Prenatal levonorgestrel exposure induces autism-like behavior in offspring through ERβ suppression in the amygdala

Abstract

BackgroundAutism spectrum disorder (ASD) is characterized by impairments in social communication and restricted or repetitive behaviors or interests. ASD is now diagnosed in more than one out of 100 children and is biased towards males by a ratio of at least 4:1. Many possible explanations and potential causative factors have been reported, such as genetics, sex, and environmental factors, although the detailed mechanisms of ASD remain unclear.MethodsThe dams were exposed through oral contraceptives to either vehicle control (VEH) alone, levonorgestrel (LNG) alone, ethinyl estradiol (EE) alone, or a combination of LNG/EE for 21 days during their pregnancy. The subsequent 10-week-old offspring were used for autism-like behavior testing, and the limbic tissues were isolated for analysis. In another experimental group, 8-week-old male offspring were treated by infusion of ERβ overexpression/knockdown lentivirus in the amygdala, and the offspring were analyzed after 2 weeks.ResultsWe show that prenatal exposure of either LNG alone or a LNG/EE combination, but not EE alone, results in suppression of ERβ (estrogen receptor β) and its target genes in the amygdala with autism-like behavior in male offspring, while there is a much smaller effect on female offspring. However, we find that there is no effect on the hippocampus and hypothalamus. Further investigation shows that ERβ suppression is due to LNG-mediated altered methylation on the ERβ promoter and results in tissue damage with oxidative stress and the dysfunction of mitochondria and fatty acid metabolism, which subsequently triggers autism-like behavior. Overexpression of ERβ in the amygdala completely restores LNG-induced ERβ suppression and autism-like behaviors in offspring, while ERβ knockdown mimics this effect, indicating that ERβ expression in the amygdala plays an important role in autism-like behavior development.ConclusionsWe conclude that prenatal levonorgestrel exposure induces autism-like behavior in offspring through ERβ suppression in the amygdala. To our knowledge, this is the first time the potential effect of oral contraceptives on the contribution of autism-like behavior in offspring has been discovered.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-017-0159-3) contains supplementary material, which is available to authorized users.

PGC1β regulates multiple myeloma tumor growth through LDHA‐mediated glycolytic metabolism

“…In brief, the amplified transcripts were quantified by the comparative threshold cycle method using β‐actin as a normalizer. Fold changes in gene mRNA expression were calculated as 2 −ΔΔCT with CT = threshold cycle, ΔCT = CT (target gene)‐CT (β‐actin), and the ΔΔCT = ΔCT (experimental)−ΔCT (reference) (Zhang et al ., 2017; Zou et al ., 2017). …”

“…The membrane was incubated with appropriate antibodies, washed, and incubated with HRP‐labeled secondary antibodies, and then, the blots were visualized using the ECL Plus Western Blotting Detection System (Amersham). The blots were quantitated by IMAGEQUANT, and the final results were normalized by β‐actin (Zhang et al ., 2017; Zou et al ., 2017). …”

“…DNA fragments were recovered by phenol/chloroform extraction and ethanol precipitation. A ~150‐bp fragment in the range of ‐200~0 from the transcription start site on the LDHA promoter was amplified by real‐time PCR (qPCR) using the primers provided in Table S1 (Zhang et al ., 2017; Zou et al ., 2017). …”

“…The primers for ATP6 were as follows: forward 5′‐ cat tta cac caa cca ccc aac ‐3′ and reverse 5′‐ tat ggg gat aag ggg tgt agg ‐3′. The mitochondrial DNA copies were obtained from relative ATP6 copies that were normalized by β‐actin copies using the ΔΔ CT method (Yao et al ., 2005; Zou et al ., 2017). …”

“…An ATP standard curve was generated at concentrations of 10 −12 –10 −3  m . Intracellular ATP levels were calculated and expressed as nmol·mg −1 protein (Yao et al ., 2005; Zou et al ., 2017). …”