Mechanism Study of p300 Maintaining Primordial Follicle Activation by Inhibiting VEGFA Transcription

Academic Background

In the female reproductive system, primordial follicles (PFs) are the earliest formed follicles in the ovary, remaining dormant and awaiting activation to enter the growth phase. The activation of primordial follicles is a key factor in determining female reproductive lifespan, with abnormal activation or premature depletion potentially leading to diseases such as Premature Ovarian Insufficiency (POI). Although previous studies have shown that signaling pathways like PI3K and mTOR play important roles in primordial follicle activation, the upstream regulatory mechanisms remain unclear. p300, a histone acetyltransferase, is widely involved in gene transcription regulation and cellular function control, but its role in primordial follicle activation has not been extensively studied. Therefore, this study aims to explore the function of p300 in primordial follicle activation and its regulatory mechanisms, providing new theoretical insights for the treatment of ovarian diseases.

Source of the Paper

This paper was co-authored by Meina He, Yaoyun Liang, Xiaoran Nie, and others from Guizhou Medical University and its affiliated hospitals. It was published on November 8, 2024, in the journal American Journal of Physiology-Cell Physiology, with the DOI: 10.1152/ajpcell.00198.2024.

Research Workflow

1. Expression Dynamics of p300 During Primordial Follicle Activation

The study first analyzed published RNA-seq data to examine the expression changes of p300 at different developmental stages in mouse ovaries. The results showed that p300 expression gradually decreased with ovarian aging, but significantly increased during the critical period of primordial follicle activation (3–14 days). Immunohistochemical experiments further confirmed that p300 is primarily expressed in the nuclei of oocytes and granulosa cells, with its expression increasing during primordial follicle activation.

2. Regulation of Primordial Follicle Activation by p300

To determine the role of p300 in primordial follicle activation, the study treated newborn mouse ovaries with the p300 inhibitor C646 and activator CTPB. The results showed that C646 treatment significantly increased the activation rate of primordial follicles, while CTPB treatment inhibited follicle activation. Additionally, Western blot and real-time quantitative PCR (qPCR) experiments demonstrated that p300 influences follicle activation by regulating H3K27ac acetylation levels.

3. Regulation of Granulosa Cell Proliferation and Apoptosis by p300

Primordial follicle activation is accompanied by granulosa cell proliferation. By detecting proliferating cell nuclear antigen (PCNA) and apoptosis markers (TUNEL), the study found that the p300 inhibitor C646 significantly promoted granulosa cell proliferation and inhibited apoptosis, whereas the p300 activator CTPB suppressed granulosa cell proliferation.

4. Regulation of Primordial Follicle Activation by p300 via PI3K and mTOR Signaling Pathways

The study further explored the molecular mechanisms by which p300 regulates primordial follicle activation. Western blot results showed that the p300 inhibitor C646 significantly activated the PI3K and mTOR signaling pathways, while the p300 activator CTPB inhibited these pathways. Additionally, immunofluorescence experiments indicated that p300 affects the PI3K signaling pathway by regulating FOXO3a nucleocytoplasmic translocation.

5. Regulation of Primordial Follicle Activation by p300 Through Inhibition of VEGFA Transcription

Through RNA-seq analysis, the study found that the p300 inhibitor C646 significantly upregulated the expression of VEGFA and its receptor VEGFR1. Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP-qPCR) experiments further confirmed that p300 directly binds to the VEGFA promoter region and inhibits VEGFA transcription by regulating H3K27ac acetylation levels.

6. Potential Clinical Applications of p300 in In Vitro Activation of Primordial Follicles

The study also explored the potential clinical applications of p300 inhibitors in activating primordial follicles in vitro. Using renal subcapsular transplantation experiments, it was found that short-term treatment with C646 significantly increased the activation and further development of primordial follicles in transplanted mouse ovaries.

Research Results

1. Relationship Between p300 Expression and Primordial Follicle Activation: p300 expression increases during primordial follicle activation, with its inhibitor C646 significantly promoting follicle activation, while its activator CTPB inhibits follicle activation.
2. Regulation of Granulosa Cell Proliferation and Apoptosis by p300: The p300 inhibitor C646 promotes granulosa cell proliferation and inhibits apoptosis, indicating that p300 plays an important role in maintaining granulosa cell function.
3. Regulation of Primordial Follicle Activation by p300 via PI3K and mTOR Signaling Pathways: p300 influences primordial follicle activation by regulating the activity of the PI3K and mTOR signaling pathways.
4. Regulation of Primordial Follicle Activation by p300 Through Inhibition of VEGFA Transcription: p300 directly binds to the VEGFA promoter region and inhibits VEGFA transcription by regulating H3K27ac acetylation levels.
5. Potential Clinical Applications of p300 Inhibitors: Short-term use of the p300 inhibitor C646 significantly increased primordial follicle activation and further development in vitro.

Conclusions and Significance

This study reveals for the first time the critical role and regulatory mechanisms of p300 in primordial follicle activation. p300 maintains the dormant state of primordial follicles by inhibiting VEGFA transcription, thereby regulating the PI3K and mTOR signaling pathways. This discovery not only deepens our understanding of the mechanisms of primordial follicle activation but also provides new potential targets for treating diseases such as premature ovarian insufficiency. Additionally, the application prospects of p300 inhibitors in activating primordial follicles in vitro offer new ideas for the development of assisted reproductive technologies.

Research Highlights

1. First Revelation of p300’s Function in Primordial Follicle Activation: This study demonstrates for the first time that p300 plays a key role in primordial follicle activation by regulating VEGFA transcription and the PI3K/mTOR signaling pathways.
2. Multilevel Molecular Mechanism Study: Through RNA-seq, ChIP-qPCR, Western blot, and other experimental methods, the regulatory network of p300 is comprehensively revealed.
3. Potential Clinical Application Value: Research on p300 inhibitors in activating primordial follicles in vitro provides new strategies for treating premature ovarian insufficiency.

Other Valuable Information

The RNA-seq data from this study has been made publicly available and can be accessed upon reasonable request. Additionally, the study provides detailed experimental methods and data analysis processes, serving as a reference for future research.

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Through this study, we not only gain a deeper understanding of the molecular mechanisms of p300 in primordial follicle activation but also provide new insights for the treatment of ovarian diseases and the development of assisted reproductive technologies. In the future, based on the regulatory network of p300, more effective strategies for ovarian function protection and repair may be developed.