Microarray profiling of gene expression patterns in adult male rat brain following acute progesterone treatment

BR A IN RE S EA RCH 1 0 67 ( 20 0 6 ) 5 8 –66

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Microarray profiling of gene expression patterns in adult male rat brain following acute progesterone treatment Catherine J. Auger a,⁎, Heather M. Jessen b , Anthony P. Auger b a

Department of Zoology, Birge Hall, 430 Lincoln Drive, University of Wisconsin, Madison, WI 53706, USA Department of Psychology, W.J. Brogden Hall, 1202 West Johnson Street, Madison, WI 53706, USA

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Progesterone can influence various behaviors in adult male rats, however, little is known

Accepted 16 October 2005

about which particular genes are regulated by progesterone in the male rat brain. Using

Available online 27 December 2005

focused microarray technology, we where able to define a subset of genes that are responsive to progesterone. Nylon membrane-based cDNA microarrays were used to profile

Theme:

gene expression patterns in the preoptic area/mediobasal hypothalamus (POA/MBH) of male

Endocrine and autonomic regulation

rat brain 7 h following a single injection of progesterone. RNA was isolated from the brains of

Topic:

6 male rats injected with progesterone and 6 male rats injected with sesame oil. Next, we

Neuroendocrine regulation: other

hybridized the RNA from each animal to individual cDNA microarrays that contained more than 100 target genes, all of which are involved in cAMP and or calcium signaling pathways.

Keywords:

Direct side-by-side comparison of all 12 arrays revealed differences in the expression

Progesterone

patterns of 12 different genes. We confirmed the data gathered from the arrays on 4 different

Microarray

genes using Real-Time PCR. These data begin to outline the important role played by

Gene array

progesterone in mediating changes in gene expression within the male brain. © 2005 Elsevier B.V. All rights reserved.

Male rat Mediobasal hypothalamus Real-Time PCR

1.

Introduction

Progesterone has customarily been thought of as the hormone that influences female reproductive behavior and physiology (Pfaff et al., 1994). However, progesterone also appears to be involved in modulating some physiological and behavioral functions in males. These include influencing anxiety behavior in rats and mice (Frye et al., 1992, 2004; Picazo and FernandezGuasti, 1995; Gomez et al., 2002) sexual behavior in male lizards (Witt et al., 1994) and rats (Witt et al., 1995; Phelps et al., 1998), sexual differentiation of the brain in male rats (Wagner et al.,

1998; Quadros et al., 2002; Lonstein et al., 2001), and infantrelated aggression in male mice (Schneider at el., 2003). Also, progesterone is involved in an assortment of physiologic and pathophysiologic functions in human men (Oettel and Mukhopadhyay, 2004). Based on previous research, it is clear that progesterone can influence a variety of behaviors in adult male rats (Witt et al., 1994; Schneider at el., 2003; Phelps et al., 1998); however, little is known about which particular genes are regulated by progesterone in the male rat brain. Progesterone acts within the brain by binding to intracellular steroid receptors (Tsai and O'Malley, 1994). We, and

⁎ Corresponding author. Fax: +1 608 262 4029. E-mail address: [email protected] (C.J. Auger).

0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.033

59

BR A IN RE S E A RCH 1 0 67 ( 20 0 6 ) 5 8 –6 6

others, have shown that males express progestin receptors in various regions of the brain in a similar pattern to that observed in females (Auger and De Vries, 2002; Blaustein et al., 1980; Brown et al., 1987). Additionally, the highest concentration of progestin receptors in both the male and female rat brain is in the preoptic area and hypothalamus (Blaustein et al., 1980; Brown et al., 1987). While progesterone action within the brain of females is widely studied, the action of progesterone in males is less clear. In males, progesterone is secreted by both the adrenal glands and the testes (Dohler and Wuttke, 1975; Kalra and Kalra, 1977; Fenske, 1997) and is regulated in blood and brain by a number of factors, including stress and social conditions (Serra at al., 2000). Our understanding of what genes are influenced by progesterone in the female brain is quite comprehensive contrasted to males; for that reason the screening technique employed in this study is important because it will provide a solid direction in which to proceed when examining the potential role that progesterone plays in the male brain (Fig. 1). Recently, microarray technology has been used to examine progesterone mediated gene expression in mouse uterus (Jeong et al., 2005), however, it has not been used to detail progesterone's influence on gene expression in the male rat brain. In order to identify genes regulated by progesterone within the adult male rat brain, we used membrane-based focused microarray technology. The microarray technique is very powerful in that it allows one to screen over a hundred genes at one time. This screening technique will allow us to further focus our research question based on the changes in gene expression patterns in male rat brain following physiological doses of progesterone. In this study, we identify a subset of genes that are responsive to progesterone in adult male rat brain (Tables 1, 2A and 2B).

2.

Results

2.1.

Progesterone enzyme immunoassay (EIA)

In order to examine the levels of progesterone that were in the serum 7 h following 1 mg progesterone injection, we used a progesterone EIA kit to measure the concentration of proges-

Table 1 – Sequences for primers used in Real-time PCR amplification Gene name Arc c-fos Calr Smst 18S rRNA

Primers (forward and reverse) F5′-ATGGAGCTGGACCATATGACGACC-3′ R5′-CTATTCAGGCTGGGTCCTGTCACT-3′ F5′-CCCGTAGACCTAGGGAGGAC-3′ R5′-CAATACACTCCATGCGGTTG-3′ F5′-ACGAGCCAAGATTGATGACC-3′ R5′-TCCCACTCTCCATCCATCTC-3′ F5′-GCCACCGGGAAACAGGAACT-3′ R5′-GAACGGGCTCCAGGGCATCG[FAM]TC-3′ F5′-GTCCCCCAACTTCTTAGAG-3′ R5′-CACCTACGGAAACCTTGTTAC-3′

terone found in the serum of progesterone- and oil-treated rats. The levels of progesterone in the progesterone-treated animals (3.8 ± 0.3; P = 0.002) were significantly higher than that of oil-treated animals (0.5 ± 0.3) 7 h after treatment (Fig. 2). These levels of progesterone are similar to what is found to be the physiological range for stressed and non-stressed animals, respectively (Andersen et al., 2004, 2005).

2.2. Gene expression profiles in the preoptic area/mediobasal hypothalamus (POA/MBH) of male rats Among the 109 target genes that were profiled in male rats treated with oil or progesterone, 12 genes were influenced by progesterone treatment (Tables 2A and 2B). In male rat brain, ten genes were found to be up-regulated by progesterone treatment (Table 2B), and two genes were found to be downregulated by progesterone treatment (Table 2A).

2.3.

Real-Time PCR confirmation of microarray results

We selected a subset of the genes that were influenced by progesterone for confirmation using quantitative Real-Time PCR. Of the twelve genes found to be regulated by progesterone treatment, we selected two genes that were up-regulated (somatostatin and calreticulin) and two genes that were downregulated (Arc and c-fos) following progesterone treatment. Confirming the microarray data, we found that both somatostatin (Fig. 3; P = 0.036) and calreticulin (Fig. 4; P = 0.049) mRNA were up-regulated by progesterone treatment, and Arc (Fig. 5;

Fig. 1 – Drawing of a sagittal view of the rat brain. The areas shaded in grey and encompassed by the rectangle represent tissue collected for RNA isolation and microarray hybridization.

60

BR A IN RE S EA RCH 1 0 67 ( 20 0 6 ) 5 8 –66

Table 2B – Genes found to be significantly up-regulated by progesterone treatment in male POA/MBH area based on chemiluminescent data gained from microarray hybridization Gene name

Accession no.

Calmodulin 1

NM 009790

Calr

NM 007591

Hspa4

NM 008300

Plf

NM 031191

Fig. 2 – Histogram representing progesterone levels in the serum of male rats 7 h after they were treated with either 1 mg progesterone or oil.

Pln

AK 002622

Ppp2ca

NM 019411

P = 0.018) and c-fos mRNA (Fig. 6; P = 0.044) were downregulated by progesterone treatment.

DUS1

NM 013642

Scg2

NM 009129

Smst GAPDH

NM 009215 M32599

3.

Discussion

Our data identify several genes that are regulated by progesterone within the POA/MBH of adult male rat brain. While progesterone is more commonly thought of as a female hormone, a role for progesterone in influencing physiology and behavior in males is becoming increasingly clear. This study used membrane-based focused microarrays to examine the effect of an acute, physiological, increase in serum progesterone levels on gene expression in the POA/MBH of male rats. These data are important as they pinpoint some of the genes that are regulated by progesterone, a component of progesterone signaling that is not widely understood for males, and suggest a broad impact on behavioral functions. We found that out of the 109 genes that we probed, progesterone was effective at influencing 12 of these genes. We found that progesterone can influence a diverse set of genes that include immediate early genes, calcium binding proteins, and steroid receptor binding proteins.

Description Calmodulin 1—Calcium binding protein Calreticulin—Calcium binding protein Heat Shock 70 kDa protein 4—Molecular chaperone Mus musculus proliferin— Promotes cell growth Phospholamban—Ca2+ handling protein Protein phosphotase 2a, catalytic subunit, alpha isoform—Signal transduction Tyrosine phosphotase, nonreceptor type 16— Signal transduction Secretogranin 2—Encodes secretory vesicle protein Somatostatin— Glyceraldehyde-3phosphotase dehydrogenase— Housekeeping gene

P value 0.020 0.037 0.010 0.037 0.023 0.001

0.040

0.001 0.026 0.029

progesterone. Regulation of the protein product of the c-fos gene by steroid hormones confirms previous reports that showed Fos protein levels are influenced by treatment with progesterone and/or estradiol (Auger and Blaustein, 1995; Insel, 1990), sometimes in a phasic manner (Rudick and Woolley, 2000, 2003). In addition to c-fos, we also observed a decrease in levels of another IEG, Arc, which is more tightly linked with some learning paradigms and can lead to changes in synaptic plasticity (Link et al., 1995; Yin et al., 2002; Lyford et al., 1995). As progesterone and its metabolites can interfere

3.1. Progesterone regulation of immediate early genes: potential role in learning and anxiety Our data indicated that both c-fos and Arc, two immediate early genes (IEG), are down-regulated following treatment with

Table 2A – Genes found to be significantly down-regulated by progesterone treatment in male POA/ MBH area based on chemiluminescent data gained from microarray hybridization Gene Accession name no. Arc

AF162777

c-fos

V00727

Description

P value

Activity regulated cytoskeletalassociated protein—Immediate early gene c-fos oncogene—Immediate early gene

0.039

0.043

Fig. 3 – Histogram representing the influence of progesterone on the relative mRNA levels of somatostatin within the preoptic area/mediobasal hypothalamus (POA/ MBH) of male rat brain. Relative mRNA levels, as obtained through Real-Time PCR amplification, confirmed results acquired from microarray analysis.

BR A IN RE S E A RCH 1 0 67 ( 20 0 6 ) 5 8 –6 6

61

Fig. 4 – Histogram representing the influence of progesterone on the relative mRNA levels of calreticulin within the POA/MBH of male rat brain. Relative mRNA levels, as obtained through Real-Time PCR amplification, confirmed results acquired from microarray analysis.

Fig. 6 – Histogram representing the influence of progesterone on the relative mRNA levels of the immediate early gene, c-fos, within the POA/MBH of male rat brain. Relative mRNA levels, as obtained through Real-Time PCR amplification, confirmed results acquired from microarray analysis.

with some learning paradigms (Chesler and Juraska, 2000; Warren and Juraska, 2000; Johansson et al., 2002) and Arc is associated with learning and synaptic plasticity, perhaps progesterone decreases some types of state dependent learning by decreasing the expression of Arc and possibly Fos protein. Another physiological link between progesterone and the IEGs, Arc and c-fos, is in the regulation of anxiety-related behavioral responses. For example, stress is known to increase both Fos and Arc expression in rat brain (Ons et al., 2004), and it is well known that progesterone in males has an anxiolytic effect (Gomez et al., 2002); therefore, progesterone may act in the male brain to reduce physiological and behavioral responses to stressors by decreasing stress-induced Fos and Arc.

calreticulin, which are involved in calcium signaling and in steroid receptor-mediated signaling. This regulation is in agreement with other reports indicating that steroid hormones can influence the expression pattern of calcium binding proteins, and that there are sex differences in the expression of calcium binding proteins (Gannon and McEwen, 1994; Rodriguez-Medina et al., 1998; Brager et al., 2000). While little is known about progesterone regulation of calcium binding proteins, previous studies indicate that glucocorticoids and testosterone can influence the levels of the calcium binding protein, calmodulin, within the hippocampus and the hypothalamus (Gannon and McEwen, 1994; Gannon et al., 1994; Rodriguez-Medina et al., 1998). Furthermore, the modulation of calmodulin levels in neonatal rat has profound effects on sexual differentiation of the brain and on adult sexual behavior (Rodriguez-Medina et al., 2002). Although calreticulin is commonly thought to play a role as a calcium binding chaperone in the endoplasmic reticulum, it has also been implicated in nuclear hormone receptor signaling (Roderick et al., 1997; Burns et al., 1994; Dedhar et al., 1994; Wheeler et al., 1995). Recently, calreticulin has been shown to interfere with the transactivation of a number of nuclear hormone receptors, it is for this function that it is also considered a steroid receptor corepressor (Wang et al., 2005). Another steroid receptor-associated protein influenced by progesterone is Hsp70. Consistent with previous reports; progesterone treatment increased the expression of Hsp70 within adult male rat brain. Hsp70 is a molecular chaperone that is involved in a variety of cellular functions. One important function of Hsp70 is its contribution to steroid receptor activity as it relates to signal transduction (Pratt and Toft, 2003). Hsp70 mRNA is up-regulated in the hypothalamus and pituitary within the rat brain following progesterone injection in female rats (Krebs et al., 1999). Therefore, progesterone regulation of calmodulin, calreticulin, and Hsp 70 is likely to impact steroid receptor-dependent cellular responses to subsequent steroid hormone exposure. These data suggest that acute increases in progesterone levels, which mimic the levels observed under brief stressful or

3.2. Progesterone regulation of signaling proteins known to influence steroid-receptor mediated responses We also observed that progesterone increased the expression of genes for two calcium binding proteins, calmodulin and

Fig. 5 – Histogram representing the influence of progesterone on the relative mRNA levels of the immediate early gene, Arc, within the POA/MBH of male rat brain. Relative mRNA levels, as obtained through Real-Time PCR amplification, confirmed results acquired from microarray analysis.

62

BR A IN RE S EA RCH 1 0 67 ( 20 0 6 ) 5 8 –66

anxiety provoking conditions, can quickly modulate how the brain responds to steroid hormones.

3.3. Progesterone regulation of somatostatin: potential role in arousal and analgesia Somatostatin, originally identified as an inhibitor of growth hormone secretion, has been shown to have varied effects on behavior and physiology (Blake et al., 2004; Murray et al., 2004). A dosage dependant effect of somatostatin in the nervous system can either stimulate or suppress general arousal of behavior and analgesia (Raynor et al., 1993; Tashev et al., 2001a, b; Schindler et al., 1998). Furthermore, somatostatin neurons are steroid responsive, as gonadectomy results in changes in somatostatin mRNA content in the hypothalamus of both male and female rats, and express receptors for estrogen, progesterone and androgens (Herbison, 1994; Herbison and Theodosis, 1993; Kalra and Kalra, 1977). Progesterone is also known to influence arousal as well as analgesia (Mong and Pfaff, 2004). Therefore, it is possible that progesterone mediates some of these changes by altering somatostatin levels in male brain.

3.4. Progesterone regulation of proliferin and secretogranin: influence on endocrine function Our array data indicate that progesterone up-regulates two genes within the male brain that are known to impact endocrine function. Proliferin is a hormone within the prolactin/growth hormone family that is known to promote growth and development of cells in a number of different tissue types (Wilder and Linzer, 1986; Linzer and Nathans, 1983, 1984; Corbacho et al., 2002). Proliferin is very important in normal control of fetal/maternal chemical communication and development of maternal reproductive, placental and fetal tissues during pregnancy (Fang et al., 1999; Corbacho et al., 2002; Jackson and Linzer, 1997). As both proliferin and progesterone are increased during pregnancy, these data are consistent with the idea that progesterone alters proliferin expression. Our data may suggest a novel role for proliferin in steroid hormone signaling in the adult male brain. Secretogranin is a member of the granin family of proteins and is mainly found in dense-core vesicles of neurons, endocrine, and neuroendocrine cells (Fischer-Colbrie et al., 1995; Rosa et al., 1989; Laslop and Mahata, 2002). Secretogranin is commonly found to be associated with the gonadotrophins, and it may play a role in the differential secretion of the gonadotrophins (Nicol et al., 2002, 2004; Crawford et al., 2002). Secretogranin also appears to be important in releasing the behaviorally-relevant peptide, vasopressin, in male rat brain (Ang et al., 1997). Like proliferin, our data suggest a novel role for secretogranin in potentially mediating progesterone responses in male rat brain.

3.5. Identification of additional progesterone responsive genes in male rat brain Finally, our array data indicate that GAPDH and two protein phosphatases are up-regulated in the brain following pro-

gesterone treatment. GAPDH, a so-called housekeeping gene, which is commonly used to normalize protein or mRNA between samples in different assays is known to be sexually dimorphic during development and regulated by hormones in adult female rats (Perrot-Sinal et al., 2001; Funabashi et al., 1994). We report that GAPDH is also regulated by progesterone in adult male brain. We also observed that progesterone up-regulated two genes involved in cellular signal transduction, protein phosphatase 2A (PP2A) and tyrosine phosphatase nonreceptor type 16 (DUS 1). PP2A is known to be involved in the regulation of sex steroid biosynthesis (Pandey et al., 2003), and tyrosine phosphorylation is known to be modulated by steroids (Revelli et al., 1998). We also observed that phospholamban is regulated by progesterone exposure in male rat brain. Phospholamban is a protein that plays a role in calcium signaling and is known to be important for cardiac contraction and relaxation in mammals (Simmerman and jones, 1998). The functional significance of these genes in response to progesterone in male rat brain is currently unknown. In summary, our array data indicate that progesterone action can regulate a variety of target genes in the adult male rat brain. These data identify several genes that are impacted by physiological changes in serum progesterone levels, which mimic the levels that males show in response to anxietyprovoking situations. Our data appear to be consistent with the concept that progesterone, like estrogen, impacts brain function and behavior and influences gene sets that modulate social, sexual, and anxiety-related functions (Mong and Pfaff, 2004). As the progesterone levels achieved in the animals in this study were similar to that of animals in stressful and/or anxious situations, this study may have revealed additional genes involved in modulating an organism's ability to cope with anxiogenic events.

4.

Experimental procedures

4.1.

Animals

Intact adult male Sprague–Dawley rats were obtained from our breeding colony and maintained under a 14:10 h light/dark cycle with food and water available ad libitum. Rats were held under these conditions for approximately six months. Rats were given sub-cutaneous injections of either 1 mg progesterone in 0.2 ml sesame oil (n = 6) or 0.2 ml control oil (n = 6). Seven hours later, animals were rapidly decapitated and the whole brain was removed from the skull. The dosage of 1 mg of progesterone was chosen as this dose was previously reported to decrease vasopressin mRNA expression within the hypothalamus of male rats (Patchev et al., 1996). Also a similar dosage was used to influence anxiety-related behavior of male rats in an elevatedplus maze (Gulinello and Smith, 2003). We choose to examine changes in mRNA expression 7 h after treatment as this time course was previously used to detect progesterone-induced changes of pituitary adenylate cyclase-activating polypeptide within the mediobasal hypothalamus (Ha et al., 2000). The progestin receptor-rich POA/MBH was rapidly dissected out of the brain (Fig. 1), snap frozen, and stored frozen at −80° until processed for RNA extraction. We choose the POA/MBH as this area expresses high levels of progestin receptors contrasted to other regions of the male brain in order to maximize our chances of observing changes in gene expression following progesterone treatment.

BR A IN RE S E A RCH 1 0 67 ( 20 0 6 ) 5 8 –6 6

4.2.

Hormone assay

Trunk blood was collected 7 h after animals were treated with either 1 mg progesterone in 0.2 ml sesame oil or 0.2 ml sesame oil for testosterone enzyme immunoassay (EIA) (Cayman Chemical Company, Ann Arbor, MI). At least 500 μl of blood was collected from treated animals and centrifuged at ∼9700 RPMs or 10,000 × g for 10 min. Next, serum was removed and stored in a clean tube at −20 °C until used in progesterone EIA. This progesterone EIA is based on the competition between progesterone and a progesterone–acetylcholinesterase conjugate for a limited number of progesterone specific binding sites. Progesterone standards were prepared according to the manufacturer's instructions. Following the preparation of progesterone standards, these and the serum samples from our treated animals were loaded into a 96-well plate, as well as the necessary controls. Each well of a 96-well plate was coated with mouse anti-rabbit IgG. Progesterone specificacetylcholinesterase (ACHe) tracer was added to most of the wells followed by addition of the rabbit anti-progesterone antiserum to most of the same wells. The plate was then left to incubate for 1 h at room temperature on an orbital shaker to allow for competitive binding. After this incubation, the plate was washed to discard unbound reagents, 5 times with wash buffer supplied by the manufacturer. Following these rinses, the concentration of progesterone was determined by measuring the enzymatic activity of ACHe with Ellman's Reagent (which contains the substrate for ACHe). The product of this enzymatic reaction has a distinct yellow color that absorbs at 412 nm. The plate was left to develop in the dark for about 1 h or until the absorbance of the maximum binding wells equaled 0.3 A.U., as instructed by the manufacturer. Following the developing process, the plate was read at a wavelength between 405 and 420 nm with a plate reader. All samples for hormone measurement were quantified in the same assay. The assay specificity is 100% for progesterone, and intra-assay coefficient was 8.7%. The detection limit of this assay was 10 pg/ml. Results were calculated using a computer spreadsheet program provided by Cayman Chemicals (www. caymanchem.com/eiatools/promo/kit). 4.3.

RNA extraction

RNA extraction protocol was similar to that provided by Qiagen RNeasy Kit (Qiagen, Valencia, CA); however, we found that some modification of the protocol was necessary for the most efficient RNA yield. Frozen POA/MBH tissue samples (∼30 mg) were homogenized in buffer containing β-mercaptoethanol using a Dremel digital rotary tool (5000 RPM) and then centrifuged at 20,817 × g for 30 min. The supernatant was collected and mixed with equal amounts of 70% ETOH, loaded onto mini spin column, and then centrifuged for 15 s at 14,000 × g. Next, the samples were run through 3 wash spins with the buffer provided by the Qiagen RNeasy kit to remove contaminants. The first and second wash was centrifuged for 15 s, at 14,000 × g, and the third wash was centrifuged for 2 min at 16,000 × g. RNA was eluted by loading 50 μl of UV-treated nanopure water into the mini spin column and incubated for 5 min so the water would saturate the membrane. Then the spin column was centrifuged for 1 min at 14,000 × g. The concentration of RNA was determined using a BioMate 3 spectrophotometer (Thermo Spectronic, Rochester, NY, USA). Known concentrations of total RNA were then used to probe focused nylon membrane based microarrays, GEArray™ Q Series cAMP/Calcium PathwayFinder Gene Array, obtained from SuperArray Bioscience Corporation (Frederick, MD, USA). 4.4.

cAMP/calcium focused microarrays

The expression of progesterone-regulated brain mRNAs were investigated using a commercial DNA microarray (SuperArray Bioscience Corp). Of signal transduction pathways, we know a

63

substantial amount about cAMP and calcium regulated pathways within the hypothalamus, especially those of PKC and PKA signaling pathway altered by steroid hormones. To maximize the number of genes detected, we chose this array based upon the information that progesterone is known to influence many of these genes in females. The microarrays used in this study employ an easily recognizable tetra-spot pattern and each array contains up to 96 target gene spots plus 13 housekeeping genes and negative controls. Each microarray probe tetra-spot contains gene-specific double-stranded 250–600 bp cDNA fragments. The protocol used was similar to that provided by SuperArray Bioscience Corp. Twelve arrays were processed at the same time, and each of the arrays were hybridized with the RNA obtained from 1 rat. Therefore, 6 arrays were probed with RNA from progesterone treated rats (n = 6) and 6 arrays probed with RNA from oil treated rats (n = 6). Membranes were prehybridized in GEAhyb Hybridization Solution (provided by SuperArray Bioscience Corp) and salmon sperm DNA (100 μg/ml) at 60 °C under constant rotation in individual vials to block non-specific binding sites on the array membrane. During this time, total RNA was converted to cDNA using an GEArray™ Probe Synthesis Kit (SuperArray Bioscience Corp, Frederick, MD, USA), and labeled with Biotin-16-dUTP (Roche, Indianapolis, IN, USA) for chemiluminescent detection of hybridized cDNA. Probe synthesis was accomplished using a standard thermal cycler (Mastercycler®Personal, Eppindorf North America, Westbury, NY, USA). After the probe was labeled, hybridization buffer was prepared by combining labeled cDNA with the prehybridization buffer. Hybridization occurred overnight at 60 °C under constant rotation (5–10 RPM). On the following day, hybridization buffer was rinsed from the membranes using 2× SSC containing 1% SDS and then 0.1× SSC containing 0.5% SDS at room temperature under constant rotation (15 RPM). Membranes were then incubated in GEAblocking solution (provided by SuperArray Bioscience Corp) for 40 min with constant rotation (10 RPM). Membranes were then incubated for 10 min in alkaline phosphatase-conjugated steptavidin. Following this incubation, the membranes were washed four times, five min each rinse, in washing buffer provided with the SuperArray kit, and then incubated in 1 ml of CDP-Star Solution for 2 min. Membranes were exposed to X-ray film for 2 min then developed using an automatic film processor. 4.5.

Image acquisition and analysis

X-ray films were converted to digitized TIFF images with a scanner and then converted to numerical data using the ScanAlyze software developed by Dr. Michael Eisen. Numerical data were then analyzed using the GEArray Analyzer software, which is available on the SuperArray website (http://www.superarray. com). Data from this analysis was then subjected to a Two Tailed t test using the Sigma Stat statistical analysis software for Windows v 3.11 (Systat Software, Inc., Point Richmond, CA). Differences were only considered significant if the P value was 0.05 or less. 4.6.

Real-time PCR confirmation

We then confirmed the data obtained from the gene expression microarrays using quantitative Real-Time PCR. A StrataScript First Strand Synthesis system kit (Stratagene, Cedar Creek, TX, USA) was used to reverse transcribe 5 μg of dissected POA/MBH total RNA to cDNA in an Eppindorf Mastercycler®Personal PCR machine (Eppindorf North America, Westbury, NY, USA). The cDNA was then amplified using two different methods. For the somatostatin gene, Invitrogen LUX™ primers (Invitrogen Corporation, Carlsbad, CA, USA) were used in combination with 18S rNA. Primers labeled with either the fluorogenic reporter dyes 6-carboxy-fluorescein (FAM) or 6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE) were multiplexed in the same tube during the Real-Time

64

BR A IN RE S EA RCH 1 0 67 ( 20 0 6 ) 5 8 –66

PCR reaction. Multiplexing allowed us to use different sets of primers with different fluorogenic labels to amplify separate genes within the same reaction tube. Somatostatin primers were labeled with FAM and 18S rRNA (Cat. #115HM-02; Invitrogen Corporation, Carlsbad, CA, USA), which was used as a reference gene, was labeled with JOE. FAM is read at 492 nm–516 nm, and JOE is read at 535 nm–555 nm wavelength. In order to more tightly control for variation in the amplification procedure, we performed a dilution curve using the 18S primers so that the range of amplification more closely resembled that of the gene of interest, i.e., somatostatin. For the Arc, Calerticulin, and c-fos genes, products were amplified using a Brilliant Sybr Green QPCR MasterMix kit (Stratagene, Cedar Creek, TX, USA) in separate reaction tubes and contrasted to 18S rRNA labeled with Brilliant Sybr Green in separate reaction tubes using a Real-Time PCR thermo cycler. Sybr Green was detected at 492 nm–516 nm wavelength. For primer sequences see Table 1. Quantitative Real-Time PCR was carried out in a Stratagene Mx3000 Real-Time PCR system. The amplification protocol was as follows: an initial denaturing step at 95 °C for 5 min followed by 30 cycles of a 95 °C denaturing step for 1 min, a 60 °C annealing step for 1 min, and a 72 °C elongation step for 1 min. Following amplification, a dissociation curve analysis was performed to insure purity of PCR products. All primers used were synthesized by Invitrogen (Carlsbad, CA) with standard purity. 18S rRNA was used as a reference gene in this method, as it is considered to be a housekeeping gene, and was found not to be regulated by the treatment conditions in this study. Data were analyzed using the ΔΔCT method (Livak and Schmittgen, 2001). Briefly, the CT for each sample was determined by obtaining the difference between the average CT of the reference gene (18S rRNA) and the average CT of the gene of interest (i.e., ARC, somatostatin). The ΔCT of the calibrator (an untreated control) is then subtracted from the ΔCT of each of the samples to determine the ΔΔCT. This number is then used to determine the amount of mRNA relative to the calibrator and normalized by 18s rRNA, or the n-fold difference. The n-fold difference was calculated by the equation 2(−ΔΔCT). Statistical comparisons were carried out using Sigma Stat statistical analysis software for Windows v 3.11 (Systat Software, Inc., Point Richmond, CA). A one-tailed t test was used, as we were able to predict direction based on data from the microarrays.

REFERENCES

Andersen, M.L., Bignotto, M., Tufik, S., 2004. Hormone treatment facilitates penile erection in castrated rats after sleep deprivation and cocaine. J. Neuroendocrinol. 16, 154–159. Andersen, M.L., D'Almeida, V., Martins, P.J., Antunes, H.K., Tufik, S., 2005. Effects of paradoxical sleep deprivation and cocaine on genital reflexes in hyperlipidic-fed rats. Pharmacol. Biochem. Behav. 81, 758–763. Ang, C.W., Dotman, C.H., Winkler, H., Fischer-Colbrie, R., Sonnemans, M.A., van Leeuwen, F.W., 1997. Specific expression of secretogranin II in magnocellular vasopressin neurons of the rat supraoptic and paraventricular nucleus in response to osmotic stimulation. Brain Res. 765, 13–20. Auger, A.P., Blaustein, J.D., 1995. Progesterone enhances an estradiol-induced increase in Fos-immunoreactivity in localized regions of female rat forebrain. J. Neurosci. 15, 2272–2279. Auger, C.J., De Vries, G.J., 2002. Progestin receptor immunoreactivity within steroid-responsive vasopressin-immunoreactive cells in the male and female rat brain. J. Neuroendocrinol. 14, 561–567. Blake, A.D., Badway, A.C., Strowski, M.Z., 2004. Delineating somatostatin's neuronal actions. Curr. Drug Targets CNS Neurol. Disord. 3, 153–160. Blaustein, J.D., Ryer, H.I., Feder, H.H., 1980. A sex difference

in the progestin receptor system of guinea pig brain. Neuroendocrinology 31, 403–409. Brager, D.H., Sickel, M.J., McCarthy, M.M., 2000. Developmental sex differences in calbindin-D(28K) and calretinin immunoreactivity in the neonatal rat hypothalamus. J. Neurobiol. 42, 315–322. Brown, T.J., Clark, A.S., MacLusky, N.J., 1987. Regional sex differences in progestin receptor induction in the rat hypothalamus: effects of various doses of estradiol benzoate. J. Neurosci. 7, 2529–2536. Burns, K., Duggan, B., Atkinson, E.A., Famulski, K.S., Nemer, M., Bleackley, R.C., Michalak, M., 1994. Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367, 476–480. Chesler, E.J., Juraska, J.M., 2000. Acute administration of estrogen and progesterone impairs the acquisition of the spatial morris water maze in ovariectomized rats. Horm. Behav. 38, 234–242. Corbacho, A.M., Martinez, D.L.E., Clapp, C., 2002. Roles of prolactin and related members of the prolactin/growth hormone/ placental lactogen family in angiogenesis. J. Endocrinol. 173, 219–238. Crawford, J.L., McNeilly, J.R., Nicol, L., McNeilly, A.S., 2002. Promotion of intragranular co-aggregation with LH by enhancement of secretogranin II storage resulted in increased intracellular granule storage in gonadotrophs of GnRH-deprived male mice. Reproduction 124, 267–277. Dedhar, S., Rennie, P.S., Shago, M., Hagesteijn, C.Y., Yang, H., Filmus, J., Hawley, R.G., Bruchovsky, N., Cheng, H., Matusik, R.J., 1994. Inhibition of nuclear hormone receptor activity by calreticulin. Nature 367, 480–483. Dohler, K.D., Wuttke, W., 1975. Changes with age in levels of serum gonadotropins, prolactin, and gonadal steroids in prepubertal male and female rats. Endocrinology 97, 898–907. Fang, Y., Lepont, P., Fassett, J.T., Ford, S.P., Mubaidin, A., Hamilton, R.T., Nilsen-Hamilton, M., 1999. Signaling between the placenta and the uterus involving the mitogen-regulated protein/proliferins. Endocrinology 140, 5239–5249. Fenske, M., 1997. Role of cortisol in the ACTH-induced suppression of testicular steroidogenesis in guinea pigs. J. Endocrinol. 154, 407–414. Fischer-Colbrie, R., Laslop, A., Kirchmair, R., 1995. Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin. Progr. Neurobiol. 46, 49–70. Frye, C.A., Bock, B.C., Kanarek, R.B., 1992. Hormonal milieu affects tailflick latency in female rats and may be attenuated by access to sucrose. Physiol. Behav. 52, 699–706. Frye, C.A., Walf, A.A., Rhodes, M.E., Harney, J.P., 2004. Progesterone enhances motor, anxiolytic, analgesic, and antidepressive behavior of wild-type mice, but not those deficient in type 1 5 alpha-reductase. Brain Res. 1004, 116–124. Funabashi, T., Brooks, P.J., Weesner, G.D., Pfaff, D.W., 1994. Luteinizing hormone-releasing hormone receptor messenger ribonucleic acid expression in the rat pituitary during lactation and the estrous cycle. J. Neuroendocrinol. 6, 261–266. Gannon, M.N., McEwen, B.S., 1994. Distribution and regulation of calmodulin mRNAs in rat brain. Brain Res. Mol. Brain Res. 22, 186–192. Gannon, M.N., Akompong, T., Billingsley, M.L., McEwen, B.S., 1994. Adrenalectomy-induced alterations of calmodulin-dependent hippocampal adenylate cyclase activity: role of guanine nucleotide-binding proteins. Endocrinology 134, 853–857. Gomez, C., Saldivar-Gonzalez, A., Delgado, G., Rodriguez, R., 2002. Rapid anxiolytic activity of progesterone and pregnanolone in male rats. Pharmacol. Biochem. Behav. 72, 543–550. Gulinello, M., Smith, S.S., 2003. Anxiogenic effects of neurosteroid exposure: sex differences and altered GABAA receptor pharmacology in adult rats. J. Pharmacol. Exp. Ther. 305, 541–548.

BR A IN RE S E A RCH 1 0 67 ( 20 0 6 ) 5 8 –6 6

Ha, C.M., Kang, J.H., Choi, E.J., Kim, M.S., Park, J.W., Kim, Y., Choi, W.S., Chun, S.Y., Kwon, H.B., Lee, B.J., 2000. Progesterone increases mRNA levels of pituitary adenylate cyclaseactivating polypeptide (PACAP) and type I PACAP receptor (PAC(1)) in the rat hypothalamus. Brain Res. Mol. Brain Res. 78, 59–68. Herbison, A.E., 1994. Somatostatin-immunoreactive neurons in the hypothalamic ventromedial nucleus possess oestrogen receptors in the male and female rat. J. Neuroendocrinol. 6, 323–328. Herbison, A.E., Theodosis, D.T., 1993. Absence of estrogen receptor immunoreactivity in somatostatin (SRIF) neurons of the periventricular nucleus but sexually dimorphic colocalization of estrogen receptor and SRIF immunoreactivities in neurons of the bed nucleus of the stria terminalis. Endocrinology 132, 1707–1714. Insel, T.R., 1990. Regional induction of c-fos-like protein in rat brain after estradiol administration. Endocrinology 126, 1849–1853. Jackson, D., Linzer, D.I., 1997. Proliferin transport and binding in the mouse fetus. Endocrinology 138, 149–155. Jeong, J.W., Lee, K.Y., Kwak, I., White, L.D., Hilsenbeck, S.G., Lydon, J.P., Demayo, F.J., 2005. Identification of murine uterine genes regulated in a ligand-dependent manner by the progesterone receptor. Endocrinology 146, 3490–3505. Johansson, I.M., Birzniece, V., Lindblad, C., Olsson, T., Backstrom, T., 2002. Allopregnanolone inhibits learning in the Morris water maze. Brain Res. 934, 125–131. Kalra, P.S., Kalra, S.P., 1977. Circadian periodicities of serum androgens, progesterone, gonadotropins and luteinizing hormone-releasing hormone in male rats: the effects of hypothalamic deafferentation, castration and adrenalectomy. Endocrinology 101, 1821–1827. Krebs, C.J., Jarvis, E.D., Pfaff, D.W., 1999. The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus. Proc. Natl. Acad. Sci. U. S. A. 96, 1686–1691. Laslop, A., Mahata, S.K., 2002. Neuropeptides and chromogranins: session overview. Ann. N. Y. Acad. Sci. 971, 294–299. Link, W., Konietzko, U., Kauselmann, G., Krug, M., Schwanke, B., Frey, U., Kuhl, D., 1995. Somatodendritic expression of an immediate early gene is regulated by synaptic activity. Proc. Natl. Acad. Sci. U. S. A. 92, 5734–5738. Linzer, D.I., Nathans, D., 1983. Growth-related changes in specific mRNAs of cultured mouse cells. Proc. Natl. Acad. Sci. U. S. A. 80, 4271–4275. Linzer, D.I., Nathans, D., 1984. Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family. Proc. Natl. Acad. Sci. U. S. A. 81, 4255–4259. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods 25, 402–408. Lonstein, J.S., Quadros, P.S., Wagner, C.K., 2001. Effects of neonatal RU486 on adult sexual, parental, and fearful behaviors in rats. Behav. Neurosci. 115, 58–70. Lyford, G.L., Yamagata, K., Kaufmann, W.E., Barnes, C.A., Sanders, L.K., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Lanahan, A.A., Worley, P.F., 1995. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 14, 433–445. Mong, J.A., Pfaff, D.W., 2004. Hormonal symphony: steroid orchestration of gene modules for sociosexual behaviors. Mol. Psychiatry 9, 550–556. Murray, R.D., Kim, K., Ren, S.G., Chelly, M., Umehara, Y., Melmed, S., 2004. Central and peripheral actions of somatostatin on the growth hormone–IGF-I axis. J. Clin. Invest. 114, 349–356. Nicol, L., McNeilly, J.R., Stridsberg, M., Crawford, J.L., McNeilly, A.S., 2002. Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to

65

LH release in LbetaT2 gonadotroph cells. J. Endocrinol. 174, 473–483. Nicol, L., McNeilly, J.R., Stridsberg, M., McNeilly, A.S., 2004. Differential secretion of gonadotrophins: investigation of the role of secretogranin II and chromogranin A in the release of LH and FSH in LbetaT2 cells. J. Mol. Endocrinol. 32, 467–480. Oettel, M., Mukhopadhyay, A.K., 2004. Progesterone: the forgotten hormone in men? Aging Male 7, 236–257. Ons, S., Marti, O., Armario, A., 2004. Stress-induced activation of the immediate early gene Arc (activity-regulated cytoskeleton-associated protein) is restricted to telencephalic areas in the rat brain: relationship to c-fos mRNA. J. Neurochem. 89, 1111–1118. Pandey, A.V., Mellon, S.H., Miller, W.L., 2003. Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17. J. Biol. Chem. 278, 2837–2844. Patchev, V.K., Hassan, A.H., Holsboer, D.F., Almeida, O.F., 1996. The neurosteroid tetrahydroprogesterone attenuates the endocrine response to stress and exerts glucocorticoid-like effects on vasopressin gene transcription in the rat hypothalamus. Neuropsychopharmacology 15, 533–540. Perrot-Sinal, T.S., Davis, A.M., McCarthy, M.M., 2001. Developmental sex differences in glutamic acid decarboxylase (GAD(65)) and the housekeeping gene, GAPDH. Brain Res. 20 (922), 201–208. Pfaff, D.W., Schwartz-Giblin, S., McCarthy, M.M., Kow, L.-M., 1994. Cellular and molecular mechanisms of female reproductive behaviors. In: Knobil, E., Neill, J.D. (Eds.), Physiology of Reproduction. Raven Press, Ltd., New York, pp. 107–220. Phelps, S.M., Lydon, J.P., O'Malley, B.W., Crews, D., 1998. Regulation of male sexual behavior by progesterone receptor, sexual experience, and androgen. Horm. Behav. 34, 294–302. Picazo, O., Fernandez-Guasti, A., 1995. Anti-anxiety effects of progesterone and some of its reduced metabolites: an evaluation using the burying behavior test. Brain Res. 680, 135–141. Pratt, W.B., Toft, D.O., 2003. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. (Maywood) 228, 111–133. Quadros, P.S., Lopez, V., De Vries, G.J., Chung, W.C., Wagner, C.K., 2002. Progesterone receptors and the sexual differentiation of the medial preoptic nucleus. J. Neurobiol. 51, 24–32. Raynor, K., Lucki, I., Reisine, T., 1993. Somatostatin receptors in the nucleus accumbens selectively mediate the stimulatory effect of somatostatin on locomotor activity in rats. J. Pharmacol. Exp. Ther. 265, 67–73. Revelli, A., Massobrio, M., Tesarik, J., 1998. Nongenomic actions of steroid hormones in reproductive tissues. Endocr. Rev. 19, 3–17. Roderick, H.L., Campbell, A.K., Llewellyn, D.H., 1997. Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors. FEBS Lett. 405, 181–185. Rodriguez-Medina, M.A., Vergara, M., Chavarria, M.E., Rosado, A., Reyes, A., 1998. Changes in hypothalamic calmodulin concentration induced by perinatal hormone manipulation in the rat. Pharmacol. Biochem. Behav. 61, 445–450. Rodriguez-Medina, M.A., Reyes, A., Chavarria, M.E., VergaraOnofre, M., Canchola, E., Rosado, A., 2002. Asymmetric calmodulin distribution in the hypothalamus: role of sexual differentiation in the rat. Pharmacol. Biochem. Behav. 72, 189–195. Rosa, P., Weiss, U., Pepperkok, R., Ansorge, W., Niehrs, C., Stelzer, E.H., Huttner, W.B., 1989. An antibody against secretogranin I (chromogranin B) is packaged into secretory granules. J. Cell Biol. 109, 17–34. Rudick, C.N., Woolley, C.S., 2000. Estradiol induces a phasic Fos response in the hippocampal CA1 and CA3 regions of adult female rats. Hippocampus 10, 274–283. Rudick, C.N., Woolley, C.S., 2003. Selective estrogen receptor

66

BR A IN RE S EA RCH 1 0 67 ( 20 0 6 ) 5 8 –66

modulators regulate phasic activation of hippocampal CA1 pyramidal cells by estrogen. Endocrinology 144, 179–187. Schindler, M., Holloway, S., Hathway, G., Woolf, C.J., Humphrey, P.P., Emson, P.C., 1998. Identification of somatostatin sst2(a) receptor expressing neurones in central regions involved in nociception. Brain Res. 798, 25–35. Schneider, J.S., Stone, M.K., Wynne-Edwards, K.E., Horton, T.H., Lydon, J., O'Malley, B., Levine, J.E., 2003. Progesterone receptors mediate male aggression toward infants. Proc. Natl. Acad. Sci. U. S. A. 100, 2951–2956. Serra, M., Pisu, M.G., Littera, M., Papi, G., Sanna, E., Tuveri, F., Usala, L., Purdy, R.H., Biggio, G., 2000. Social isolation-induced decreases in both the abundance of neuroactive steroids and GABA(A) receptor function in rat brain. J. Neurochem. 75, 732–740. Simmerman, H.K., Jones, L.R., 1998. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol. Rev. 78, 921–947. Tashev, R., Belcheva, S., Milenov, K., Belcheva, I., 2001a. Antinociceptive effect of somatostatin microinjected into caudate putamen. Peptides 22, 1079–1083. Tashev, R., Belcheva, S., Milenov, K., Belcheva, I., 2001b. Behavioral effects of somatostatin microinjected into caudate putamen. Neuropeptides 35, 271–275.

Tsai, M.J., O'Malley, B.W., 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63451–63468. Wagner, C.K., Nakayama, A.Y., De Vries, G.J., 1998. Potential role of maternal progesterone in the sexual differentiation of the brain. Endocrinology 139, 3658–3661. Wang, L., Hsu, C.L., Chang, C., 2005. Androgen receptor corepressors: an overview. Prostate 63, 117–130. Warren, S.G., Juraska, J.M., 2000. Sex differences and estropausal phase effects on water maze performance in aged rats. Neurobiol. Learn Mem. 74, 229–240. Wheeler, D.G., Horsford, J., Michalak, M., White, J.H., Hendy, G.N., 1995. Calreticulin inhibits vitamin D3 signal transduction. Nucleic Acids Res. 23, 3268–3274. Wilder, E.L., Linzer, D.I., 1986. Expression of multiple proliferin genes in mouse cells. Mol. Cell, Biol. 6, 3283–3286. Witt, D.M., Young, L.J., Crews, D., 1994. Progesterone and sexual behavior in males. Psychoneuroendocrinology 19, 553–562. Witt, D.M., Young, L.J., Crews, D., 1995. Progesterone modulation of androgen-dependent sexual behavior in male rats. Physiol. Behav. 57, 307–313. Yin, Y., Edelman, G.M., Vanderklish, P.W., 2002. The brain-derived neurotrophic factor enhances synthesis of Arc in synaptoneurosomes. Proc. Natl. Acad. Sci. U. S. A. 19 (99), 2368–2373.