Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type...

Area of Science:

• Molecular Biology
• Cell Biology
• Neuroendocrinology

Background:

• Immediate-early response genes (IEGs) are crucial for cellular responses to extracellular signals.
• Studying IEG patterns in single cells (SCs) necessitates high-throughput, accurate methods that minimize experimental artifacts.

Purpose of the Study:

• To develop and validate a novel RNA stabilization method for preserving single-cell RNA integrity.
• To investigate single-cell heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) in pituitary gonadotropes.

Main Methods:

• Development and validation of RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best) for single-step cell and tissue preservation.
• High-accuracy single-cell RNA profiling of pituitary gonadotropes subjected to varying concentrations of GnRH stimuli.

Main Results:

• RNA-Best effectively stabilizes RNA in intact SCs without altering transcription patterns.
• A gene-specific, hierarchical pattern of all-or-none transcript induction was identified in response to increasing GnRH concentrations.
• This quantal gene activation suggests IEG induction may operate via binary switch-like mechanisms at the single-cell level.

Conclusions:

• RNA-Best is a versatile tool for accurate single-cell RNA analysis, particularly for studying dynamic gene expression.
• The study reveals a novel, binary-switch-like mechanism governing IEG activation in response to GnRH in pituitary cells.
• Findings advance the understanding of gene regulation and cellular signaling at the single-cell resolution.