Sex Differences in Epigenetic Regulation of the Estrogen Receptor-α Promoter within the Developing Preoptic Area

Animal breeding and tissue collection

Adult female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were mated in our animal facility and allowed to deliver normally. Cages were checked regularly to determine the day of birth. All animals were killed by rapid decapitation, alternating among male, female, and treated female pups. Brains were extracted, and the POA was immediately dissected and snap frozen in isopentane on dry ice. These samples were stored frozen at −70 C until homogenization for nucleic acid isolation or Western immunoblotting. This research was approved by the University of Wisconsin-Madison Animal Care and Use Committee.

Simulated maternal grooming (SMG)

Maternal grooming was simulated by stroking the anogenital region with a small nylon-bristled paintbrush. Each animal received three stints of 10 strokes with about 1 min between stints. SMG was performed three times per day, once during the light cycle and twice during the dark cycle. Control pups were handled similarly but did not receive simulated grooming. Pups were kept with the dam until just before treatment, kept under a warming lamp while away from the dam, and then returned to the dam immediately after treatment. All pups were treated and returned to the dam within 10–15 min. SMG performed for CpG methylation analysis was performed on postnatal d (PN) 5–7 and included five animals from each group (control male, control female, and SMG female). We chose PN5-7 because this time course appears to be when sex differences in maternal grooming starts occurring (21). SMG performed for ERα mRNA measurements occurred on PN0-10 and included 10 animals in each group (control female and SMG female). We chose a longer period of SMG stimulation because we did not know whether a change in DNA methylation would result immediately in a significant change in mRNA levels. It is likely that it may take several hours to days to register a difference in mRNA levels. Because DNA methylation during development appears to be somewhat stable, we chose to extend the duration of SMG stimulation; therefore, we examined ERα mRNA expression levels around the end of the neonatal sensitive period for sexual differentiation of the brain, which is typically considered to occur around PN10. Animals were pooled across multiple litters, assigned to treatment groups, and placed back with dams such that each litter contained no more than 10 animals with mixed treatment groups.

Hormone administration

Female neonatal rats received sc injections of 100 μg estradiol benzoate (EB) (n = 7) or sesame oil (n = 7) on the day of birth (PN0) and PN1. Administration of this amount of EB is capable of mimicking typical estradiol levels in the developing male brain (31). Animals were then left undisturbed until PN8, when they were killed for DNA or RNA extraction from the POA.

Quantitative PCR (qPCR)

Total RNA was isolated from snap-frozen POA sections using the GenElute mammalian total RNA Miniprep kit (catalog item RTN-70; Sigma-Aldrich, St. Louis, MO). RNA concentrations were determined using a BioMate 3 spectrophotometer (Thermo Spectronic, Lanham, MD), and cDNA was generated with the StrataScript first-strand synthesis system kit (catalog item 200420; Stratagene, Cedar Creek, TX) according to the manufacturer. Samples were stored at −70 C until used in qPCR.

qPCR was done with a Stratagene Mx3000P real-time PCR system. All reactions were multiplexed with ERα and the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (hprt1) amplified together (32). Amplifications were done with Platinum qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and ROX as a reference dye. Uracil-N-glycosylase (UDG) removes uracil residues from single- or double-stranded DNA, preventing deoxyuridine-containing DNA from serving as template in future PCR. Hprt1 primers were JOE (6-carboxy-40,50-dichloro-20,70-dimethyoxyfluorescein) labeled (Invitrogen; catalog item 105M-02), and ERα primers were FAM (6-carboxyfluorescein) labeled (Invitrogen; catalog item RLUX3601174). JOE, FAM, and ROX fluorescence was simultaneously measured in each well with optimal absorbance of 535–555, 492–516, and 584 nm, respectively. Primer and sample concentrations were optimized to ensure linearity of amplification near 25 and 30 cycles for hprt1 and ERα, respectively. The amplification protocol was as follows: an initial UDG carryover prevention step at 50 C for 2 min and an initial UDG inactivation/denaturing step at 95 C for 2 min followed by 50 cycles of a 95 C melting step for 15 sec, 55 C annealing step for 30 sec, and a 72 C elongation step for 30 sec. After amplification, a dissociation melt curve analysis was performed to ensure the purity of PCR products.

Data were analyzed with the following program term settings based on Invitrogen recommendations: 1) amplification-based threshold, 2) adaptive baseline set version 1.00 to version 3.00 algorithm, and 3) smoothing moving average with amplification averaging three points. Relative cDNA levels were calculated using the ΔΔCT method (33). Group averages were compared using t tests. All statistics were computed using Sigmastat version 3.1.

Western immunoblotting

Snap-frozen POA samples were homogenized after adding 300 μl ice-cold lysis buffer consisting of 50 mm Tris-HCl, 1% Na-deoxycholate, 0.25% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, protease inhibitor cocktail (Sigma-Aldrich; catalog item P-8340), and phosphatase inhibitor cocktail (Sigma-Aldrich; catalog item P-2850). Homogenized samples were centrifuged at 14,000 rpm for 10 min at 4 C to remove cellular debris and nuclei. The supernatant was collected, and protein concentration was determined by a Lowry assay. Total protein (15 μg) from each animal was gel electrophoresed using a precast SDS-PAGE 4–20% Tris-glycine gel (Cambrex Bio Science Rockland, Inc., Rockland, ME; catalog item 58645) and transferred to a polyvinyl difluoride membrane (Immobilon-P, catalog item IPVH20200; Millipore, Bedford, MA). Membranes were blocked for 1 h in 0.1 m Tris-buffered saline (TBS) containing 5% nonfat dry milk with constant agitation at room temperature. Membranes were then incubated overnight at 4 C with agitation in TBS containing 0.05% Tween 20 (TBS-T), 2% nonfat dry milk, and 1:2000 diluted rabbit polyclonal anti-ERα antibody (Millipore; catalog item C1355). Membranes were then washed quickly three times, followed by four 5-min washes in TBS-T. After washes, membranes were incubated in a goat antirabbit horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Inc., Beverly, MA; catalog item 7074) at a 1:3000 dilution and horseradish peroxidase-conjugated antibiotin antibody (Cell Signaling; catalog item 7075) at a 1:5000 dilution for 30 min at room temperature. After this incubation, the membranes were washed three times for 5 min each with TBS-T and twice for 5 min each with TBS. Immunoreactive bands were detected using a chemiluminescence kit (Cell Signaling; catalog item 7003) and exposed to x-ray film (Eastman Kodak Co., Rochester, NY; catalog item 1788207). Membranes were then incubated with agitation in Ponceau S solution (Sigma-Aldrich; catalog item P-7170) for 5 min at room temperature and then washed twice for 2 min each in distilled water. Ponceau S stains a protein band at approximately 45 kDa and allows for accurate quantification of the amount of total protein bound to a membrane (34,35). The analysis of multiple proteins reduces the possibility of confounds due to sex differences that are known to occur in the expression of a single housekeeping gene [e.g. GAPDH (36), actin (37), and tubulin (36)] and hormone-sensitive housekeeping genes (i.e. GAPDH (38), β-actin (39), and β-tubulin (39)]. Ponceau S solution-stained membranes and the x-ray films were digitized using a scanner and analyzed using the Western blot macro gel plot 2 within the Scion Image program. Ratios of ERα immunoreactive protein to the nonspecific protein band intensities were obtained for each animal. Group averages were compared with t tests.

Sodium bisulfite sequencing of the ERα exon 1b

Genomic DNA was isolated from snap-frozen POA sections using the GenElute mammalian genomic DNA Miniprep kit (Sigma-Aldrich; catalog item G1N-70). Sodium bisulfite treatment of DNA was done with the methyl detector kit (Active Motif, Carlsbad, CA; catalog item 55001) according to manufacturer instructions. Exon 1b in the promoter of the ERα gene (accession X98236) was amplified with a protocol modified from a previous report (17). The amplification cycles included an initial denaturation cycle of 5 min at 94 C and then 40 cycles of 1 min at 94 C (denaturing), 3 min at 56 C (annealing), and a 1-min extension at 72 C. A final extension of 7 min at 72 C followed cycling. The initial PCR was done with the following primers: forward 5′-TAGTATATTTTGATTGTTATTTTAT-3′ and reverse 5′-TCTCAAACCACTCAAACTAC-3′. This generated a 504-bp product, which was used as template in a subsequent nested PCR using the same cycling program, the two 5′-phosphorylated primers, forward 5′-pAGCTTTTTATTTGTGGTTTATAGATATTT-3′ and reverse 5′-pGATCTACAAAAAAAAAAAAATCAAAACAC-3′, and the Director Universal PCR system (Sigma-Aldrich; catalog item RDC1). Short sequences compatible with HindIII and BglII were added to the 5′ ends of previously published nested forward and reverse primers, respectively. These sequences were added to enable directional cloning into a double-digested pT7-MAT-2 vector, which enabled greater efficiency for cloning and sequencing. After exonuclease digestion according to manufacturer directions, the nested PCR product (489 bp) was gel purified and ligated into the above vector following manufacturer recommendations for the Director-Ready pT7-MAT-2 set (Sigma-Aldrich; catalog item T-6825). Ligated vectors were transfected into GC10 competent cells (Sigma-Aldrich; catalog item G2794) by heat shock at 42 C for 45 sec followed directly by incubation for 5 min on ice. Cells were then grown in superoptimal catabolite medium for 90 min while vigorously shaking (225 rpm) at 37 C. Cells were then spread on agar plates containing 100 μg/ml ampicillin and allowed to grow overnight at 37 C. The next day, individual well-isolated colonies were picked from the plate and allowed to grow further by shaking in 3 ml Luria-Bertani broth containing 100 μg/ml ampicillin at 37 C for 8 h. Cells were harvested by centrifugation, and cloned vectors were isolated using the Purelink quick plasmid Miniprep kit (Invitrogen; catalog item K2100-11). Vector inserts were sequenced with the supplied Director-Ready pT7-MAT-2 set reverse sequencing primer (verification primer T7R). Reactions included 2 μl BigDye Terminator version 3.1 mix (Applied Biosystems, Carlsbad, CA), 3 μl dilution buffer (Applied Biosystems), 5 pmol primer, and 0.2 μg vector DNA in a final reaction volume of 20 μl. Cycling conditions included an initial denaturation at 94 C for 3 min followed by 50 cycles of 94 C for 10 sec, 50 C for 15 sec, and 60 C for 3 min and then a final 7-min extension at 72 C. Reactions were cleaned and sequenced at the University of Wisconsin Biotechnology Center Sequencing facility. Reactions were cleaned there using the CleanSeq magnetic bead sequencing reaction clean-up kit (Agencourt Biosciences, Beverly, MA). Reactions were then sequenced with an Applied Biosystems 3730xl automated DNA sequencing instrument and analyzed using PE Biosystems (Foster City, CA) Sequencing Analysis version 3.7. This protocol was repeated until 10 clones containing the expected region of exon 1b of the ERα gene were sequenced for each animal. Percent CpG methylation for each clone was calculated, and an average percent CpG methylation across all clones was determined for each animal. Group differences in CpG methylation at each CpG site was analyzed by two-way repeated-measures ANOVA. Group differences in average percent CpG methylation across the entire region were compared by ANOVA with Holm-Sidak pairwise post hoc tests for the sex difference and SMG comparisons. A t test was used to compare average percent CpG methylation for the EB treatment experiment.

Sex difference in ERα expression

Previous reports show that ERα gene expression is sexually dimorphic in the POA during preweaning development (13,15,27) and in adulthood (28,29,30). As expected, we confirmed that ERα gene expression is sexually dimorphic in the POA of Sprague Dawley rats with males expressing less ERα mRNA and protein than females on PN10 (Fig. 1, A and B).

Early social experience alters a sex difference in ERα promoter methylation and expression

Methylation of exon 1b of the ERα promoter site was recently associated with maternal licking and grooming of female rat pups (17). Interestingly, male pups experience more anogenital region stimulation than females during the first week of postnatal life (21,22). This suggested that the dam’s behavior might contribute to sexual differentiation of ERα expression through methylation of the genome. To test this, we investigated whether increased SMG of females could be associated with masculinization of the ERα promoter. We found that SMG given to females on PN5-7 was associated with increased methylation compared with control females on PN8; remarkably, this methylation was similar to methylation of the male promoter in the POA (Fig. 2, A and B). Average methylation across the 17 CpG sites analyzed (Fig. 2B, inset) was lower in females compared with males (P < 0.05) and SMG females (P < 0.05), whereas methylation across this region was similar between males and SMG females. Two-way repeated-measures ANOVA indicated an effect of CpG site (F = 62.5; P < 0.001) and a group by site interaction (F = 1.5; P = 0.05). Specifically, females had less methylation than males (P < 0.001) and SMG females (P < 0.001) at CpG site 1. Males had greater methylation at CpG site 2 compared with SMG (P < 0.05) and control females (P < 0.05). Interestingly, although statistical significance was not reached, methylation between sites 4 and 9 occurred only in males and SMG-treated females, whereas methylation was similar among all groups across the remainder of the sites analyzed.

In a separate experiment, we found that SMG decreased ERα gene expression in the female POA, again to levels comparable to those found in males (P < 0.05) (Fig. 2C). Because peripheral estradiol exposure may be related to increased methylation during this period of development, we also measured plasma estradiol or testosterone levels after anogenital stimulation. As expected, SMG did not change circulating levels of these hormones (data not shown). These data suggest that a component of maternal care may partly influence sexual differentiation of the brain through DNA methylation.

ERα promoter methylation and expression are altered by perinatal estradiol exposure

Because many of the sex differences in rodent brain are primarily associated with estradiol exposure and because estradiol exposure can lead to down-regulation of ERα expression, we treated female pups with EB on the first 2 d of postnatal life to investigate whether the estradiol-induced down-regulation of ERα expression correlated with changes in DNA methylation patterns. On PN8, percent methylation across the 17 CpG sites analyzed (Fig. 3A, inset) was greater in EB-treated females than control (oil-treated) females (P < 0.05). Specifically, methylation was higher in EB-treated compared with oil-treated females at CpG sites 1 (P < 0.001) and 6 (P < 0.05) (Fig. 3A). In a separate experiment, EB-treated females were also found to express less ERα mRNA (P < 0.05) (Fig. 3B).