Epigenetic control of expression is maintained by steroid hormones in the adult male rat

Catherine J. Augera,b,1, Dylan Cossa, Anthony P. Augera,b, and Robin M. Forbes-Lormana

aDepartment of Psychology and bNeuroscience Training Program, University of Wisconsin, Madison, WI 53706

Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved February 2, 2011 (received for review January 6, 2011) Although some DNA patterns are altered by steroid AVP expression. Estradiol regulates AVP expression mainly by hormone exposure in the developing brain, less is known about acting upon neuronal estrogen receptors. Interestingly, castra- how changes in steroid hormone levels influence DNA methylation tion is known to increase estrogen receptor α (ERα) mRNA (14, patterns in the adult brain. Steroid hormones act in the adult brain 19). Therefore, castration decreases AVP but increases ERα to regulate expression. Specifically, the expression of the expression. As ER activation by estradiol is known to down- socially relevant peptide vasopressin (AVP) within the bed nucleus regulate ERα expression (14, 19), it is not surprising that cas- of the stria terminalis (BST) of adult brain is dependent upon tration increases ERα due to lowered ERα activation. This testosterone exposure. Castration dramatically reduces and tes- suggests that testosterone inversely regulates these two in tosterone replacement restores AVP expression within the BST. As the adult male brain, and possibly within the same . decreases in mRNA expression are associated with increases in As AVP expression is exquisitely responsive to steroid hor- DNA promoter methylation, we explored the hypothesis that AVP mones within the BST of the adult brain (14, 20), and as changes expression in the adult brain is maintained through sustained in DNA methylation patterns can influence , we epigenetic modifications of the AVP gene promoter. We find that hypothesized that the steroid dependence of AVP expression castration of adult male rats resulted in decreased AVP mRNA within the adult brain is maintained through epigenetic modifi- expression and increased methylation of specific CpG sites within cation (i.e., DNA methylation) of the AVP promoter region. In NEUROSCIENCE the AVP promoter in the BST. Similarly, castration significantly additionally, we expect that ERα promoter methylation will increased estrogen receptor α (ERα) mRNA expression and de- follow a pattern opposite to that of AVP promoter methylation. creased ERα promoter methylation within the BST. These changes Therefore, we assessed whether testosterone removal by castra- were prevented by testosterone replacement. This suggests that tion differentially altered AVP and ERα promoter methylation the DNA promoter methylation status of some steroid responsive patterns. The data here suggest that steroid hormones actively genes in the adult brain is actively maintained by the presence of maintain DNA methylation patterns in the adult brain, and that circulating steroid hormones. The maintenance of methylated or changes in steroid hormone levels result in changes in mRNA demethylated states of some genes in the adult brain by the pres- expression and DNA methylation promoter patterns of AVP and ence of steroid hormones may play a role in the homeostatic reg- ERα in the adult brain. ulation of behaviorally relevant systems. Results pigenetic modification of (e.g., changes in DNA Castration Effect on AVP mRNA Expression and Methylation. AVP Emethylation status), by either steroid hormone exposure or mRNA in BST. Our data replicate previous findings on testosterone changes in the social environment, can create changes in tran- regulation of AVP mRNA levels in adult male rats. That is, scription rates of a number of genes within the developing brain, castration (CX) significantly reduces AVP expression within the and in some cases these changes extend into adulthood (1–6). In BST of male rats compared with sham controls. We also con- adulthood, steroid hormones act to regulate the expression of firmed that testosterone replacement (CX+T) can reverse this steroid receptors as well as other factors in a fairly transient effect in adult male rats [F (2, 18) = 5.380, P = 0.015] (Fig. 1). manner (7). Recent findings in vitro have shown that DNA AVP methylation in BST. We then examined the methylation profile methylation patterns on some genes cycle back and forth from of the AVP promoter within the BST using two different methylated to unmethylated states within minutes (8, 9). This methylation sensitive restriction enzymes (MSREs) based on the supports the emerging concept that not all methylation patterns specific CpG sites bound by these enzymes on the AVP promoter are stable. Whether a change in methylation status plays a role in (Fig. 2, gray-shaded bases in each sequence). Using four differ- the regulation of gene expression in the adult brain is not well ent primer sets, we were able to target four individual CpG sites understood. We begin to elucidate this possibility by examining on the AVP promoter. We used the HpaII enzyme to bind three the hormonal maintenance of expression and DNA promoter distinct CCGG sequences on the AVP promoter. For the first methylation status of the socially relevant neuropeptide vaso- and third CCGG sequence that we targeted, located at bases pressin (AVP) in the adult brain. 1392 and 3895, using AVP promoter primer-targeted CpG site 1 The extrahypothalamic AVP system in the rat brain is highly and 3, respectively, we did not detect any change in relative sexually dimorphic and steroid responsive (10). Adult male rats have two times more AVP-expressing cells in the bed nucleus of the stria terminals (BST) compared with females (11, 12). AVP Author contributions: C.J.A. designed research; C.J.A., D.C., and R.M.F.-L. performed re- expression within this area is dependent upon gonadal hor- search; C.J.A. and A.P.A. contributed new reagents/analytic tools; C.J.A., D.C., and A.P.A. mones, as castration results in a significant decrease in AVP analyzed data; and C.J.A. wrote the paper. mRNA and expression, and testosterone replacement The authors declare no conflict of interest. restores AVP expression (13–15). The AVP cells within the BST This article is a PNAS Direct Submission. express receptors for androgens (16), estrogens (17), and pro- Freely available online through the PNAS open access option. gestins (18). Although testosterone reinstates AVP expression in 1To whom correspondence should be addressed. E-mail: [email protected]. the BST following castration (10), it is essentially estradiol, a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metabolite of testosterone, that is the major factor regulating 1073/pnas.1100314108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100314108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 the second CCGG site. Using AVP promoter primer-targeted CpG site 4, we found that relative methylation was significantly increased following castration, and importantly, this increase was prevented by replacement with testosterone 2 wk after castration [F (2, 25)=17.26, P ≤ 0.001; Figs. 2D and 3D]. Increased methylation of the AVP promoter region is associated with de- creased AVP mRNA levels. AVP mRNA in paraventricular nucleus of (PVN), and AVP methylation in PVN. As AVP in the BST is highly steroid re- sponsive, we examined AVP mRNA in another brain area that is less steroid responsive than the BST. We chose to examine AVP expression within the PVN; in this area, there was no statistically significant effect of castration or testosterone replacement on AVP mRNA (P > 0.05). Fig. 1. Castration effect on AVP mRNA in BST. Relative AVP mRNA expression As expected from the mRNA data, we did not find any sig- within the BST in each treatment group. As expected, AVP is significantly re- nificant changes in methylation profiles of the AVP gene (P > fi duced by CX and replacement with testosterone- lled capsules reinstates AVP 0.05; Fig, S1). We used the same restriction enzymes in the PVN expression in the BST (*P =0.015;n ≥ 6). Error bars represent SEM. as we did in the BST, and we also used the same primer sets corresponding to each enzyme cut site. There was no difference methylation in any of the groups [F (2, 26) = 0.0987, P = 0.906; in the methylation status of the AVP gene in the PVN using Figs. 2A and 3A; F (2, 26) = 0.251, P = 0.780; Figs. 2C and 3C]. primers for AVP promoter primer-targeted CpG site 1, 2, 3, or 4 P > With the second CCGG sequence that we targeted, located at ( 0.05 for each primer set). base 2243, using AVP promoter primer-targeted CpG site 2, we α ERα fi Castration Effect on ER mRNA Expression and Methylation. found that our treatment had a signi cant effect on methylation mRNA in BST. Previous studies have reported that castration of fi status. Methylation at this site was signi cantly increased in re- male rats, results in a significant increase in ER mRNA levels sponse to long-term castration, and returned to control levels in within the BST (19). Also, treatment with testosterone (21) or animals that were castrated and 2 wk later treated with testos- estradiol (19) reduces ER mRNA expression in the hypothala- terone [F (2, 24) = 4.681, P = 0.019; Figs. 2B and 3B]. Using the mus and BST, respectively. Our data replicate these previous BstUI enzyme, which binds to a CGCG sequence at base 4970 on findings. Castration results in significantly higher levels of ERα the AVP promoter, we found a similar methylation pattern to mRNA expression within the BST. Treatment with testosterone

Fig. 2. Primer sequences used to assess relative methylation levels. For all primers forward and reverse primers are underlined, targeted CpG sites are highlighted and bolded. Base numbering from GenBank accession no. AF112363.1. HpaII restriction enzyme was used to bind highlighted CCGG site encompassed by primers in A–C. BstUI restriction enzyme was used to bind highlighted CGCG site encompassed by primer in D.(A) AVP promoter primer- targeted CpG site 1. (B) AVP promoter primer-targeted CpG site 2. (C) AVP promoter primer-targeted CpG site 3. (D) AVP promoter primer- targeted CpG site 4. This CGCG site is near a number of transcriptional regulators. Bases in bold lower-case type signify an ERE-half site (*ggtca*), GRE/PRE (*tgtcacaactgtcct*), and CRE (*tcccgtca*). (E) Forward and reverse primers for ERα promoter region are underlined; and the CGCG site at which the BstUI restriction enzyme binds is highlighted and bold. Base numbering from GenBank accession no. NM_012689.1.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1100314108 Auger et al. Downloaded by guest on September 29, 2021 NEUROSCIENCE

Fig. 3. Effects of CX and testosterone replacement on methylation of the AVP gene promoter in BST. (A) Relative methylation a region of the AVP promoter. Real-time PCR using primer, AVP promoter target site 1, did not reveal any difference in relative methylation levels between any of the treatment groups. (*P = 0.906; n ≥ 9). (B) Primer that encompassed the second target site did reveal a difference between groups, with relative methylation at low levels in control animals (Sham), relative methylation levels increasing with testosterone removal (CX), and relative methylation levels returning back to control levels when hormones were replaced (CX+T) (*P = 0.019; n ≥ 7). (C) Much like the methylation profile using the primer to target site 1, with the AVP promoter primer that encompassed a different target site, target site 3, no differences in relative methylation levels between groups were observed (*P = 0.780; n ≥ 9). (D) With the primer, AVP promoter-target site 4, robust differences in relative methylation between the controls (Sham), castrated (CX), and CX animals replaced with testosterone (CX+T) were observed. Relative methylation levels appeared to be low in control animals, but removal of hormones caused these levels to increasel replacement with hormones caused a decrease in relative methylation levels, which could be suggestive of possible demethylase activity increasing with hormonal replacement (*P < 0.001; n ≥ 8). Error bars represent SEM.

2 wk after castration lowers ERα mRNA expression back to the same samples that castration decreased ERα promoter control levels [F (2, 25) = 4.683, P = 0.019) (Fig. 4A)]. methylation. This decrease in ERα promoter methylation was ERα methylation in BST. Methylation of the ERα promoter was prevented in animals by testosterone replacement 2 wk after examined using the MSRE BstUI. The BstUI enzyme binds to a castration [F (2, 24) = 4.14, P = 0.028; Fig. 4B forprimerse- CGCG site, located at base 2215, on the ERα promoter that is quence, and Fig. 2E]. The decrease in CGCG ERα promoter near the Stat 5 binding region. As opposed to the increase in methylation following castration was associated with increased methylation we observed for the AVP promoter, we found in ERα mRNA levels.

Fig. 4. Castration (CX) effects on ERα expression and methylation in the BST. (A) Relative expression of ERα mRNA is significantly increased by CX and reduced with testosterone (CX+T) treatment (*P =0.019;n ≥ 8). (B) Relative methylation of ERα promoter. Methylation of ERα promoter mirrors pattern observed in mRNA levels. CX reduces methylation, and testosterone replacement reinstates methylation to sham CX levels (*P =0.028;n ≥ 9). Error bars represent SEM.

Auger et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 ERα mRNA in PVN, and ERα methylation in PVN. In contrast to the regions by steroid hormones. It remains to be elucidated how this changes in ERα mRNA levels and ERα promoter methylation specificity in promoter targeting is regulated. While studies have within the BST following castration and testosterone replace- identified several methylating factors within the brain, it is less ment, we did not detect any statically significant changes in ERα clear how demethylation occurs within the brain. mRNA levels or ERα promoter methylation within the PVN Previous studies have suggested that transitions of histone (P > 0.05). acetylation and decactylation as well as DNA methylation and demethylation states are important for gene expression as well as Discussion for physiological function (28, 29). Indeed, recent studies report We report that the methylation status of some CpG sites on the that memory enhancing genes must be turned on and memory AVP gene promoter within the BST can be altered in the adult inhibiting genes must be turned off for proper memory consoli- brain in response to changes in testosterone levels. Specifically, dation to occur in adult brain (30). Our current data suggest that testosterone withdrawal following castration, which leads to a the presence of testosterone actively maintains the promoter decrease in AVP expression (14), results in an increase in meth- methylation status of AVP and ERα in the adult male brain, and ylation of CpG sites within the AVP promoter. These data are in that testosterone withdrawal leads to increased methylation of agreement with the repressive function of methylation on gene the AVP promoter and demethylation of the ERα promoter. expression (22). The increase in methylation of the AVP gene in Therefore, the maintenance of methylation patterns by the the BST can be prevented in castrated animals by testosterone presence of testosterone may be part of a potential homeostatic replacement. This suggests that the methylation status of the mechanism by which testosterone sustains gene specific expres- AVP gene is maintained by the hormonal environment, and that sion profiles and subsequent behavior. As decreases in testos- changes in hormonal status lead to changes in AVP mRNA ex- terone and AVP expression occur with normal aging in male rats pression and methylation patterns. These data also support the (31), it is possible that decreases in testosterone levels also co- emerging concept that, in some cases, DNA methylation patterns incide with altered DNA methylation profiles during aging. in the brain may not be stable, and further suggest that some Although the number of AVP cells is a small minority of all methylation patterns may need to be actively maintained. There cells within the BST, AVP expression is highly regulated by was no significant change in mRNA levels or methylation status steroid hormones (13–15) and therefore shows dramatic changes of the AVP gene within the PVN following castration. As the in mRNA levels and promoter methylation in response to AVP cells in the PVN are less steroid-responsive compared with changes in steroid hormones. In contrast, steroid hormones ap- AVP cells in the BST, and that the PVN is not rich in ERα (23), pear to have a less dramatic impact on ERα mRNA levels and the lack of hormone responsiveness in AVP mRNA or promoter promoter methylation relative to the large population of ERα methylation status is not surprising. However, this suggests that cells within the BST. As castration results in a near elimination steroid hormone maintenance of AVP promoter methylation of AVP within the BST, it is possible that most AVP cells in this patterns and expression is region specific in the adult brain. area undergo a change in promoter methylation and a near We also report that ERα is epigenetically regulated by cas- silencing of that gene. As castration results in less dramatic tration in the adult brain; however the pattern is opposite to that changes in ERα levels (19), changes in ERα promoter methyla- of AVP. Previous studies show that gonadectomy results in in- tion in response to castration may occur in a subset of ERα cells creased ER expression and hormone treatment decreases ERα or on CpG sites that only decrease ERα transcription rates. expression (19, 21, 24, 25), as activation of ER leads to a subtle These data suggest a difference in the homeostatic maintenance down-regulation of its own receptor. We extend these findings to of DNA promoter methylation of AVP versus ERα in response show that the increase in ERα mRNA expression in response to to a decline in hormone levels. adult castration corresponds to a reduction in ERα promoter It is important to note that the locations of the CpG sites being methylation, supporting previous findings indicating that ERα altered are near transcriptional response elements within the promoter methylation correlates inversely with changes in ERα AVP promoter region. For example, changes in AVP promoter mRNA levels (2, 3, 26). Interestingly, the directional change in methylation were confirmed in two of the four CpG sites that we promoter methylation of AVP and ERα are opposing, as cas- targeted. One of these sites is located near a number of binding tration increases AVP promoter methylation and decreases ERα sites for transcriptional regulators (i.e., response elements for promoter methylation, and testosterone replacement prevents CREB, glucocorticoid/progestin receptors, and an estrogen re- these effects. While ERα is coexpressed in AVP cells, and is an sponse element half site) within the AVP promoter (Fig. 2D) important regulator of AVP expression, it is unknown if ERα (32, 33). Although the consequences of methylation on gene plays a direct role in AVP promoter methylation. Nevertheless, expression are being investigated (34), it is possible that meth- this suggests that testosterone can act to either increase or de- ylation at this site would have a higher probability of suppressing crease gene expression within the same brain region and poten- AVP gene transcription, as methylation at this site could reduce tially within the same cell by altering DNA methylation patterns. the accessibility of the DNA to several transcription factors. We recently suggested a model by which the same hormone Much of the study of epigenetic phenomena in the mammalian signal can have opposing outcomes on the epigenetic regulation systems focus on methylation events that occur early on in de- of gene expression during development to program juvenile and velopment, and have lasting effects well into adulthood [i.e., adult behavior (27). We now report a bidirectionality in the early-life stress (35) and sexual differentiation (6, 27, 36)]. In- methylation status of the ERα and AVP gene promoters in re- deed, early-life stress in mouse pups results in an increased ex- sponse to changes in gonadal hormones in adulthood. When the pression of AVP in the parvocellular cells of the PVN. This up- major source of testosterone is removed, the methylation and regulation of AVP mRNA is associated with hypomethylation gene expression profile of both these genes are reversed. It is of the AVP promoter, an effect that is likely achieved through intriguing that hormonal withdrawal can elicit methylation and phosphorylation of methyl CpG-binding protein 2 (MeCP2) (5). demethylation of these steroid responsive genes. As to how this This suggests that the epigenetic control of AVP cells in the occurs is unclear; however, it may suggest that different meth- PVN is at least sensitive to stress hormones during early de- ylating or demethylating factors are being directed to ERα pro- velopment, it is unclear if stress hormones alter DNA methyla- moter regions versus those being directed to AVP promoter tion patterns in adulthood within these cells. Other examples of regions in response to changes in steroid hormone levels. Al- long term epigenetic modification of chromatin also come from ternatively, the activity of the same methylating or demethylating studies examining sexual differentiation of the brain. Indeed, factor may be increased or decreased at the different promoter sexual differentiation of the BST in mice appears to require his-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1100314108 Auger et al. Downloaded by guest on September 29, 2021 tone acetylation, as inhibiting histone acetylation blocks the annealing step for 30 s, and a 72 °C elongation step for 30 s. Relative mRNA ΔΔ masculinization of the BST, which is typically larger in males levels were calculated using the 2- CT method (39). contrasted to females (37). Furthermore, sexually dimorphic ERα promoter methylation appears to be partly responsible for the Methylation-Sensitive Restriction Enzyme Assay. Methylation of samples was sexually dimorphic expression of this receptor (2, 3). Although assessed using the methylation sensitive restriction enzyme (MSRE) process (40). This method uses a restriction enzyme that will bind a specific CpG site DNA methylation patterns may change during the perinatal pe- and cut DNA unless it has been methylated. Therefore, designing primers riod, methylation of some genes in the brain is considered a surrounding the targeted CpG site will produce a PCR product only if that somewhat stable event that may require maintenance throughout site has been methylated. This is a powerful technique as it can assess the the life span. It is assumed that the maintenance of some meth- relative methylation of all DNA in isolated from the tissue sample. To ex- ylation patterns occur through relatively passive processes; how- amine DNA methylation, 2 μg of DNA from each rat was equally divided into ever, our data suggest that in the adult brain, steroid hormones two tubes: an enzyme treated + buffer and a no-enzyme + buffer control play an active role in maintaining methylation profiles. tube. These tubes were then processed using the same primers surrounding In conclusion, the present study suggests a homeostatic nature the targeted CpG site with the no-enzyme control serving as the normalizer α control during Real-Time PCR. Of the two enzymes used in this study, HpaII of AVP and ER promoter methylation patterns that are main- cleaves unmethylated DNA at any CCGG sequence and BstUI cleaves tained by steroid hormones in the adult brain, and that reducing unmethylated DNA at any CGCG sequence. For examining methylation at this signal leads to altered DNA methylation patterns of these CCGG sites, 1 μg of DNA from each animal was diluted in 1 μl of NEB buffer 1 two steroid responsive genes in opposing directions. This indi- in separate tubes, and was incubated at 37 °C for three hours with 2 μlof cates that steroid hormones actively maintain the methylation or HpaII restriction enzyme (New England Biolabs, Ipswich, MA). For examining demethylation status of these two genes. methylation at CGCG sites, 1 μg of DNA from each animal was diluted in 1 μl of buffer R in separate tubes, and was incubated at 37 °C for one hour with 1 Materials and Methods μl of BstUI restriction enzyme (Fermentas Inc., Glen Burnie, MD). No-enzyme controls for each sample were also run, as well as no-DNA with enzyme and Animals and Treatment. Adult male Sprague–Dawley rats were obtained from no-DNA and no-enzyme controls. Omission of DNA resulted in no PCR our breeding colony and group housed (usually two animals per cage) until product. HpaII and BstUI were inactivated by incubation at 65 °C for 20 min. sacrifice. At ∼3 mo of age, all animals underwent surgery. We assessed if To assess relative levels of methylation in the promoter regions of the castration altered AVP promoter methylation in 3 groups of adult male rats. AVP and ERα genes, all enzyme-treated and no-enzyme controls were sub- One (n = 10) was castrated (CX) for 5 wk until sacrifice, another (n = 9) was jected to Real-Time PCR. The primers were designed to encompass each one sham castrated for the same amount of time, and a third (n = 11) was cas- of three CCGG sites on the AVP gene promoter (Fig. 2A–C), and to encompass trated for 2 wk and then implanted with testosterone-filled Silastic capsules the CGCG site on the AVP gene promoter that is near a CRE binding site, NEUROSCIENCE (Dow Corning; 2.5 cm long, 1.5 mm i.d., 2.4 mm o.d; CX+T) for 3 wk before a GRE and an ERE (Fig. 2D). Primers were also designed to amplify a por- animals were killed (Fig. 1A). All animals were killed at the same time fol- tion encompassing a CGCG site near the Stat 5 binding region of the ERα lowing the initial surgery. gene promoter (2) (Fig. 2E). All primers were found to have efficiencies of near 100%, both dissociation curve and amplification plot analysis was used μ Tissue Processing. were collected, snap frozen, and sectioned at 250 m to confirm purity of products. Real-Time PCR was optimized and conducted using a cryostat at around -10 °C. Micropunches were taken from the ap- using the Stratagene Mx3000P system (Cedar Creek) with primers designed propriate sections for BST and PVN, rapidly refrozen and stored at -80 °C. using the OligoPerfect tool (Invitrogen) and purchased from Invitrogen or BST punches were taken from an area of the BST covered by AVP expressing synthesized with standard purity by the Biotechnology Center at the Uni- cells (18). These areas encompass portions of the lateral and medial portions versity of Wisconsin. Real-time PCR was carried out as described previously of BST, ventral to the stria terminalis (plate 21 for the BST and plate 25 for (41). The following procedure was used for amplification of DNA samples: an the PVN in the atlas of Paxinos and Watson(38)). DNA and RNA were initial denaturing step at 95 °C for 25 min followed by 40–45 cycles of a 95 °C ’ extracted from each individual animal s micropunch using the AllPrep DNA/ denaturing step for 15 s, a 55 °C annealing step for 30 s, and a 72 °C elongation RNA Mini Kit (Qiagen). Individual samples were maintained throughout the step for 30 s. Relative DNA levels were calculated using the 2-ΔΔCT method (39). experiment. Concentrations of DNA and RNA in each sample were measured The ΔCT for each sample was determined by obtaining the difference between fi using the Qubit quanti cation platform (Invitrogen). the average CT of the non enzyme-treated gene and the average CT of Δ the enzyme-treated sample. The CT of the calibrator was subtracted from Δ ΔΔ Primers and qPCR for AVP and ERα mRNA. Extracted RNA was converted to the CT of each of the samples to determine the CT. This number was cDNA using the ImProm-II Reverse Transcription System (Promega U.S., then used to determine the amount of DNA relative to the calibrator, or the Madison, WI). The relative levels of AVP and estrogen receptor α (ERα) mRNA n-fold difference. The n-fold difference was calculated by the equation 2-ΔΔCT. in the samples was assessed using primers that were designed using the OligoPerfect tool (Invitrogen), and were purchased from Invitrogen or syn- Statistical Analysis. N-fold differences between the groups were compared thesized with standard purity by the Biotechnology center at the University by one-way ANOVA, using the statistical software package Sigma-Stat of Wisconsin. Primers are as follows: AVP, accession number NM_016992.2, (Systat). ANOVAs that produced significant results were followed by analysis forward TGCCTGCTACTTCCAGAACTGC, reverse AGGGGAGACACTGTCTCA- with the Student–Neuman–Keuls post hoc method. Significance was set at GCTC; ERα, accession number NM_012689.1, forward TCCGGCACATGAGT- P < 0.05. AACAAA, reverse TGAAGACGATGAGCATCCAG. Expression of HPRT was measured within the same samples and was used as a control. The Real-Time ACKNOWLEDGMENTS. This work was supported by National Institute of PCR protocol was as follows: an initial denaturing step at 95 °C for 25 min Mental Health Grant R01-MH072956 (to A.P.A.) and the Psychology De- followed by 40–45 cycles of a 95 °C denaturing step for 15 s, a 55 °C partment and Graduate School at the University of Wisconsin-Madison.

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