Transcriptome Dynamics and Cell Dialogue Between Oocytes and Granulosa Cells During Mouse Follicle Development

Overview

Follicle development and maturation is a complex, multi-stage process. During this process, dynamic gene expression in oocytes and surrounding somatic cells, as well as the dialogue between them, play crucial roles. This study revealed transcriptome changes and intercellular communication mechanisms at different developmental stages by precisely classifying oocyte and follicle developmental stages and analyzing gene expression in mouse oocytes and their surrounding granulosa and cumulus cells. The research was conducted by Wenju Liu, Chuan Chen, Yawei Gao, Xinyu Cui, et al. from institutions including Tongji University and Nanjing Medical University, and was published in the journal “Genomics, Proteomics & Bioinformatics” in 2024.

Research Background

Oocytes originate from primordial germ cells and develop within follicles. The development of oocytes and follicles is a multi-stage process, typically classified by follicle volume and the number of granulosa cell layers surrounding the oocyte. However, these methods cannot precisely define the developmental stages of oocytes and follicles. Moreover, while progress has been made in understanding oocyte-somatic cell dialogue (such as signaling mediated by cytokines and growth factors, and gap junctions allowing small molecule exchange), the specific factors involved and their regulatory mechanisms remain unclear.

Research Source and Author Affiliations

The main authors of this study include Wenju Liu, Chuan Chen, Yawei Gao, Xinyu Cui, Yuhan Zhang, Liang Gu from Tongji University, and Yuanlin He, Jing Li from Nanjing Medical University. The research findings were published ahead of print on December 6, 2024, in the journal “Genomics, Proteomics & Bioinformatics”.

Research Process and Detailed Experimental Methods

Research Process

The study was conducted in the following steps:

1. Collection and Classification of Oocytes and Granulosa Cells:

   • Collected oocytes, granulosa cells, and cumulus cells at nine stages of oocyte and follicle development.
   • Accurately classified based on oocyte and follicle size and follicle morphology.

2. RNA Sequencing:

   • Used ultra-low input RNA sequencing (RNA-seq) to determine the transcriptomes of oocytes, granulosa cells, and cumulus cells at various stages.
   • Highly reproducible replicate results supported the reliability of transcriptome data.

3. Transcriptome Clustering Analysis and Principal Component Analysis (PCA):

   • Demonstrated different trajectories of oocytes and granulosa cells during development through unsupervised hierarchical clustering and PCA.

4. Transcriptome Dynamic Analysis:

   • Compared differentially expressed genes (DEGs) between adjacent developmental stages, revealing the greatest change in gene expression at stage 4.
   • Used STEM clustering to analyze dynamic patterns of gene expression in oocytes and granulosa cells.

5. Intercellular Dialogue Analysis:

   • Revealed communication between oocytes, granulosa cells, and cumulus cells using cell-paired ligand-receptor pair methods.

Special Experimental Methods

To ensure the reliability of oocyte transcriptomes, standard RNA was added to some RNA sequencing. The consistency of gene expression patterns was verified by comparing transcriptome results with and without standard RNA. Additionally, pseudotime analysis was applied to simulate the developmental trajectory of gene expression, further validating the dynamic nature of the research results.

Main Research Findings

Overall Gene Expression Profile

1. Transcriptome Clustering and Differential Gene Expression Analysis:

   • Oocytes and granulosa cells showed significant dynamic changes in gene expression across multiple developmental stages.
   • Gene expression peaked at stage 4, followed by a sharp decline in both oocytes and granulosa cells.

2. Dynamic Changes in Cell Dialogue:

   • From stage 4 onwards, cell communication between oocytes and granulosa cells became active, including unidirectional and bidirectional signaling. TGF-β and Notch signaling pathways played important roles in granulosa cell development.

Gene Expression Differences Between Oocytes and Somatic Cells

Pairwise comparisons of gene expression between oocytes and surrounding somatic cells showed a significant increase in the number of differentially expressed genes between oocytes and granulosa cells starting from stage 4, with over half of these differentially expressed genes persisting into subsequent developmental stages. These results emphasize the significance of stage 4 as a critical turning point in oocyte and granulosa cell development.

Involvement of Important Signaling Pathways

Analysis revealed that TGF-β, Notch, and other signaling pathways play crucial roles in regulating follicle development. The construction of cell communication networks revealed complex interactions of these signaling pathways between oocytes and granulosa cells.

Dynamic Expression of Maternal Effect Genes and Imprinted Genes

Maternal effect genes play important roles in egg maturation and early embryo development. This study found that these genes begin to express significantly in eggs from stage 1 and gradually increase during subsequent follicle development. Imprinted genes (especially those marked by DNA methylation or H3K27me3) were silenced in early oocyte development but remained active in surrounding granulosa and cumulus cells.

Research Conclusions and Significance

The conclusions of this study are summarized as follows:

1. Scientific Value: This study comprehensively revealed the transcriptome dynamics and cell communication mechanisms between oocytes and granulosa cells during mouse follicle development for the first time, providing a profound understanding of oocyte development and follicle maturation.

2. Application Value: The newly discovered signaling pathways and regulatory factors provide new directions for future research and may offer new ideas and strategies for treating ovarian dysfunction and infertility.

3. Important Findings: This study discovered multiple signaling pathways that play important roles in follicle development and their multi-level intercellular interaction networks, highlighting the critical turning point significance of stage 4 in oocyte and granulosa cell development.

Research Highlights

This study significantly revealed bidirectional cell dialogue between oocytes and granulosa cells, as well as different expression patterns of maternal effect genes and imprinted genes in eggs and somatic cells. These findings not only enrich the understanding of folliculogenesis but also provide valuable references for future application research in fertility disorders and reproductive medicine.

Other Valuable Information

In addition to the new mechanisms and pathways revealed in the study, this research also provides detailed experimental methods and data processing procedures, offering strong support for other researchers to conduct further experiments and analyses in related fields.