Abstract
Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine and metabolic disorder in women of reproductive age. Epigenetic mechanisms contribute to the development of PCOS. Nevertheless, the role of DNA methylation in the development of PCOS remains unclear. To investigate the molecular mechanisms underlying the hyperandrogenic phenotype of PCOS, dihydrotestosterone (DHT)-induced prenatally androgenized (PNA) mice were used to mimic this phenotype. Ovarian samples from PNA and control mice were subjected to methyl-CpG-binding domain (MBD)-seq and RNA-seq, and validation was conducted using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (RT-qPCR). Immunohistochemical analysis (using anti-LC3II antibody) and transmission electron microscopy were conducted using ovarian tissue sections (which included granulosa cells) from PNA and control mice. There were 857 genes with differentially methylated promoter regions and 3,317 differentially expressed genes (DEGs) in the PNA mice compared to the control mice. Downregulation of Dnmt1 (which encodes DNA methyltransferase 1), accompanied by global hypomethylation, was observed in the PNA mice compared to the control mice. The promoter regions of Map3k1 (which encodes MEKK1) and Map1lc3a (which encodes LC3II) were hypomethylated, accompanied by upregulation of Map3k1 and Map1lc3a mRNA expression. The autophagy profiling results showed that LC3II protein expression and autophagosomes were significantly increased in the granulosa cells of PNA mice. Additionally, the mRNA expression of genes related to the mitogen-activated protein kinase (MAPK)/p53 pathway (Mapk14, Mapkapk3, and Trp53) and the autophagy-related gene Becn1 were significantly increased. DHT could change the DNA methylation and transcription level of Map3k1 and lead to an activation of autophagy in granulosa cells. These observations indicated that the change in autophagy may be driven by MAPK/p53 pathway activation, which may have been caused by DHT-induced transcriptional, and the methylation level changed of the key upstream gene Map3k1. Our study provides a novel genetic basis and new insights regarding the pathogenesis of PCOS.
Highlights
MATERIALS AND METHODSPolycystic ovary syndrome (PCOS) is one of the most common endocrine diseases, with features such as chronic anovulation, hyperandrogenism, and polycystic ovaries (Franks, 1995)
The sections were incubated in goat serum for 1 h at room temperature, followed by anti-LC3-phosphatidylethanolamine conjugate (LC3II) (1:300, ab48394, Abcam, United Kingdom), and the sections were incubated overnight at 4◦C with this antibody. 3,3 -Diaminobenzidine was used as the chromogen
By combining two types of sequencing data (RNAseq and methyl-CpG-binding domain (MBD)-seq) and validation, we found that the expression levels of Map3k1 and Map1lc3a were both negatively correlated with the methylation level of their respective promoter
Summary
Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases, with features such as chronic anovulation, hyperandrogenism, and polycystic ovaries (Franks, 1995). It is associated with an increased risk of metabolic aberrations and other conditions, including hyperinsulinism, insulin resistance, dyslipidemia, type 2 diabetes mellitus, and endometrial carcinoma. The abnormal DNA methylation of estrogen and androgen synthesis-related genes (such as NCOR1, PPARG1, HDAC3, and CYP19A1) is believed to be a major factor underlying the development of hyperandrogenemia in PCOS (Hosseini et al, 2019). Androgenized (PNA) mice are commonly used in serological, pathological, and omics-related studies of the hyperandrogenic phenotype of PCOS
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