Supporting Information

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Supporting Information

Hu et al. 10.1073/pnas.1507514112

SI Materials and Methods

Animal Ethics.All studies were carried out with ethical permis- sions from the Animal Ethics Committee of the University of Gothenburg (Ethical no: 53-2013 and 195-2013), in accordance with legal requirements of the European Community (Decree 86/609/EEC). All efforts were made to minimize suffering.

Drugs.Testosterone propionate (T, T1875), flutamide (an AR antagonist; Flut, F9397-1G) and tamoxifen (SERM; Tam, T5648- 1G) were purchased from Sigma. In Exp. 1, all injected substances were dissolved in a 2:1 mixture of sesame oil (S3547) and benzyl benzoate (B6630). In Exp. 2, DMSO (D2650-5) was used as the vehicle and solvent for testosterone for central microinjection. All vehicles were purchased from Sigma.

Experiments 1 and 2.

PNA treatment.The testosterone dose used for PNA treatment has previously been shown to have a masculinizing effect on the offspring (39) and to mimic the human PCOS from a maternal perspective (13, 39).

Experiment 1.

Maternal and placenta characteristics.On GD21, dams were anesthetized with thiobutabarbital sodium (130 mg/kg, i.p., Inactin;

Sigma) and killed, and maternal and placental samples were collected. Blood from dams was obtained by heart puncture and centrifuged, and the serum was frozen at–80 °C for subsequent measurement of sex steroids. Placentas were dissected and cleaned to remove umbilical cord, and weighed. Placentas were washed in cold Tris-saline buffer, placed in buffer D containing 10 mM Tris-Hepes, 250 mM sucrose, 1 mM EDTA, protease, and phosphatase inhibitors (P8340, P5726, and P0044; Sigma), and homogenized on ice with a Polytron. Homogenates were snap- frozen and stored at–80 °C until Western blot analyses.

Protein preparation.Homogenates from rat placentas were centrifuged at 10,000× g for 15 min to remove debris. Supernatants were collected, and protein concentration was determined with a spec- trometer (Direct Detect).

Western blot. For Western blot analyses, we used antibodies against total STAT3 (#9139, Cell Signaling) and phosphorylated (P) fractions of STAT3 (Tyr-705; #9145, Cell Signaling). Protein signal intensity was quantified by densitometry with MultiGauge Software v3.0 or Image Lab Software (Bio-Rad).β-actin was used as a loading control and for normalization. For each protein target, all individual density values for controls and treated subjects were expressed relative to the mean density of the controls.

Experiment 2.

Phenotyping of female offspring.

Euglycemic-hyperinsulinemic clamp.Insulin sensitivity was evaluated by euglycemic-hyperinsulinemic clamp in female and male PNA offspring. Briefly, rats were anesthetized with thiobutabarbital sodium (Inactin; Sigma). Insulin (Actrapid; Novo Nordisk) diluted in 10 mL of saline plus 0.2 mL of albumin was infused at 8 mU·min·kg. Plasma glucose was analyzed every 5 min with a OneTouch Ultra 2 Meter (LifeScan) and maintained at 6.0 mM by administration of 20% (wt/vol) glucose in saline. At steady-state, 50-μL blood samples were taken to determine plasma insulin concentrations (Mercodia). The mean glucose infusion rate was normalized to body weight. An insulin sensitivity index (M/Iclamp) was calculated by the mean glucose infusion rate/plasma insulin levels at steady-state.

Ovarian morphology.Ovaries were excised, fixed in neutral buffered 4% formaldehyde for 24 h, placed in 70% ethanol, dehydrated, and

embedded in paraffin. For assessment of ovarian morphology, two 4-μm sections of each ovary were taken 40 μm apart at the largest diameter, mounted on a glass slide, stained with H&E, and analyzed by conventional light microscopy.

Behavioral testing.Anxiety-like behavior and locomotor activity testing was carried out at 53–59 d of age. Female offspring were tested in the diestrus phase of the ovarian cycle, which was confirmed by vaginal smears.

Elevated plus maze.To investigate the presence of anxiety-like behavior in male and female offspring of PCOS dams, the EPM test was performed. The EPM is a well-established rodent model used to characterize anxiety-like behavior. The maze is comprised of two open and two closed arms (Med Associates). The rats were placed in the junction area and their movements were measured for 5 min using infrared beams installed on each arm and au- tomatically registered by the MED_PC software (Med Asso- ciates) for further analysis.

Locomotor activity was tested immediately after the EPM for 30 min in photo-cell equipped activity boxes (Kungsbacka Mätoch Reglerteknik).

Tissue collection and sex-steroid measurements.In diestrus phase, the hypothalamus, hippocampus, and amygdala were quickly dissected on ice using a brain matrix, the tissue was then frozen in liquid nitrogen and stored in −80 °C for mRNA expression analyses after performing euglycemic-hyperinsulinmic clamp.

Adrenal gland was dissected and weighed. Blood was collected to assess corticosterone and a comprehensive sex-steroid profile in serum in diestrus phase after the last behavior test. Circulating corticosterone was analyzed by ELISA (Catalog #80554, Chrystal Chem). Sex-steroids: E2, estrone, testosterone, DHT, progesterone, and androstenedione were analyzed by using the highly sensitive GC-MS/MS method recently evaluated in rodents (40). The limit of quantitation defined as lowest level that can be detected with a CV < 20% for E2, estrone, testosterone, DHT, progesterone, and androstenedione were 0.5, 0.5, 8, 2.5, 74, and 12 pg/mL, respectively.

RNA isolation and mRNA expression.Individual samples were homogenized in Qiazol lysis reagent (Qiagen) using TissueLyzer (Qiagen). Total RNA was extracted with RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA quantification and quality were assessed by spectrophotometric measurements (Nanodrop 1000, NanoDrop Technologies) and labchip microfluidic technology (Experion automated electro- phoresis system; Bio-Rad). cDNA synthesis was carried out using the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR was performed using TaqMan Custom Arrays (Applied Biosystems). TaqMan probe sets for target genes and reference genes were chosen from the online catalog (Table S1). The arrays were run according to the manufacturer’s protocol with Biomek FX robot and QuantStudio 12K Flex Real-Time PCR System (Applied Biosystem).

The relative gene expression was measured using the com- parative critical threshold (Ct) method (Table S2). Gapdh and Actb were used as endogenous controls and identified by Ex- pressionSuite Software v1.0.4 (Life Technologies).

Experiment 3.

Brain cannula surgery. Adult female rats, age 7–8 wk old, were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (2.7 mg/kg) and implanted with a guide cannula targeting the amygdala (26 gauge; Plastics One). The following coordinates were chosen for the amygdala:−2.0 mm posterior to bregma, ±4.2 mm

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from the midline, and−7.2 mm from the skull surface on which it was based. Cannulae were attached to the skull with dental acrylic and jeweler’s screws and closed with an obturator. Cannula placement was verified histologically postmortem by injection of 0.5μL of India ink (volume matched drug delivery in the experiments). Rats whose dye injections were not located in the amygdala were excluded from the data analysis.

Behavioral testing.

Elevated plus maze.All behavioral tests were performed in the diestrus phase at 9–10 wk of age. One hour and 24 h after the first intra-amygdala microinjection of testosterone (10μg/0.5 μL) (n= 13) or vehicle (0.5 μL DMSO) (n = 12), animals were tested for 5 min on the EPM. After the first testing day, microinjections of testosterone (10 μg/0.5 μL) or vehicle were repeated daily until the following diestrus phase (∼4–5 d), behavioral testing was then repeated. During the second estrus cycle, the dose of daily microinjections was increased to 20μg/0.5 μL testosterone followed by behavioral testing in the following diestrus phase.

Open-field test.Immediately following the EPM the rats were tested in an open-field arena for 30 min (MED-OFA-RS, Med Associates). Animals were placed in the center of an open field and exploration was assessed for 30 min. The dimensions of the arena were 40 cm× 40 cm, of which the peripheral 10 cm were considered as the peripheral zone and the central 20 cm were considered as the central zone. The open-field test was repeated during the following diestrus phase subsequent to EPM testing.

Results for Experiment 2.

Phenotyping of female offspring.Female offspring of T-treated dams had irregular cycles (vehicle 3.90± 0.10 vs. testosterone 4.33 ± 0.14, P= 0.022) and polycystic ovary morphology (Fig. S2 A and B). Circulating E2, estrone, testosterone, DHT, progesterone, and androstenedione did not differ between the groups with one ex- ception; DHT was lower in female PNA offspring receiving AR blocker (Table S4). Female, but not male, offspring of T-treated dams were insulin-resistant, as measured by euglycemic hyperinsulinemic clamp compared with vehicle offspring (Fig. S2 C and D). Thus, the model mimics the human PCOS, and reproduces the irregular cycles and PCO morphology found in PCOS patients.

Postnatal body weight, food intake, adrenal weight, and circulating corticosterone.

Female PNA offspring had an increased body weight 4 d after birth with no difference in male offspring (Fig. S3 A and B).

However, at the time of behavioral testing, female and male offspring of T-treated dams did not differ in body weight compared

with the vehicle-treated group (Fig. S3 C and D). Both female and male offspring of T+Flut-treated dams weighed significantly more than the vehicle-treated group (Fig. S3 C and D). Male offspring of T+Tam-treated dams weighed significantly less than vehicle- treated pups (Fig. S3D). Food intake did not differ between the groups (Fig. S3 E and F).

Adrenal weight did not differ between the groups in female and male PNA offspring: female vehicle 43.4± 2.30 vs. T 46.0 ± 3.23, (P = 0.541); male vehicle 28.6 ± 1.89 vs. T 33.5 ± 2.56, (P = 0.132). Similarly, circulating corticosterone levels were not different in female and male offspring: female vehicle 129.9± 24.81 vs. T 89.68± 14.15 (P = 0.178); male vehicle 47.46 ± 14.99 vs.

T 85.26± 15.36 (P = 0.097).

Anxiety-like behavior (continued).When data from male and female offspring are analyzed together by two-way ANOVA (sex and treatment) there was a main effect of treatment on both time in the open and closed arm (F_3,71= 5.270, P = 0.002; F3,71 = 6.539, P= 0.001, respectively) but no effect for sex (F1,71= 0.001, P = 0.971; F1,71 = 1.953, P = 0.167, respectively), and no in- teraction between the two factors (F_3,71= 0.209, P = 0.890; F3,71= 0.495, P= 0.687, respectively). Post hoc Dunnett’s tests indicated the offspring spend less time in the open arm (P= 0.006) and more time in the closed arm (P= 0.003). This effect was reversed by the AR blockade (time in open arm and closed arm P= 0.883 and P= 0.838, respectively) and the SERM (time in open arm and closed arm P= 0.547 and P = 0.992, respectively). Thus, the PNA effect on anxiety may not be restricted to female offspring. The total number of open-arm entries were not different (female: vehicle 15.33± 2.87 vs. T 13.89 ± 2.47; male: vehicle 11.00 ± 1.27 vs.

T 14.17± 2.45).

Gene-expression analysis.To address whether prenatal testosterone masculinizes the female brain, vehicle-treated male offspring was compared with vehicle-treated females. The expression of Htr1a in hippocampus and amygdala was higher in vehicle-treated females compared with vehicle-treated male offspring, whereas the expression in T-treated female offspring did not differ from vehicle-treated male offspring (Fig. S4 D and E). These data indicate that the maternal testosterone dose used masculinizes the brain in female offspring. The Ar and Gper1 mRNA expression in the hypothalamus, hippocampus and amygdala in vehicle- treated females differed from vehicle-treated male offspring and T-treated female offspring followed the same pattern as control females (Fig. 4 A–C).

Fig. S1. Phosphorylation and total protein expression of placental signal transducer and activator of transcription 3 (STAT3) (Tyr-705). Values represent mean±

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Fig. S2. Ovarian morphology and insulin sensitivity of offspring. (A) Two representative pictures of ovaries from vehicle-treated and (B) two from T-treated offspring. The 4-μm sections of each ovary were taken 40 μm apart at the largest diameter, mounted on a glass slide, stained with H&E, and analyzed by conventional light microscopy (5× magnification). The insulin sensitivity index (M/I clamp) in (C) female and (D) male offspring of T-treated dams obtained during the euglycemic-hyperinsulinemic clamp. Values represent means± SEM, *P < 0.05 vs. vehicle analyzed by Student t test. (Graphpad prism 6). Females:

vehicle, n = 6; T, n = 9; T+Flut, n = 8; T+Tam, n = 11, and males vehicle, n = 10; T, n = 9; T+Flut, n = 9; T+Tam, n = 9.

Fig. S3. Body weight and food intake of female and male offspring of T-treated dams. Body weight on day 4 in (A) female and (B) male offspring; body weight on day 51 before behavioral tests in (C) female and (D) male offspring. (E) Female and (F) male offspring food intake measured weekly per rat from weaning until the behavioral test. Values are means± SEM; *P < 0.05, **P < 0.01, vs. vehicle, analyzed by one-way ANOVA followed by Dunnett’s post hoc test.

Females: vehicle, n = 9; T, n = 9; T+Flut, n = 8; T+Tam, n = 9; and males: vehicle, n = 12; T, n = 12; T+Flut, n = 10; T+Tam, n = 10.

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Fig. S4. Gene expression of androgen and estrogen receptors in the (A) hypothalamus, (B) hippocampus, and (C) amygdala in vehicle-treated male and female offspring and T-treated female offspring. Expression of serotonergic and GABAergic genes in the amygdala (D) and hippocampus (E) and T-treated female offspring. The gene expression was quantified by quantitative RT-PCR relative to the housekeeping genesβ-actin and Gapdh. Results represent relative gene expression in vehicle-treated male offspring compared with T-treated female rats. Values are means± SEM; *P < 0.05; **P < 0.01 vs. the vehicle-treated male group analyzed using one-way ANOVA followed by Dunnett’s post hoc test.

Fig. S5. Locomotor activity after microinjection of testosterone into the amygdala in adult females. (A) Locomotor activity count 1 h, (B) 24 h, (C) during the first and (D) during second diestrus phase. Values are means ± SEM, T-injected vs. vehicle-treated group analyzed by Student t test.

Fig. S6. Anxiety-like behavior during subcutaneous testosterone (0.5 mg·kg·d) treatment. Time (%) spent in open arms and closed arms in the EPM at first diestrus phase (A). Time spent in central and peripheral area (B) and locomotor activity (C) in the open-field test at second diestrus phase, and (D) body weight change. Values are means± SEM; * P < 0.05; **P < 0.01, *** P < 0.001, vs. vehicle-treated group analyzed by Student t-test and repeated measure ANOVA.

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Table S1. Genes evaluated in this study

Gene symbol Assay ID Gene name Reference sequence Alias

Ar Rn00560747_m1 Androgen receptor NM_012502.1 Andr; Tfm

Esr1 Rn01640372_m1 Estrogen receptor-α NM_012689.1 ERα; Esr; RNESTROR

Esr2 Rn00562610_m1 Estrogen receptor-β NM_012754.1 ERβ; Erb2

Gper1 Rn01643280_s1 G protein-coupled estrogen receptor 1 NM_133573.1 GPR41; Gpr30

Htr1a Rn00561409_s1 5-hydroxytryptamine (serotonin) receptor 1A NM_012585.1 5HT1A; RAT5HT1A Htr2c Rn00562748_m1 5-hydroxytryptamine (serotonin) receptor 2C NM_012765.3 5-HT2C;5-HTR2C; 5HT-1C

Gad1 Rn00690300_m1 Glutamate decarboxylase 1 NM_017007.1 GAD67

Gad2 Rn00561244_m1 Glutamate decarboxylase 2 NM_012563.1 GAD65

Gabbr1 Rn00578911_m1 γ-Aminobutyric acid (GABA) B receptor 1 NM_031028.3

Actb Rn00667869_m1 Actin-β NM_031144.2 Actx

Gapdh Rn01775763_g1 Glyceraldehyde-3-phosphate dehydrogenase NM_017008.3 Gapd

Table S2. The mean of target gene CTvalue in the hypothalamus, hippocampus and amygdala

Gene

Female Male

Hypothalamus Hippocampus Amygdala Hypothalamus Hippocampus Amygdala

Ar 28.65 29.71 28.31 28.83 29.58 30.16

Esr1 29.29 33.93 31.63 30.79 34.43 32.38

Esr2 31.54 34.52 32.71 32.44 35.63 33.23

Gper1 30.89 32.17 30.57 30.65 31.66 31.53

Htr1a NA 27.24 28.73 NA 27.97 29.95

Htr2c NA 30.81 28.64 NA 32.42 28.30

Gad1 NA 28.52 27.93 NA 28.67 25.72

Gad2 NA 26.22 25.19 NA 26.57 27.48

Gabbr1 NA 24.94 25.53 NA 25.23 25.63

Actb 20.65 20.71 20.12 22.10 21.79 22.82

Gapdh 19.93 20.30 19.87 21.36 21.35 21.89

NA, not applicable.

Table S3. Maternal circulating sex steroids and placental characteristics

Sex steroids and

plcacental characteristics Vehicle (n = 13) T (n = 13) T+Flut (n = 10) T+Tam (n = 9)

P value

T vs. vehicle

T+Flut vs. vehicle

T+Tam vs. vehicle Sex steroids

Estrone (pg/mL) 43.06± 11.19 34.14± 5.50 44.34± 4.80 45.44± 5.12 0.607 0.315 0.258 Estradiol (pg/mL) 26.12± 5.19 24.24± 3.32 28.51± 2.28 30.40± 2.64 0.689 0.211 0.161 Testosterone (pg/mL) 268.68± 76.50 727.24± 64.55* 734.61 ± 73.24* 927.70 ± 132.15* 0.005 0.001 <0.001 Androstenedione (pg/mL) 495.31± 81.24 370.40± 52.66 437.14± 63.06 442.88± 51.79 0.456 0.842 0.796

DHT (ng/mL) 10.63± 1.83 18.40± 1.65* 25.94± 4.61* 23.23± 5.04* 0.018 0.001 0.040

Placental characteristics

Placental weight (g) 0.60± 0.02 0.53± 0.02* 0.56± 0.01 0.65± 0.03 0.029 0.115 0.324 Fetal/placental weight ratio 7.37± 0.35 7.83± 0.30 7.19± 0.25 7.07± 0.42 0.125 1.000 0.601 Values are mean± SEM; *significant differences (P < 0.05). P values were determined with the nonparametric Kruskal–Wallis test followed by Mann–

Whitney U test. Bold entries indicate significant values.

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Table S4. Circulating sex steroid in female PNA offspring collected in diestrus phase

Steroid Vehicle (n = 5) T (n = 7) T+ Flut (n = 6) T + Tam (n = 6)

P value

T vs. vehicle T+Flut vs. vehicle T+Tam vs. vehicle

Estrone (pg/mL) 2.86± 0.79 2.30± 0.23 2.07± 0.30 3.55± 1.13 0.884 0.761 0.824

Estradiol (pg/mL) 5.62± 1.99 3.53± 0.38 3.40± 0.44 5.92± 2.33 0.617 0.599 0.998

Progesterone (ng/mL) 18.54± 1.57 19.14± 0.78 24.93± 1.70 17.36± 4.23 0.996 0.202 0.974 Testosterone (pg/mL) 51.64± 8.72 33.34± 2.76 41.30± 7.38 44.55± 13.99 0.347 0.759 0.900 Androstenedione (pg/mL) 197.60± 30.78 108.43 ± 11.11 144.67 ± 18.51 161.17 ± 40.86 0.072 0.401 0.668

DHT (pg/mL) 13.46± 1.02 10.90± 0.83 7.68± 0.88* 11.08± 1.89 0.343 0.013 0.424

Values are mean± SEM; *significant differences (P < 0.05). P values were determined with one-way ANOVA followed by Dunnett’s post hoc test. Bold entries indicate significant values.