’s least significant difference test (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neurochemical characterization of GHRH-eGFP neurons
In naive and colchicine-treated GHRH-eGFP transgenic mice, GFP fluorescence was almost exclusively restricted to Arc cell bodies and median eminence (ME) nerve terminals. The number of GHRH-eGFP neurons was not affected by colchicine treatment, whereas eGFP fluorescence decreased in the ME. The localization of eGFP neurons coincided with that of endogenous GHRH visualized by immunocytochemistry (Fig. 1Go). The total number (1138 ± 73 in female vs. 1112 ± 40 in male mice) or size (101.7 ± 4.9 vs. 103.9 ± 5.5 µm2) of GHRH-eGFP neurons did not differ between the sexes (three animals per group).


Figure 1
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  		FIG. 1. Comparison of eGFP labeling and GHRH immunoreactivity in the Arc and ME of GHRH-eGFP mice. Immunofluorescence histochemistry was performed on 30-µm sections of colchicine-treated GHRH-eGFP mice with a GHRH antibody. eGFP labeling colocalizes with GHRH immunoreactivity (IR), as visualized by confocal microscopy in perikarya (a–f) of Arc and fibers of the external layer of ME (g–i). Arrow in A indicates the neuron that is enlarged in d–f.

 
Sexual dimorphism of sst2A receptor distribution on GHRH-eGFP neurons
sst2A receptor distribution in the GHRH-eGFP population was studied according to sex in animals not treated with colchicine (see Materials and Methods). The number of GHRH-eGFP cells immunoreactive for sst2A was quantified by confocal laser scanning microscopy on 10 sections throughout the Arc along its entire rostrocaudal axis in male and female mice (Fig. 2Go). The peak location of double-labeled cells occurred at 2.0 interaural in males, more rostrally than in females (1.8 interaural). On the 10 individual sections of the Arc, a striking (F = 29.760; P < 0.0001) sexual dimorphism was observed: in females, 78 ± 6% of GFP-positive neurons also expressed sst2A receptor immunostaining, whereas in males, only 26 ± 5% were also positive for the sst2A receptor (Fig. 2Go). The total area of sst2A receptor immunostaining in the Arc was similar in both sexes (54,300 ± 3710 µm2 in male vs. 60,735 ± 8155 µm2 in female mice).


Figure 2
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  		FIG. 2. Sexual dimorphism of sst2A receptor distribution on arcuate GHRH-eGFP neurons. A, Confocal micrographs of GHRH-eGFP-positive (a, d, g, j) and sst2A receptor-immunoreactive (sst2A-IR) (b, e, h, k) cells. sst2A immunolabeling was performed on three GHRH-eGFP mice of both sexes. Note that sst2A receptor labeling surrounds green GHRH cell bodies. Overlay staining (c, f, i, l) and scale bars (10 µm) are indicated in right columns. B, Quantification of GHRH-eGFP (black symbols) and GHRH-eGFP/sst2A-positive (white symbols) cells. The number of neurons was assessed by confocal microscopy using a x40 magnification on 30-µm sections (taken every 90 µm) along the entire rostrocaudal axis of the Arc. Data are given as mean ± SEM of three animals. The proportion of GHRH-eGFP/sst2A-positive cells is indicated in the black portion of the pie graphs.

 
sst2, GHRH, and SRIH expression
Because of the individual variation within groups, no significant difference in sst2 and SRIH mRNA levels in VMH-Arc and PeV regions and GHRH mRNA in the VMH-Arc region was found between male and female mice (Table 1Go).


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  		TABLE 1. Expression of sst2 receptor, GHRH, and SRIH mRNAs in VMH-Arc and PeV regions of male and female mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present work was undertaken to determine whether SRIH afferents on GHRH neurons are involved in the sexually dimorphic GH secretory pattern in mice, taking advantage of a transgenic model expressing eGFP in GHRH neurons (19). A major difference was the distribution of sst2 receptors, which, in female Arc, decorated almost 80% of GHRH neurons and, in male, remained less than 30%.

The distribution of GHRH-eGFP neurons was originally described in the male (19), with labeled cells mainly in the Arc and a few positive neurons in more dorsomedial hypothalamic regions. Green fluorescent somata were also immunolabeled in colchicine-treated animals with an antiserum against mouse GHRH, raised in this laboratory and tested for its specificity (see Materials and Methods). The superimposition of eGFP fluorescence and GHRH immunoreactivity allowed us to use the former to count GHRH. Aldehyde fixation of brains for immunocytochemistry may reduce the ability to detect eGFP in neurons, and it is theoretically possible that those neurons that express GHRH to a lower level would not be visualized using eGFP fluorescence. However, available data in the literature, obtained by conventional microscopic methods, provided a lower estimate of the total number of immunolabeled GHRH neurons in C57BL/6J mice, with no significant differences between males and females (24). Results presented herein, obtained by accurate counting of eGFP neurons by confocal microscopy (stacks analyzed over 20 µm to check for neuronal integrity in the depth of the section), do confirm the lack of sex difference in GHRH neuron number.

In the adult male rat, about 30% of GHRH cells bear sst receptors (15), later identified in equal proportions as sst1 and sst2 (17), but the distribution of sst receptors was unknown in the mouse. However, in this species, sst2 is necessary for GH-negative feedback on Arc GHRH neurons (25). In the mouse, sst2 mRNA can be spliced in two isoforms, sst2A and sst2B, both expressed in the rat brain and pituitary, except in the Arc/ME complex in which no sst2B was detected, indicating that sst2A is the isoform associated with GHRH neurons (26). Using a specific sst2A antiserum (21), almost 80% of female and less than 30% of male GHRH-eGFP neurons bear sst2A on their somata. This difference is observed all along the rostrocaudal axis of Arc. In the mouse, there is also evidence for a dimorphic pattern of ultradian GH secretion (Bluet-Pajot, M. T., and I. C. A. F. Robinson, unpublished observation), although technical difficulties in chronic blood sampling in nonrestrained animals have limited the reports of direct evidence for this (6, 7).

In contrast with GHRH neurons expression of sst2 immunoreactivity, no significant difference between males and females could be found in mean levels of sst2, GHRH, and SRIH transcripts assayed by real-time PCR on VMH-Arc and PeV extracts, perhaps due to the variation within individual mice in each groups. Variations in hypothalamic SRIH and GHRH mRNA levels have been related to ultradian oscillations in GH secretory episodes in the male rat (27). In the mouse, Low et al. (7) recently reported that male SRIH mRNA levels, in the whole hypothalamus, were 2-fold those of female mice. Such results were obtained in 129/Sv x C57BL animals backcrossed over five generations. The absence of dimorphism reported in SRIH hypothalamic expression in the GHRH-eGFP transgenic strain herein is also observed for SRIH hypothalamic content in 3-month-old C57BL/6J animals (data not shown). In addition, Kuwahara et al. (24) reported no significant sex differences in the immunoreactivity of SRIH PeV neurons in C57BL/6J mice up to 1 yr of age, after which a major increase is observed.

In the adult rat, short-term exposure to estradiol demasculinizes the male pattern of spontaneous and GHRH-induced GH secretion, as well as the rate of somatic growth (28); conversely, testosterone or dihydrotestosterone administration to adult female rats leads to a male-like secretory pattern (29, 30). In view of the sex-dependent distribution of sst2A receptors on mice GHRH neurons, the effects of gonadal steroids on this distribution remain to be assessed in conjunction with GH secretory patterns.


   Acknowledgments
 
We greatly acknowledge Dr. K. Toyama, P. M. Sinet, and P. Zizzari for technical assistance, and Drs. S. Pincus, P. Dournaud, and M. T. Bluet-Pajot for their advice and helpful discussions.


   Footnotes
 
This work was supported by Institut National de la Recherche Médicale and the French government (Action concertée incitative Neurosciences Grant 7AKO2H00A). K.B. was supported by a fellowship from Ministère de l’Education Nationale de la Recherche et de la Technologie (2001–2004).

K.B., C.L., I.C.A.F.R., J.E., and A.F.-B. have nothing to declare.

First Published Online February 23, 2006

Abbreviations: Arc, Arcuate nucleus; CT, threshold cycle; Cy3, cyanine 3; eGFP, enhanced green fluorescent protein; ME, median eminence; NDS, normal donkey serum; PeV, periventricular nucleus; sst, somatostatin; TBS, Tris-buffered saline; VMH, ventromedial hypothalamic nucleus.

Received November 17, 2005.

Accepted for publication February 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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