Postnatal resveratrol treatment reverses prenatal progestin exposure-induced ERβ suppression and autism-like behavior

We first investigated the potential effect of prenatal progestin exposure on ERβ expression in the amygdala and autism-like behavior in 10-week-old male offspring, and then evaluated whether postnatal resveratrol treatment could reverse this effect. In Table 1 (left panel of first row), several steroids, 17β-estradiol (E2), progesterone (P4), and eight different kinds of clinically relevant progestins, including levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nestorone (NES), norethindrone (NET), norethindrone acetate (NETA), norethynodrel (NEN), norgestimate (NGM), and hydroxyprogesterone caproate (OHPC), were used for prenatal exposure to different 3-month-old pregnant dams for 21 days (n = 8). The 5-week-old male offspring were then treated by either control (CTL) or resveratrol (RSV) for 4 weeks, and the ERβ mRNA in the amygdala was evaluated by real-time PCR and the autism-like behavior was evaluated by social interaction time. Our results showed that E2 had no effect, while P4 slightly decreased ERβ expression but had no significant effect on social interaction time, and almost all the clinically relevant progestins significantly decreased ERβ expression (except the NGM), and decreased social interaction time (except the NEN) compared to the vehicle group. Furthermore, resveratrol treatment almost completely reversed the suppression effect on ERβ expression and social interaction time (partly for the NES and NGM). Our results indicate that postnatal resveratrol treatment reverses prenatal progestin exposure-induced ERβ suppression and autism-like behavior.

Table 1 Resveratrol reverses and prevents prenatal progestin exposure-induced ERβ suppression and autism-like behavior Full size table

Prenatal resveratrol treatment prevents prenatal progestin exposure-induced ERβ suppression and autism-like behavior

We then evaluated the potential effect of prenatal resveratrol treatment on prenatal progestin exposure-induced ERβ suppression and autism-like behavior. In Table 1 (right panel of first row), prenatal exposure of P4 and all the eight different kinds of clinically relevant progestins significantly decreased ERβ expression and social interaction time (except the NEN), while prenatal resveratrol treatment (oral administration of 20 mg/kg RSV for 21 days) almost completely reversed prenatal progestin exposure-induced suppression effect on ERβ expression (partly for NEN) and social interaction time. Our results indicate that prenatal resveratrol treatment prevents prenatal progestin exposure-induced ERβ suppression and autism-like behavior.

Postnatal resveratrol treatment reverses prenatal norethindrone exposure-induced suppression of ERβ and its target genes in the amygdala

In Table 1, the prenatal exposure of norethindrone (NET) in pregnant dams showed the most dramatic suppression effect on ERβ expression and social interaction time in offspring compared to other progestins. The NET was chosen for the prenatal progestin exposure in the later experiments to investigate the detailed mechanism and effect of resveratrol on autism-like behavior. The animal was treated as shown in Fig. 1b. We first measured the gene expression of ERβ, SOD2, and ERRα in the hypothalamus and hippocampus (see Additional file 1: Figure S1). We found that prenatal NET exposure showed no effect on gene expression. Also, RSV treatment showed no effect on the expression of ERβ and SIRT1, while the expression of SOD2 and ERRα was significantly increased in both the hypothalamus and hippocampus by RSV treatment compared to the control (CTL) group. We then measured the mRNA expression of ERβ (see Fig. 2a), SOD2 (see Fig. 2b), and ERRα (see Fig. 2c) in the amygdala. We found that RSV treatment completely reversed prenatal NET exposure-induced suppression of ERβ, SOD2, and ERRα in both male and female offspring. Furthermore, in female offspring, RSV significantly increased expression of SOD2 and ERRα in both the VEH and NET groups, while in male offspring there was much less gene activation (see Fig. 2b, c), indicating that female offspring seem more responsive to RSV treatment compared to male offspring. We also measured the protein levels in the amygdala (see Fig. 2d–g). The results showed that RSV completely reversed the NET-mediated suppression effect on the expression of ERβ, SOD2, and ERRα. Furthermore, RSV increased ERRα expression in VEH treatment compared to the CTL group in male offspring (see Fig. 2d, f), and in female offspring, RSV significantly increased ERRα expression in both VEH and NET treatment compared to CTL group (see Fig. 2e, g). We also measured the SIRT1 mRNA expression (see Figure S2a) and SIRT1 activity (see Figure S2b), which showed no difference in any of the treatments, indicating that SIRT1 may not be involved in RSV-mediated effect (see detailed statistical information in Additional file 1: Data S1). Our results suggest that RSV reverses prenatal NET exposure-induced suppression of ERβ and its target genes, and female offspring seem more responsive than male offspring.

Fig. 2 Postnatal resveratrol treatment reverses prenatal norethindrone exposure-induced suppression of ERβ and its target genes. Three-month-old pregnant dams were exposed to NET (20 μg norethindrone) or VEH (vehicle, 5% ethanol in organic sesame oil) by subcutaneous daily injection of 0.1 ml for 21 days until pup delivery. Both male and female offspring were then treated with either control (CTL) or resveratrol (RSV) for 4 weeks starting from 5 weeks old. The offspring were sacrificed at 10 weeks of age to isolate the amygdala for further analysis. a–c mRNA levels in the amygdala for genes of ERβ a, SOD2 b, and ERRα c, n = 5. d Representative pictures of protein levels in male offspring by western blotting. e Representative pictures of protein levels in female offspring by western blotting. f Protein expression quantitation for d, n = 5. g Protein expression quantitation for e, n = 5. *, P < 0.05, vs VEH/CTL group; ¶, P < 0.05, vs NET/CTL group. Results are expressed as mean ± SEM Full size image

Postnatal resveratrol treatment diminishes prenatal norethindrone exposure-induced methylation of DNA and histone on the ERβ promoter

In Fig. 3a, b, the DNA methylation on the ERβ promoter was significantly increased by prenatal NET exposure, and RSV treatment completely diminished this effect compared to the CTL group. We then measured the histone methylation on the ERβ promoter by ChIP techniques. In male offspring (see Fig. 3c), prenatal NET exposure increased H3K9 di-methylation (H3K9me2) by 1.80-fold and H3K27 tri-methylation (H3K27me3) by 2.13-fold, while in female offspring (see Fig. 3d), it increased H3K9 di-methylation (H3K9me2) by 2.14-fold and H3K27 tri-methylation (H3K27me3) by 1.56-fold, but had no effect on H3K9 tri-methylation (H3K9me3). RSV treatment completely diminished the effect on H3K9me2, but partly diminished the effect on H3K27me3 in male offspring compared to the CTL group, while in female offspring, RSV partly diminished the effect on H3K9 di-methylation, but completely diminished the effect on H3K27 tri-methylation (see detailed statistical information in Additional file 1: Data S2). Our results indicate that postnatal RSV administration diminishes prenatal levonorgestrel exposure-induced methylation of DNA and histone on the ERβ promoter in the amygdala.

Fig. 3 Postnatal resveratrol treatment diminishes prenatal norethindrone exposure-induced methylation of DNA and histone on the ERβ promoter. The amygdala neurons were isolated from 10-week-old male/female offspring for in vitro cell culture analysis. a The representative bands for ERβ methylation in amygdala neurons from both male (upper panel) and female (lower panel) offspring. b DNA methylation on ERβ by real-time PCR-based methylation-specific PCR (MSP) analysis in amygdala neurons, n = 5. c ChIP analysis on the ERβ promoter in male amygdala neurons, n = 5. d ChIP analysis on the ERβ promoter in female amygdala neurons, n = 5. *, P < 0.05, vs VEH/CTL group; ¶, P < 0.05, vs NET/CTL group. Results are expressed as mean ± SEM Full size image

Postnatal resveratrol administration ameliorates prenatal norethindrone exposure-induced oxidative stress and dysfunction of mitochondria and lipid metabolism

We evaluated the effect of RSV treatment on prenatal NET exposure-induced ERβ suppression and the subsequent molecular consequences in the amygdala, including ROS generation, DNA damage, mitochondrial function and lipid metabolism. We found that prenatal NET exposure significantly increased superoxide anion (O 2 −) release by 2.27-fold (in male) and 1.88-fold (in female) respectively (see Fig. 4a); it increased 3-nitrotyrosine formation by 2.03-fold (in male) and 1.54-fold (in female) respectively (see Fig. 4b); it increased 8-OHdG formation by 2.65-fold (in male) and 2.27-fold (in female) respectively (see Fig. 4c); it increased γH2AX formation by 1.94-fold (in male) and 1.48-fold (in female) respectively (see Fig. 4d, e); it decreased mitochondrial DNA copies by 38% (in male) and 24% (in female) respectively (see Fig. 4f); it decreased intracellular ATP level by 45% (in male) and 25% (in female) respectively (see Fig. 4g). Furthermore, it decreased in vivo fatty acid oxidation by 47% (in male) and 18% (in female) respectively (see Fig. 4h), and it decreased in vitro fatty acid uptake by 42% (in male) and 26% (in female) respectively (see Fig. 4i). RSV treatment partly reversed this effect in male offspring, while in female offspring, RSV completely diminished this effect. Furthermore, in VEH group, RSV treatment significantly increased mitochondrial function (see Fig. 4f, g) and in vivo fatty acid oxidation metabolism (see Fig. 4h) in both male and female offspring. On the other hand, RSV treatment increased in vitro fatty acid uptake (see Fig. 4i) in female offspring but not in male offspring from VEH group (see detailed statistical information in Additional file 1: Data S3). Our results indicate that RSV ameliorates prenatal NET exposure-induced oxidative stress and dysfunction of mitochondria and lipid metabolism, and that female offspring were less responsive to NET exposure than male offspring.

Fig. 4 Postnatal resveratrol treatment ameliorates prenatal norethindrone exposure-induced oxidative stress, dysfunction of mitochondria, and lipid metabolism. a–h The amygdala tissues were isolated from 10-week-old male/female offspring for further analysis. a In vivo superoxide anion release, n = 5. b Quantitation of 3-nitrotyrosine (3-NT) formation, n = 5. c 8-OHdG formation, n = 5. d Representative γH2AX western blotting band for both male (upper panel) and female (down panel) offspring. e Quantitation of γH2AX formation for d, n = 5. f Mitochondrial DNA copies, n = 4. g Intracellular ATP levels, n = 5. h The in vivo palmitate oxidation rate, n = 5. i The amygdala neurons were isolated from 10-week-old male/female offspring for in vitro 14C-OA fatty acid uptake, n = 5. *, P < 0.05, vs VEH/CTL group; ¶, P < 0.05, vs NET/CTL group. Results are expressed as mean ± SEM Full size image

Postnatal resveratrol treatment ameliorates prenatal norethindrone exposure-induced autism-like behavior

We measured the effect of RSV on prenatal NET exposure-induced autism-like behavior. In male offspring, prenatal NET exposure decreased buried marbles by 52% (see Fig. 5a), while there was no difference in female offspring. We then evaluated the social interaction time. In male offspring, the NET treatment decreased by 32% in sniffing, 48% in mounting, no difference in grooming partner, and 33% in total social interaction (see Fig. 5b), while RSV treatment completely diminished this effect. In female offspring, the NET treatment decreased by 20% in sniffing, no difference in mounting or grooming partner, and 22% in total social interaction (see Fig. 5c). Furthermore, the RSV treatment completely diminished this effect in both male and female offspring (see detailed statistical information in Additional file 1: Data S4). Our results indicate that RSV completely reverses prenatal NET exposure-induced autism-like behavior and that male offspring seem more sensitive to NET-induced effect compared to female offspring.

Fig. 5 Postnatal resveratrol treatment ameliorates prenatal norethindrone exposure-induced autism-like behavior. Three-month-old pregnant dams were exposed to NET (20 μg levonorgestrel) or VEH (vehicle, 5% ethanol in organic sesame oil) by subcutaneous daily injection of 0.1 ml for 21 days until pup delivery. Both male and female offspring were then treated by either control (CTL) or resveratrol (RSV) for 4 weeks starting from 5 weeks old. Both male and female offspring were used for autism-like behavior tests at 10 weeks old. a Buried marble tests, n = 9. b, c Social interaction time in both male (b) and female (c) offspring, n = 9. *, P < 0.05, vs VEH/CTL group; ¶, P < 0.05, vs NET/CTL group. Results are expressed as mean ± SEM Full size image

Postnatal resveratrol treatment ameliorates prenatal norethindrone exposure-induced autism-like behavior through ERβ activation

Prenatal NET exposure treated male offspring received lentivirus infusion of shERβ shown in Fig. 1b. We first measured the gene expression of ERβ, SOD2, and ERRα. The results showed that prenatal NET exposure significantly decreased mRNA levels (see Fig. 6a) and protein levels (see Fig. 6b, c) of those genes. We then measured prenatal NET exposure-mediated molecular consequences in the amygdala. The results showed that postnatal resveratrol treatment ameliorates prenatal NET exposure-induced oxidative stress (see Additional file 1: Figure S3a, b), DNA damage (see Additional file 1: Figure S3c, e), dysfunction of mitochondria (see Additional file 1: Figure S3f, g), and lipid metabolism (see Additional file 1: Figure S3 h, i) through ERβ activation. RSV treatment completely reversed this suppression effect, while ERβ knockdown (shERβ) significantly diminished the effect of RSV (see Additional file 1: Figure S3). Next, we investigated the potential effect of RSV treatment and ERβ expression on prenatal NET exposure-induced autism-like behavior in male offspring. We found that prenatal NET exposure (NET/EMP/CTL) decreased buried marbles by 32% (see Fig. 6d) compared to the control group. RSV treatment completely reversed prenatal NET exposure-induced autism-like behavior, while ERβ knockdown (shERβ) completely diminished the effect of RSV. We also measured the social interaction time (see Fig. 6e). The results showed that prenatal NET exposure resulted in a 38% decrease in sniffing and a 37% decrease in total social interaction, while it showed no difference in mounting and grooming partner. RSV treatment partly diminished this effect, while ERβ knockdown (shERβ) completely diminished the effect of RSV (see detailed statistical information in Additional file 1: Data S5). Our results indicate that RSV ameliorates prenatal norethindrone exposure-induced autism-like behavior in offspring through ERβ activation.

Fig. 6 Postnatal resveratrol treatment ameliorates prenatal norethindrone exposure-induced autism-like behavior through ERβ activation. The 8-week-old male offspring from VEH or NET group received either empty (EMP) or ERβ knockdown (shERβ) lentivirus infusion and were treated by either control (CTL) or resveratrol (RSV) for 4 weeks, and the offspring were sacrificed at 13 weeks of age for further analysis. a–c The amygdala tissues were isolated from 13-week-old treated male offspring for gene expression analysis. a The mRNA levels for gene expression, n = 4. b The quantitation of protein levels, n = 5. c Representative bands for western blots. d Buried marble tests, n = 9. e Interaction time, n = 9. *, P < 0.05, vs VEH/EMP/CTL group; ¶, P < 0.05, vs NET/EMP/CTL group; #, P < 0.05, vs NET/EMP/RSV group. Results are expressed as mean ± SEM Full size image

Prenatal resveratrol treatment prevents prenatal norethindrone exposure-induced autism-like behavior

Three-month-old pregnant dams were exposed to either NET (20 μg norethindrone) or VEH (vehicle) for ~ 21 days during the whole pregnancy, and they also received either control (PreCTL) or resveratrol (PreRSV) prenatal treatment by oral administration. Both male and female offspring at 10 weeks of age were used for biomedical analysis and autism-like behavior testing. We first measured the ERβ gene expression (see Fig. 7a) and found that prenatal RSV treatment completely reversed prenatal NET exposure-induced ERβ suppression. We also measured the epigenetic changes with histone methylation on the ERβ promoter in both male (see Additional file 1: Figure S4a) and female offspring (see Additional file 1: Figure S4b). We found that prenatal RSV treatment completely normalized prenatal NET exposure-induced histone methylation, including H3K9me2 and H3K27me3. We also measured the oxidative stress, including superoxide anion (see Additional file 1: Figure S4c) and 8-OHdG formation (see Additional file 1: Figure S4d); mitochondria functions, including mitochondrial DNA copies (see Additional file 1: Figure S4e) and intracellular ATP level (see Additional file 1: Figure S4f); as well as lipid metabolism, including palmitate oxidation (see Additional file 1: Figure S4 g) and fatty acid uptake (see Additional file 1: Figure S4 h). It showed that prenatal RSV treatment completely (in female offspring) or partly (in male offspring) reversed NET-induced effect compared to control group. Finally, we measured autism-like behavior, including buried marble testing (see Fig. 7b) and social interaction time testing for both male (see Fig. 7c) and female offspring (see Fig. 7d). We found that prenatal RSV treatment completely reversed prenatal NET exposure-induced autism-like behavior in both male and female offspring (see detailed statistical information in Additional file 1: Data S6). Our results indicate that prenatal RSV treatment prevents prenatal NET exposure-induced autism-like behavior.