Background
Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of reproductive age. It is characterized by hyperandrogenism, ovulatory dysfunction, and the formation of polycystic ovaries. Its prevalence ranges from 8 to 13%. Most women with PCOS have clinical symptoms such as abnormal menstruation, hirsutism, obesity, and infertility, which can affect the physical and mental health of women with PCOS [
1‐
3].
Many studies have shown that the number of antral follicles in patients with PCOS is abnormally increased [
4‐
6]. The activation of the Hippo pathway (which is involved in overcoming the growth control property of normal cells) is related to an increased proliferation of small follicles [
7]. In patients with PCOS, the abnormal follicular proliferation may be related to the Hippo pathway.
The Hippo pathway, which is composed of a group of conserved kinases, inhibits tissue overgrowth. In mammals, receptors on the plasma membrane sense growth inhibitory signals from the extracellular environment and then act on the downstream effector protein yes-associated protein 1 (YAP) via a series of phosphorylation events. Phosphorylated YAP then interacts with cytoskeletal proteins and becomes trapped in the cytoplasm, where it can be degraded by proteases. Thus, YAP is unable to enter the nucleus to exercise its transcriptional activation function, thereby regulating organ size.
The Hippo pathway involves a core signaling axis. First, Hippo (Hpo; MTS in mammals) is activated under physiological or non-physiological stress. It phosphorylates and thereby activates the protein kinase Warts (Wts; LATS1/2 in mammals). This results in a series of changes in the downstream signaling pathway, including YAP phosphorylation [
8]. LATS can thereby regulate cell differentiation, proliferation, apoptosis, and migration, regulate the transcription and translation of genetic material, and maintain the stability of genetic material.
Our previous research showed that when S1P, a LATS blocker, was administered to 3-day-old female rats, the number of small follicles increased with increased S1P concentrations. However, whether the Hippo pathway is activated or inhibited during the development of PCOS, and whether it is involved in the increase in small follicles in patients with PCOS need to be further studied. Therefore, this study established a mouse model of PCOS to explore the changes in the number of small follicles and Hippo pathway activation in the ovarian tissue of PCOS mice. Changes in follicular development after administration of the YAP inhibitor verteporfin (VP) were also assessed in order to further analyze the relationship between the Hippo pathway and follicular development. This exploration of the pathogenesis of PCOS provides a theoretical and practical basis for developing new PCOS therapies.
Materials and methods
Establishment of mouse model of PCOS
Thirty 3-week-old mice were randomly divided into control, dehydroepiandrosterone (DHEA), and DHEA + VP groups (10 mice per group). The control mice were injected with 0.2 ml/day sesame oil for 4 weeks. To establish the PCOS model, the DHEA and DHEA + VP mice were subcutaneously injected with 60 mg/kg/day DHEA (dissolved in sesame oil) for 4 weeks. Lastly, the DHEA + VP mice were intraperitoneally injected with 50 mg/kg/day VP for 1 week starting from 3 weeks after the beginning of the DHEA injections.
Hematoxylin and eosin (HE) staining of ovarian tissue and follicle analysis
The mice were killed by cervical dislocation. Under an anatomical microscope, the two ovaries were removed, as were the adipose tissue on the surface and the covered capsule. Six ovaries (left or right) from six mice per group were randomly selected. The remaining ovaries were stored at − 80℃. The selected ovaries were fixed in 4% paraformaldehyde, dewaxed, embedded in paraffin, sectioned with a thickness of 5 inches, dewaxed, hydrated, stained with hematoxylin for 5 min, stained with eosin for 2 min, dehydrated, subjected to clearing, prepared using a coverslip, and then observed under a microscope to assess the morphological changes. Using a double blind method, six random HE-stained sections per group were analyzed. The ovarian tissue sections with the largest cross-sectional area of the ovaries were selected, and the numbers of follicles, cystic follicles, and corpus lutea at each level were observed.
Western blotting
The total protein was extracted from ovarian tissues and the protein concentration was determined. Next, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, blocked, incubated with primary antibody against anti-müllerian hormone (AMH), proliferating cell nuclear antigen (PCNA), YAP, p-YAP, MST, or LATS1, and incubated with secondary antibody. Images were then obtained for analysis.
Immunofluorescence assays
To assess AMH expression and localization, ovarian tissue sections were subjected to dewaxing, rehydration, inactivation of endogenous peroxidase, heat-induced antigen retrieval, blocking, incubation with primary antibody against AMH in the dark, incubation with secondary antibody, nuclear staining, and preparation using a coverslip. The slides were then placed in a wet box to avoid light and left at 4℃. The film was completed in 12 h. Immunofluorescence was assessed using X.
Statistical analysis
SPSS 22.0 software was used for the statistical analysis. The continuous data are expressed as mean ± standard deviation. Independent-samples t tests were used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.
Discussion
PCOS is a common female endocrine disease that can lead to female infertility and seriously affect the physical and mental health of patients. In patients with PCOS, the development of ovarian follicles is disrupted, resulting in a lack of large follicles [
4] and an increase in the number of small follicles [
5].
The Hippo pathway is related to cell proliferation and growth. Our previous research found that the LATS blocker S1P affected the activation of primordial follicles, changed the proportions of different follicle types in the ovaries, and increased cell proliferation. Research has shown that abnormal follicular development in PCOS is often accompanied by dysregulation of the Hippo pathway. YAP1/TAZ, downstream effectors of the Hippo pathway, are also known to play an important role in granulosa cell proliferation and estradiol synthesis [
9,
10]. Additionally, Li et al. showed that there is a difference in YAP protein between patients with PCOS and normal controls [
11]. Jiang et al. [
12] confirmed YAP methylation abnormalities in PCOS patients. Sun et al. [
13] found that Lats1 deletion leads to ovarian germ cell apoptosis and follicular cysts. Therefore, we speculate that the abnormal follicular development in patients with PCOS may be closely related to the Hippo pathway, that is, follicular growth and development may be regulated by the Hippo pathway.
First, we established a PCOS mouse model [
14] and administered VP, an inhibitor of YAP (a downstream protein in the Hippo pathway). In the DHEA group compared to the control group, AMH and PCNA in ovarian tissue significantly increased and decreased, respectively; VP significantly ameliorated the increase in AMH but did not ameliorate the decrease in PCNA. This indicated that follicular development was inhibited after the inhibition of YAP by VP, that is, the Hippo pathway affects follicular development [
15].
Second, we determined whether there were changes in the expression of Hippo pathway proteins in the ovarian tissue of PCOS mice. The results showed that the levels of YAP and p-YAP (downstream proteins in the Hippo pathway) in ovarian tissue in the DHEA group were significantly increased, indicating abnormalities in the Hippo pathway in the DHEA group. Furthermore, the decrease in the p-YAP/YAP ratio in the DHEA group compared to the control group indicated that nuclear YAP was more highly increased than cytoplasmic p-YAP. The relatively high increase in YAP in the nucleus indicated abnormal cell proliferation in the DHEA group. After administration of VP, p-YAP and YAP protein significantly decreased compared to their levels in the DHEA group, though the p-YAP/YAP ratio did not significantly change.
Third, MST and LATS1 protein expression (upstream proteins in the Hippo pathway) were significantly higher in the DHEA group than the control group, and they were decreased by VP. These results showed that the Hippo pathway was activated in PCOS model mice, which promoted the proliferation of antral follicles. In contrast, the Hippo pathway was inhibited by the YAP inhibitor VP, which decreased the proliferation of antral follicles (as indicated by the decrease in AMH).
To sum up, Hippo pathway activation is closely related to the proliferation of small antral follicles in PCOS. This study provides a theoretical and practical basis for the exploration of the pathogenesis of PCOS and the development of new treatments. Research has shown that there is an association between Hippo pathway activation and iron-dependent cell death [
16], and iron-dependent cell death is related to follicular genesis [
17]. As the Hippo pathway and iron-dependent cell death are both related to the occurrence of PCOS, there may be a relationship between the Hippo pathway and iron dependent cell death in PCOS and in the changes in ovarian follicles. Further studies on this relationship are needed.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.