Alternative splicing of the androgen receptor in polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ∼62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS.

Keywords: AR; PCOS; folliculogenesis; hyperandrogenism; splicing.

Fig. 1.

Identification and genomic origin of ins and del AR ASVs in the GCs of women with PCOS. (A and B) Representative PCR products (A) and corresponding sequencing results (B) of AR ASVs in patient GCs. Exon boundaries are indicated with vertical double arrows and the sequence of the in-frame insertion from ivs2 of ins AR is shown in detail. (C) Percent distribution of AR splicing patterns in control (n = 120) and PCOS (n = 68) women. (D–G) Relative mRNA levels of total, WT, ins, and del AR in GCs of PCOS women compared with controls (*P < 0.05). (H) Optimal predicted 3D model of the protein structures of AR variants. α-Helices are purple or red (ivs2), and loops are light blue. Data are presented as mean ± SEM.

Fig. 2.

Altered DNA methylation in PCOS women with ins AR. (A) Bisulfite genomic sequencing PCR shows the methylation status of individual DNA strands of exon 3 and its flanking region of AR including 11 CpG sites for individuals with different AR splicing patterns, with ten clones (shown as lines) per sample; CpG sites are shown as blank (unmethylated) or filled (methylated) circles. (B) The above results are summarized as the average methylation ratio at each CpG site (n = 3, *P < 0.05) with a schematic representation of exon 3 and its flanking region of the AR gene.

Fig. 3.

Expression of AR ASVs in primary GCs alters genome-wide AR recruitment patterns. (A) Heat map of the ChIP sequencing HA binding signal from −1 kb to +1 kb surrounding the center of WT AR-HA binding sites in GCs transduced with AR variants. Each line represents a single AR-HA binding site and the color scale denotes the AR-HA signal in reads encompassing each locus per million total reads. (B) Top-scoring motif in GCs expressing WT AR (Upper) and its enrichment in GCs expressing AR ASVs (Lower). (C) ChIP sequencing heat maps of ins (Left) and del (Right) AR-HA binding sites. (D) Top-ranking binding motifs enriched in GCs expressing ins (Left) or del (Right) AR. (E) Combined RNA and ChIP sequencing shows reduced AR-HA signal in regions flanking (−2 kb to +2 kb) gene transcription start sites (TSSs) in GCs expressing AR variants. The different color lines represent the genes, the expression levels of which are high (red), moderate (green), low (blue) and silent (purple). (F) Primary GCs transduced with the control vector (Vec) or AR-HA variants were treated with DHT and fluorescently stained with anti-HA (green) or DAPI (blue, nuclei). (Scale bars, 50 μm.) (G) DHT-induced transcriptional activation of (left, luciferase assay), and recruitment of AR-HA variants to (right, ChIP assay), a classical ARE-luciferase reporter in HEK293FT cells (n = 4, *P < 0.05). White columns: DHT (-); colored columns: DHT (+). Data are presented as mean ± SEM.

Fig. 4.

Roles of AR ASVs in GCs in ovarian hyperandrogenism. (A–D) In vitro effects of overexpressed AR variants in primary GC as indicated by CYP19A1 (aromatase) mRNA levels (A), estrogen to androgen ratios (B), CYP17A1 (17α-hydroxylase) mRNA levels (C), and A production (D) (n = 6, *P < 0.05). (E–H) In vivo effects of AR variants on androgen metabolism in control and PCOS women as indicated by GC CYP19A1 expression (E), follicular fluid estrogen to androgen ratios (F), CYP17A1 expression (G), and follicular fluid A levels (H) (n = 28, 12, 17 and 5 in the control WT, PCOS WT, ins, and del groups, respectively, *P < 0.05). (I) ChIP assay measuring the recruitment of HA-tagged AR to the U1 ARE site in the CYP19A1 promoter of primary GCs transduced with AR variants (n = 9, *P < 0.05). (J) Schematic diagram of the roles of AR ASVs in GCs in the context of hyperandrogenism and abnormal follicular development. Green (upward) and red (downward) triangles denote increases and decreases, respectively. Data are presented as mean ± SEM.