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Endocrinology Vol. 148, No. 3 1144-1149
Copyright © 2007 by The Endocrine Society

Transient Transcription of the Somatostatin Gene at the Time of Estrogen-Dependent Organization of the Sexually Dimorphic Nucleus of the Rat Preoptic Area

Chitose Orikasa, Yasuhiko Kondo and Yasuo Sakuma

Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Dr. Chitose Orikasa, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo, Tokyo 113-8602, Japan. E-mail: orikasa{at}nms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In situ hybridization detected a transient, sex-specific transcription of somatostatin gene in the central part of the rat medial preoptic nucleus, which coincides with the sexually dimorphic nucleus of the preoptic area (SDN-POA), during, but not after, the establishment of sex difference. On postnatal d 1 (day of birth), somatostatin mRNA was detected in the SDN-POA of both sexes. On d 8 through 35, the area of somatostatin mRNA-positive cells was significantly larger in males than in females. In males the area attained its maximum size on d 15 and diminished gradually thereafter. In females the area did not change in size during this period. On d 60 expression of somatostatin mRNA was low and not different between sexes. Throughout the observed period, Nissl staining and calbindin immunohistochemistry enabled visualization of the typical SDN-POA in the same region. As with Nissl staining and calbindin immunohistochemistry, somatostatin mRNA hybridization on d 15 revealed a reversal of the sexual dimorphism in the size of the SDN-POA in males that had been neonatally orchidectomized or females given estrogen as pups, showing that sex steroid milieu during the organizational period determines the area occupied by somatostatin mRNA-positive cells. Sex-specific, transient transcription of the somatostatin gene may causally relate to the estrogen-dependent organization of the SDN-POA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADAL-STEROID HORMONE action at a particular stage of brain development, termed the critical period for brain sex differentiation, determines sexually dimorphic features of certain brain structures (1). Gonadal steroids, estrogen in particular, have affected neurogenesis, migration, or survival (2, 3, 4, 5, 6). Although alterations in any of these processes during neuronal development would eventually culminate in brain sexual dimorphism, the molecular cascades that mediate hormone action are yet to be understood. Several neuropeptides, including somatostatin among others, also influence the developmental processes (7, 8). Somatostatin, originally identified as a hypothalamic peptide that inhibits the secretion of pituitary GH (9), is widely distributed in the central nervous system and periphery (10, 11), and is implicated in neuronal survival or neurogenesis (12). Marked changes in brain or cerebrospinal fluid levels of somatostatin are observed in a multitude of neurodegenerative diseases (13, 14). Furthermore, the number of somatostatin neurons is sexually dimorphic in the human (15) or rat (16) bed nucleus of the stria terminals. In the rat, this sexual dimorphism depends on the gonadal-steroid milieu during the critical period. Thus, administration of gonadal steroids to female pups or orchidectomy of male neonates could reverse the sexual dimorphism (16).

In the rat, one of the most prominent brain sex differences is found in the volume of the central part of the medial preoptic nucleus, often referred to as the sexually dimorphic nucleus of the preoptic area (SDN-POA), and which is significantly larger in the male than the female (17, 18). The difference also depends on, and could be manipulated by, the gonadal-steroid milieu during the critical period. Nissl staining (19) and immunohistochemistry against calbindin (20), the latter of which has become established as a specific marker for the SDN-POA, revealed that the SDN-POA is crammed with neurons, which may provide a good opportunity for neuropeptides like somatostatin to act in a paracrine fashion.

We report here the sexually dimorphic expression of somatostatin mRNA in the rat SDN-POA at the time of estrogen-dependent development of this structure. Neonatal orchidectomy diminished the expression of somatostatin mRNA, whereas administration of estradiol benzoate to female pups enhanced it. Sex-specific, transient transcription of the somatostatin gene may causally relate to the estrogen-dependent organization of the SDN-POA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley rats were obtained from CLEA Inc. (Tokyo, Japan) and housed at 23 C under a 14-h light/10-h dark cycle with free access to laboratory chow and water. Pregnant rats were checked daily before the expected date of parturition, and the day that pups were born was designated postnatal d 1.

Experiment 1: development of the somatostatin within the SDN-POA
Somatostatin mRNA expression was examined on d 1 (four males/four females), d 3 (three males/three females), d 8 (three males/four females), d 15 (seven males/four females), d 21 (three males/four females), d 35 (five males/five females), and in the adult aged 60 d (three males/three females). Days 1, 3, and 8, pups were placed on ice to accomplish hypothermal anesthesia. Days 15, 21, 35, and 60, rats were given an overdose (70 mg/kg) of sodium pentobarbital. All animals were perfused transcardially with 0.01 M PBS (pH 7.4), followed by ice-cold 4% paraformaldehyde fixative buffered by 0.1 M phosphate (pH 7.4). After perfusion, the brain was taken out and postfixed in the same solution for 2 d, then placed in phosphate-buffered 30% sucrose until they settled. Serial frontal sections (20-µm thick) that encompassed the organum vasculosum of the lamina terminals rostrally and the anterior hypothalamic area caudally were cut by a cryostat and mounted onto Silan-coated slides glasses (Shinetsu Silicon Chemicals, Tokyo, Japan). Six series of slides were prepared for each brain, and serial sections were distributed to them in sequence, so that every sixth section, 120-µm apart, was mounted on each slide in a given series. Slides were stored at –80 C until further processing for in situ hybridization histochemistry.

Experiment 2: neonatal manipulation of gonadal steroid milieu
Male and female neonates underwent endocrine manipulations. Males were castrated on d 1 under hypothermal anesthesia; females were given daily sc injections of 17ß-estradiol benzoate (Sigma-Aldrich, St. Louis, MO) (10 µg dissolved in 0.02 ml sesame oil) (21) on postnatal d 1–10. As in experiment 1, brains were collected under anesthesia on d 15 and processed.

In situ hybridization histochemistry
In situ hybridization was performed as previously described (21). Briefly, an antisense somatostatin cRNA probe was prepared from rat somatostatin cDNA (323 nt) (gift from Dr. M.R. Montminy) ligated into plasmid Bluescript KS II(+), which was linearized with HindIII and transcribed with T7 RNA polymerase in the presence of digoxigenin (dig)-11-UTP (Roche Molecular Biochemicals, Penzberg, Germany). For preparation of a sense probe, somatostatin cDNA was linearized with EcoRI and transcribed with T3 RNA polymerase.

Sections were treated with 20 µg/ml proteinase K digestion (Roche) in 0.1 M PBS. Each probe was dissolved (final concentration, 20 ng/100 µl) in hybridization buffer containing 50% formamide, 20 mM Tris-HCl, 10% dextran sulfate (Sigma), Escherichia coli tRNA (0.5 mg/ml; Roche), 1x Denhardt’s solution, 2.5 mM EDTA (Sigma), and 0.3 M NaCl. Processed sections were hybridized with the dig-labeled probe at 50 C overnight. Slides were rinsed in 2x SSC (1x SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7) in 50% formamide at 50 C for 1 h, and digested with RNase A (20 µg/ml; Roche) at 37 C for 30 min and in 1x SSC in 50% formamide at 50 C for 1 h, then incubated in a 1% blocking reagent in buffer I (0.1 M Tris-HCl; and 0.15 M NaCl, pH 7.5). For visualization the sections were exposed to an alkaline-phosphatase conjugate of anti-dig-Fab (1:500), and developed by nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate-toluidine. CrystalMount (Biomeda, Foster City, CA) covered the sections.

Immunohistochemistry
Sections were washed three times in 0.05 M PBS for 15 min and then incubated in 3% H2O2/methanol to remove endogenous peroxidases. After further washes in PBS, sections were incubated for 3 d at 4 C in 0.3% PB/T (0.3% Triton-X buffered by 0.1 M phosphate), 1% BSA, and 1:10,000 monoclonal antibody against calbindin-D-28k (Clone CB-955; Sigma). On the following day, sections were washed in PBS and incubated with a horse antimouse secondary antibody (5 µl/ml; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Sections were washed three times in PBS for 15 min and then incubated for 1 h at room temperature in ABC reagent (20 µl/ml; Vector Laboratories). After a final set of washes in PBS, sections were reacted with 1% diaminobenzidine/0.03% H2O2 in phosphate buffer. Sections were dehydrated in a series of ethanol washes and soaked in three changes of xylene.

Determination of the SDN-POA area and statistics
The SDN-POA was identified in alternate sections stained by cresyl violet and in situ histochemistry for somatostatin mRNA. Calbindin immunohistochemistry (20) was used as an additional clue to identify this structure. An arbitrary 200-µm square area, including the entire SDN-POA, which was visualized by one of three methods, was set to determine the size of the identified area using a Leica DC 100 digital imaging system and its software (Leica Microsystems, Wetzlar, Germany). The section with the largest identifiable SDN-POA was selected for comparison. The digitized microscopic images were converted to binary pictures that represented areas most approximated to the staining by adjusting the threshold value.

Two-way factorial ANOVA (sex x age) with the least significant difference (LSD) was used to analyze sex differences and developmental changes in the SDN-POA, as visualized by somatostatin mRNA in situ hybridization histochemistry and calbindin immunohistochemistry. When interaction effects were detected, a t test was used to analyze sex differences. One-way ANOVA (four groups) with the LSD was used to examine the effects of neonatal hormonal manipulation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Somatostatin mRNA-positive cells in the SDN-POA
Somatostatin mRNA-positive cells were found in the SDN-POA of both sexes (Fig. 1Go). The area occupied by somatostatin mRNA-positive cells agreed with that visualized by cresyl violet staining in adjacent sections. Analyses of developmental changes revealed faint but distinctive staining of somatostatin mRNA on d 1, which intensified by d 3, peaked on d 15, and was maintained until d 21 (Fig. 2Go). The expression of somatostatin mRNA in the SDN-POA diminished between d 35 and 60, whereas that in the periventricular nucleus was maintained without detectable change (Figs. 2Go and 3Go).


Figure 1
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  		FIG. 1. The sex differences in somatostatin mRNA expression (A and B) or showing the adjacent, Nissl-stained sections (C and D). All micrographs are from the central part of the medial preoptic nucleus, or the SDN-POA, of 15-d-old rats. Male, A and C. Female, B and D. Bar, 250 µm.

 

Figure 2
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  		FIG. 2. The lack of sex difference in somatostatin mRNA expression in the SDN-POA of adult male (A) and female (B) rats when visualized on d 60. Note that cells in the periventricular nucleus are intensely stained for somatostatin mRNA in both sexes at this age. Bar, 250 µm.

 

Figure 3
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  		FIG. 3. Developmental changes in the area occupied by somatostatin mRNA-expressing cells in the male and female SDN-POA. A significant sex difference (*, P < 0.05; **, P < 0.01) was established by postnatal d 8 and maintained until d 35. This difference was not detected on d 60.

 
In males the area positive for somatostatin mRNA signals at postnatal d 15 was significantly larger than that at postnatal d 1 or 3 [LSD, P < 0.05 following two-way ANOVA, F (6, 22) = 7.16; P < 0.05]. However, the area decreased gradually in size at subsequent ages. On postnatal d 60, the area was significantly smaller than that at d 15 (P < 0.05). In contrast, in females the area positive for somatostatin mRNA signals showed no significant change throughout development.

Sex difference in somatostatin mRNA expression
Through postnatal d 8–35, the area of the SDN-POA identified by somatostatin mRNA expression was significantly larger in males than females (t = 2.65–6.49; df = 5–12; Fig. 3Go). Earlier, on d 1 and 3, no sex difference was observed [t = 1.05 and 0.12; df = 6, 4, not significant (ns)]. A significant sex difference was established by postnatal d 8 and maintained until postnatal d 35 (Fig. 2Go). No sex difference was detected on d 60 (t = 1.81, ns).

Calbindin staining in the SDN-POA
Calbindin immunoreactivity was detected in the SDN-POA on d 1, in both sexes, with no sex difference in its area (t = 1.11; df = 6, ns). The area increased in size in both sexes until d 15, by which time there was a significant sex difference (t = 9.31; df = 4; P < 0.01), with the calbindin-positive area in the male twice as large as that in the female. By d 60, the calbindin-positive nucleus in the male was four times larger than that of the female (Fig. 4Go) (t = 8.92; df = 5; P < 0.01).


Figure 4
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  		FIG. 4. Developmental changes in the area visualized by calbindin immunoreactivity in the male and female SDN-POA. A significant sex difference (**, P < 0.01) was detected on d 15 and 60.

 
When compared in each sex between developmental stages, the area occupied by calbindin immunoreactive cells in males was significantly larger on d 15 than d 1 [LSD, P < 0.05 following two-way ANOVA; F (2, 7) = 132.59; P < 0.05] and continued to increase in the area at subsequent ages until d 60 (P <0.05). In females, after achieving a significant increase in the area by d 15 [LSD, P < 0.05 following two-way ANOVA; F (2, 8) = 52.79; P < 0.05], the development of the area was arrested between d 15 and 60 (Fig. 4Go).

Neonatal manipulation of gonadal steroid milieu
When observed on d 15, neonatally orchidectomized males had a significantly smaller area of the SDN-POA identified by somatostatin mRNA expression than intact males at the same age [LSD, P < 0.05 following ANOVA; F (3, 23) = 7.15; P < 0.001]. On the other hand, females treated with estradiol benzoate had a significantly larger area of somatostatin mRNA expression than normal females (Figs. 5Go and 6Go) (P < 0.001).


Figure 5
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  		FIG. 5. The effects of a neonatal sex steroid milieu on somatostatin mRNA expression in the SDN-POA on d 15. A, Intact male. B, Intact female. C, Neonatally castrated male. D, Neonatally estrogen-treated female. Bar, 100 µm.

 

Figure 6
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  		FIG. 6. Somatostatin mRNA expression in the SDN-POA after neonatal hormonal manipulations. A quantitative analysis of the effect of neonatal sex steroid milieu on the area occupied by somatostatin mRNA-expressing cells in the SDN-POA on d 15 was performed. Neonatal orchidectomy on d 1 (NC male) reduced this area significantly, to a level similar to that in intact females. Estradiol treatment of female pups (EB female) increased the size, to a level equivalent to that in the intact males. Columns with different letters indicate statistical significance (P < 0.05).

 
Correlation between measured area and number of labeled cells
Because somatostatin and calbindin-positive neurons were condensed in the SDN-POA, in most cases it was impossible to determine the number of the positive cells. We then examined correlation between the measured area size and the number of cells positive for each marker in a subset of cases. The cases were quasi-randomly collected from samples having relatively distinguishable positive cells. Pearson’s correlation coefficients were calculated in these cases, revealing a high correlation between the area and number of cells (r = 0.822; n = 19; P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study reveals transient transcription of the somatostatin gene in the SDN-POA, precisely at the time of the establishment of sexual dimorphism in this structure. Apparently because of the transient nature of the expression, the occurrence of somatostatin mRNA in the SDN-POA has been overlooked in the past. The cells expressing somatostatin mRNA occupied a significantly larger area in the male SDN-POA than in the homonymous structure in females on days spanning postnatal d 8–35, which reached its maximum size by d 15. It is noteworthy that the area of cells expressing somatostatin mRNA diminished in size after this period, whereas the area of the SDN-POA, visualized by either Nissl stain (19) or calbindin immunohistochemistry (20), was maintained until 60 d of age and presumably for the rest of the life.

The development of the sexual dimorphism in the SDN-POA has been attributed to a higher incidence of apoptosis in females than males in the central division of the medial preoptic nucleus on postnatal d 8–10 (22). Testosterone (22) and estrogen (23) prevent apoptosis during this period. On the other hand, the observed sexual dimorphism in cells expressing somatostatin mRNA was a result of a gradual increase of the area of these cells in males or estrogen-treated females during the same period, while the cell area in females has remained unchanged. Increasing in the area of these cells is the possible involvement of the changes in cell size during development. In this study we measured the size of the area positive for either somatostatin mRNA or calbindin immunoreactivity. Although an increase of neuronal soma size by growth may possibly cause the increased signal-positive areas, the contribution of cell growth to the increase of total area might be quite small. We examined the relationship between the area size and number of cells positive for each marker, and found that the area size is highly correlated with the number of cells included. Our data suggest that the number of neurons in the male rat SDN-POA is possibly increased by postnatal d 15. Therefore, we suggest a mechanism other than apoptosis for generating sexual dimorphism in the SDN-POA. A study of Bax-null mice shows that sex differences are predominantly caused by apoptosis; however, these mice still have sexual dimorphism in the dopaminergic neurons of the anteroventral periventricular nucleus of the preoptic area, suggesting the involvement of morphological sex differentiation mechanisms other than apoptosis (24).

There are three major possible mechanisms that could explain the sex difference in somatostatin expression in the SDN-POA: neuromigration, transcriptional activation, and neurogenesis. A sex-dependent influence on migratory characteristics has been shown in the mouse POA/anterior hypothalamic area (4). However, a study demonstrating the migratory development of the SDN-POA in rats showed that sex-dependent influence on the migration is still unconfirmed (3).

Little is known regarding a sex difference of steroid or age-dependent transcriptional activation in preexisting cells. In this case it is still possible that the net number of neurons is unchanged but that some cue determines whether SDN-POA neurons express somatostatin. There is also little evidence for a sex difference in neurogenesis in the SDN-POA (2). Because of the possible transient increase in somatostatin-positive cell number during a "critical period" in the developing male rat brain, we propose differences in neurogenesis as the mechanism responsible for the establishment of sexual dimorphism in somatostatin mRNA-expressing cells.

Somatostatin mRNA expression and peptide content, both of which are sexually dimorphic, have correlated in neurons in the periventricular nucleus (25). The mRNA expression level and peptide content in periventricular neurons increase during postnatal d 1–10 in both sexes, with significantly higher values in males on d 5 and 10 than females (26). In the SDN-POA we detected somatostatin mRNA on the day of birth in both sexes, and its sex difference was established on d 8. Thus, a striking resemblance exists in the onset of sex difference of somatostatin mRNA expression in the periventricular nucleus and the SDN-POA. The major difference, albeit in the mice, is that somatostatin neurons in the periventricular nucleus maintain the sexual dimorphism up to 60 d of age (27). The maintenance of the sexual dimorphism in the adult rat periventricular nucleus has been attributed to androgen actions (28).

Hormonal manipulation of female pups or male neonates revealed the now classic pattern of estrogen-dependent masculinization in the sex-specific transcription of the somatostatin gene and establishment of the SDN-POA as visualized by Nissl stain or calbindin immunohistochemistry. The diffuse and extensive distribution of estrogen receptor (ER) {alpha}-positive neurons in the preoptic area (5) has up till now hindered the specific localization of these cells in the SDN-POA. In a transgenic rat that expresses enhanced green fluorescent protein under the control of ER {alpha} promoter 0/B (29), we detected green fluorescence colocalized in ER {alpha}-positive neurons in the SDN-POA (30). Intrahypothalamic infusion of an antisense oligodeoxynucleotide to ER {alpha} in female pups before systemic testosterone resulted in a significantly smaller SDN-POA in the adult when compared with those that received scrambled oligos and testosterone (31). Castrated neonatal males treated with estradiol benzoate have increased levels of somatostatin mRNA compared with intact males, whereas dihydrotestosterone has no effect on the level of somatostatin mRNA (26). Thus, estrogen, presumably through the ER {alpha}, triggers the observed transient transcription of the somatostatin gene. Although the somatostatin gene does not contain an estrogen response element sequence, the somatostatin promoter contains a cAMP response element (32). Phosphorylation of a cAMP response element-binding protein is one of the signal transduction pathways by which estrogen regulates gene activation via ER {alpha} (33).

The SDN-POA identified by calbindin immunoreactivity has been described as a subdivision of the SDN-POA stained by cresyl violet (20). In the present study we noted a close agreement in the distribution of somatostatin mRNA-positive cells and those visualized by cresyl violet staining in adjacent sections. This may partially be caused by difficulty in delineating the SDN-POA, particularly during development, when somatostatin signals were emerging. Double staining for calbindin immunoreactivity and somatostatin mRNA would solve this conflict. Despite differences in the temporal expression of somatostatin and calbindin in the developing rat SDN-POA, in adults, the two peptides colocalize in many neurons in the basolateral amygdala (34) or those in the endopiriform cortex (35). On d 8–15 the morphology of the SDN-POA coincided when visualized by either in situ hybridization of somatostatin mRNA or calbindin immunohistochemistry. Therefore, somatostatin might be colocalized with calbindin during the development of the SDN-POA. Calbindin is a gene target of several cytokines and neurotrophic factors, which interrupt apoptotic biochemical cascades (36). IGF-II, which is present in high concentrations in the developing brain, activates the ER in embryonic brain nuclei, as observed in a neuroblastoma cell line (37). This growth factor not only increases the number of calbindin-immunoreactive neurons but also the number of neurites in individual neurons (38).

As a hypothalamic neuropeptide, somatostatin can be released from dendrites, like oxytocin and vasopressin, and function as an autocrine or paracrine signal at its site of origin (39). Because all subtypes of somatostatin receptors are functionally coupled to inhibition of adenylyl cyclase via Gi proteins (40), and reduced cyclic AMP culminates in neuronal apoptosis in many in vitro (41, 42) and in vivo (43) systems, it may seem unlikely that a paracrine action of somatostatin could have contributed to the enlargement of the male SDN-POA. However, that could be the case if some specific receptor subtype, such as estrogen-regulated somatostatin receptor subtype 1 or 2 (44) or somatostatin receptor subtype 4 coupled to mitogen-activated protein kinase, which is a convergence target for signaling pathways regulated by either ER {alpha} or IGF-I receptor to promote neuronal survival (45), was involved. Based on observations that differentiating and migrating neurons express somatostatin in the teleost brain, in which neurogenesis continues during adulthood, a role for somatostatin in neuronal differentiation and survival has been suggested (46). We are now in the process of determining the site-specific expression of a particular subtype of somatostatin receptor to settle this confusion.

Conclusions
This study shows sexually dimorphic transcription of the somatostatin gene in the area of the SDN-POA at the time of the establishment of sexual dimorphism. A gradual increase in the area of somatostatin mRNA expression in response to sex steroids accomplished sexual dimorphism, while the area of somatostatin mRNA-positive cells in females remained unchanged. Thus, active neuronal elimination through apoptosis did not account for the observed sexual differentiation. Further studies on somatostatin receptor subtypes expressed in the SDN-POA will help us understand the physiological significance of the transient transcription of the somatostatin gene during brain development in general.


   Footnotes
 
{smtexp}This work was supported by grants-in-aid for scientific research no. 16086210 from the Japanese Ministry of Education, Science, Sports and Culture (to Y.S.), and nos. 14370025 (to Y.S.) and 16590182 (to C.O.) from the Japanese Society for Promotion of Science.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 30, 2006

Abbreviations: dig, Digoxigenin; ER, estrogen receptor; LSD, least significant difference; ns, not significant; SDN-POA, sexually dimorphic nucleus of the preoptic area.

Received September 5, 2006.

Accepted for publication November 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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