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Updated: Jun 2, 2026
Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland
Published on: November 16, 2017
Molecular morphology of pituitary cells, from conventional immunohistochemistry to fluorescein imaging
Akira Matsuno1, Akiko Mizutani, Hiroko Okinaga
1Department of Neurosurgery, Teikyo University Chiba Medical Center, Chiba 299-0111, Japan. akirakun@med.teikyo-u.ac.jp
Abstract:
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²+ influx or Ca²+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.
Insights
Electron microscopic in situ hybridization (EM-ISH) visualizes mRNA and protein synthesis sites. Advanced techniques like EM-ISH&IHC and quantum dot imaging reveal pituitary hormone transport and secretion dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Intracellular mRNA distribution and protein synthesis are crucial for cellular function.
- Electron microscopy-based in situ hybridization (EM-ISH) is vital for pinpointing mRNA localization and synthesis sites, particularly for pituitary hormones on the rough endoplasmic reticulum.
Purpose of the Study:
- To detail advanced techniques for visualizing pituitary hormone synthesis, transport, and secretion.
- To elucidate the roles of specific proteins in the exocytotic machinery of anterior pituitary cells.
- To develop and utilize novel imaging methods for real-time observation of hormone dynamics.
Main Methods:
- Combined electron microscopic in situ hybridization and immunohistochemistry (EM-ISH&IHC) for 2D ultrastructural localization.
- Quantum dot (Qdot) labeling with confocal laser scanning microscopy (CLSM) for 3D visualization of mRNA and protein.
- Development of a stable GH3 cell line expressing growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP) for real-time secretion studies.
Main Results:
- EM-ISH&IHC provides high-resolution 2D localization of pituitary hormones and their mRNA.
- Qdot-CLSM enables simultaneous 2D and 3D imaging of protein-mRNA relationships.
- CLSM identified rab3B and SNARE proteins (SNAP-25, syntaxin) as key components of the exocytotic machinery.
- The EYFP-GH3 cell line allows real-time tracking of GH transport and secretion.
Conclusions:
- Advanced EM-ISH and Qdot-CLSM techniques offer powerful tools for studying subcellular localization and dynamics.
- These methods facilitate a comprehensive understanding of pituitary hormone synthesis, from transcription to secretion.
- The developed GH3 cell line provides a valuable model for real-time analysis of hormone secretion processes.
