Animals and treatment.
All studies involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals
(Institute of Laboratory Animal Research 2011
) and approved by the National Institute of Environmental Health Sciences (NIEHS) Animal Care and Use Committee. Animals were treated humanely and with regard for alleviation of suffering. The generation of ERα- and ERβ-null mice is previously described (Krege et al. 1998
; Lubahn et al. 1993
). Mice were generated by breeding C57/BL6 mice heterozygous for disruption of the ERα gene (Esr1
) or the ERβ gene (Esr2
) to produce homozygous ERα-null (αERKO) or ERβ-null (βERKO) mice, respectively, and WT littermates.
Pregnant females were housed under controlled lighting (12-hr light/dark cycle) and temperature conditions and were provided with NIH 31 laboratory mouse chow (Zeigler Brothers Inc., Gardners, PA) and fresh water ad libitum
. On the day of parturition, considered day l of age, male offspring were pooled from multiple litters and randomly distributed among CD-1 foster mothers at eight per female. All offspring then received a subcutaneous injection of 2 μg (1–2 mg/kg/day) DES in corn oil or an equal volume of corn oil alone (vehicle) daily on PNDs 1–5. All offspring were weaned at 21 days of age and genotyped by polymerase chain reaction (PCR) on DNA extracted from tail biopsy using previously described methods (Couse et al. 2003
). Mice in group 1 were killed at 10 weeks of age. Mice in group 2 were castrated at 10 weeks of age and allowed to recover for 10 days. Mice then received a subdermal implant of a 1-cm length of sealed Silastic tubing (1-cm in length, 1.47 mm inner diameter, 1.95 mm outer diameter) packed with crystalline dihydrotestosterone (DHT) or nothing (placebo) (implants were kindly provided by D. Handelsman, Sydney, Australia), and then were killed 2 weeks later (Lim et al. 2008
). At necropsy, we recorded body weights, collected and heparinized whole blood from the inferior vena cava, and stored the plasma at –70°C until assayed. We collected the SV, trimmed off the coagulating gland, and recorded the wet weight. SVs were snap-frozen for RNA and protein analysis or fixed in paraformaldehyde solution for histological analysis. SV tissue sections (4 µm) were stained with hematoxylin and eosin.
Hormone serum assays. We evaluated serum estradiol and testosterone levels using Coat-A-Count radioimmunoassay kits (Siemens Healthcare Diagnostics, Los Angeles, CA) and were assayed using an APEX automatic gamma counter (ICN Micromedic Systems Inc., Huntsville, AL).
RNA and protein isolation. Frozen SV tissue from individual animals was pulverized, and the material was subdivided for either protein or RNA extraction. Total RNA was isolated using the RNeasy isolation kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. Cytoplasmic and nuclear protein was extracted from frozen pulverized SV tissue using the NE-PER Protein Extraction Kit (Pierce, Rockford, IL) according to the manufacturer’s protocol.
Reverse-transcriptase polymerase chain reaction (RT-PCR). SV RNA (0.5 μg) was reverse transcribed using the SuperScript First Strand Synthesis Kit (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. As a negative control, we used a sample containing RNA but no reverse transcriptase. Real-time RT-PCR was performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Green (Invitrogen). Primers were designed using Applied Biosystems Primer Express Software (version 2.0).
Real-time RT-PCR was performed using 2.5 ng cDNA. Samples were analyzed in duplicate, and a negative control sample was included on each plate. For all samples, the cyclophilin gene [peptidylprolyl isomerase A (Ppia
)] was used as an endogenous control for normalization. Expression ratios were calculated using the mathematical model described by Pfaffl (2001)
2–(Ctgene of interest – CtPpia) × 10,000,
where Ct is cycle threshold.
Western immunoblot analysis.
Cytoplasmic SV protein (1 µg) was immobilized to nitrocellulose membrane using the BioRad Dot Blot apparatus (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. We performed the protein analysis using a single blot that was stripped and reprobed. Equal loading was determined using MEMCode total protein stain (Pierce) and then destained before Western blotting. Nonspecific peroxidases were eliminated using 3% hydrogen peroxide, and nonspecific sites were blocked with 10% bovine serum albumin in Tris-buffered saline, pH 7.4, plus 0.1% Tween-20 (TBS-T). Blots were then incubated with primary antibodies for 1 hr at room temperature. Anti-mouse SV secretory protein IV (SVS IV) rabbit polyclonal antibody, a gift from T. Teng (NIEHS), and rabbit anti-mouse LF polyclonal antibody, isolated as described previously (Jefferson et al. 1996
), were used at 1:5,000 dilution. Blots were then incubated with secondary antibody, anti-rabbit horseradish peroxidase (Amersham, Piscataway, NJ), diluted 1:10,000 in TBS-T. Membranes were washed five times for 5 min each in TBS-T, and immunoreactive bands were visualized using WestDura Reagent (Pierce) following the manufacturer’s instructions. Blots were exposed to film for 1 min, and images were captured by a camera (model c8484-54-03G) using LabWorks s46 software (both from UVP BioImaging Systems, Upland, CA).
Statistical analysis. The data were analyzed using JMP software (version 7) and SAS software (version 9.1), both from SAS Institute Inc. (Cary, NC). For body weight and SV wet weight, parametric tests were used to compare values. We evaluated real-time RT-PCR data using analysis of variance (ANOVA) followed by Tukey’s test. For estrogen and testosterone levels, statistical significance was determined using t-test, and groups were compared using nonparametric Mann-Whitney tests. p-Values < 0.05 were considered statistically significant.