Resveratrol ameliorates polycystic ovary syndrome via transzonal projections within oocyte-granulosa cell communication

Abstract

Rationale: Polycystic ovary syndrome (PCOS) is closely linked to follicular dysplasia and impaired bidirectional oocyte-granulosa cell (GC) communication. Given that PCOS is a heterogeneous, multifactorial endocrine disorder, it is important to clarify the pathophysiology of this ovarian disease and identify a specific treatment. Methods: We generated PCOS rat models based on neonatal tributyltin (TBT) exposure and studied the therapeutic effect and mechanism of resveratrol (RSV), a natural plant polyphenol. Transcriptome analysis was conducted to screen the significantly changed pathways, and a series of experiments, such as quantitative real-time polymerase chain reaction (PCR), Western blot and phalloidin staining, were performed in rat ovaries. We also observed similar changes in human PCOS samples using Gene Expression Omnibus (GEO) database analysis and quantitative real-time PCR. Results: We first found that injury to transzonal projections (TZPs), which are specialized filopodia that mediate oocyte-GC communication in follicles, may play an important role in the etiology of PCOS. We successfully established PCOS rat models using TBT and found that overexpressed calcium-/calmodulin-dependent protein kinase II beta (CaMKIIβ) inhibited TZP assembly. In addition, TZP disruption and CAMK2B upregulation were also observed in samples from PCOS patients. Moreover, we demonstrated that RSV potently ameliorated ovarian failure and estrus cycle disorder through TZP recovery via increased cytoplasmic calcium levels and excessive phosphorylation of CaMKIIβ. Conclusions: Our data indicated that upregulation of CaMKIIβ may play a critical role in regulating TZP assembly and may be involved in the pathogenesis of PCOS associated with ovarian dysfunction. Investigation of TZPs and RSV as potent CaMKIIβ activators provides new insight and a therapeutic target for PCOS, which is helpful for improving female reproduction.

Keywords: CaMKIIβ; PCOS; TZP assembly; oocyte-GC communication; resveratrol.

Figure 1

Protective effects of RSV on impaired estrus cycles and sexual hormone disturbance induced by TBT. A, Schematic illustration of the experimental design. s.c.: subcutaneous injection, i.g.: intragastric administration, PND: postnatal day. B, Representative estrus cyclicity of adult (2-3 months) rats over 16 consecutive days. M/D: metestrus/diestrus phase, P: proestrus, E: estrus. C, The proportions of regular, irregular and rescued estrus cycles; rescued cycles were defined as regular estrus cycles observed at the later part of the study in rats in the TBT + RSV group. D, Quantitative analysis of estrus cyclicity in adult rats (Control, n = 12; TBT, n = 18; TBT + RSV, n = 14; RSV, n = 11). E, The hormone levels of adult rats (3 months) in estrus (control, n = 8; TBT, n = 9; TBT + RSV, n = 9; RSV, n = 9). Data are presented as the mean ± SEM. Comparisons between two groups were analyzed using unpaired Student's t-test and IBM SPSS Statistics 19 (IBM, USA). Statistical analyses were performed using the GraphPad Prism 7 program, and p ≤ 0.05 was considered statistically significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2

RSV ameliorated TBT-induced ovarian failure. A, Representative H&E staining images of rat ovaries. CL: corpus luteum, AF: antral follicles, Atr F: atretic follicles. B, Follicle counting results for ovaries from all four groups (Control, n = 5; TBT, n = 6; TBT + RSV, n = 4; RSV, n = 3). C, Plasma AMH levels in adult (3 months old) diestrus females in estrus (Control, n = 8; TBT, n = 9; TBT + RSV, n = 9; RSV, n = 9). D, Expression of PI3K, Akt, p-Akt, and mTOR protein in ovaries. E, The results are expressed as the fold change in the optical density of a target protein, and β-actin expression served as the control. The mean protein expression of the control is designated as 1 in the graph. Data are presented as the mean ± SEM (n = 3-6). Data were compared between two groups with Student's t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

Effects of RSV on PCNA expression in the ovaries of TBT-exposed rats. A, Immunohistochemical analysis of PCNA in rat ovaries from the control group, the TBT group, the TBT + RSV group and the RSV group. B, The number of PCNA-positive GCs was counted in randomly selected fields. C, Western blotting analysis of PCNA in ovaries. D, PCNA protein expression was analyzed statistically. Data are presented as the mean ± SEM (n = 3-6, *p ≤ 0.05).

Figure 4

RSV inhibited TBT-induced granulosa cell apoptosis. A, TUNEL staining of rat ovaries treated with TBT and RSV. B, The number of TUNEL-positive GCs was counted in randomly selected fields. C-D, Protein expression of apoptosis-related genes detected by Western blot. Data are presented as the mean ± SEM (n = 3-6). Data were compared between two groups with Student's t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5

RNA-seq analysis of ovarian tissue revealed overlapping gene expression linked to cell projection assembly and calcium ion transport. A, Functional annotation charts derived using the Cluster Profiler R package for the differentially regulated genes in the TBT group versus the control group. Significance is indicated by the -log10 p value. B, Functional annotation charts derived using the Cluster Profiler R package for the differentially regulated genes in the TBT + RSV group versus the TBT group. Significance is indicated by the -log10 p value. C, Venn diagram of the 62 upregulated genes in the TBT rats compared to the control rats and the 80 downregulated genes in the TBT + RSV-treated rats compared to the TBT rats with 14 overlapping genes. Similarly, 22 overlapping genes were downregulated in TBT rats compared to the control rats and then upregulated in TBT + RSV-treated rats. D, Biological process GO term enrichment of the 36 overlapping DEGs. E-F, Heatmap showing the expression patterns of the overlapping DEGs and the confirmatory qPCR results. G, CaMKIIβ sequesters monomeric actin to inhibit actin polymerization, and Ca²⁺ activates calmodulin, which triggers CaMKIIβ dissociation from G-actins. H, Protein expression of CaMKIIβ and phospho-CaMKIIβ (Thr286). I, Analysis of the protein expression of CaMKIIβ and p-CaMKIIβ/CaMKIIβ. Data are presented as the mean ± SEM (n = 3-6). J, CAMK2B expression in COCs from women with PCOS and control women. Data were compared between two groups using Student's t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 6

RSV repaired TBT-induced oocyte-granulosa cell communication injury. A, Phalloidin staining (green) of TZPs (arrow) formed from filamentous actin between oocytes (triangles) and GCs (asterisks). B, Oocyte-GC communication during growth. C, Protein expression of GDF9, BMP15, BMPR2 and Smad3. D, The results are expressed as the fold change in the optical density of a target protein, and β-actin expression served as the control. The mean protein expression of the control is designated as 1 in the graph. Data are presented as the mean ± SEM (n = 3-6). Data were compared between two groups with Student's t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.