title: Molecular morphology of pituitary cells, from conventional immunohistochemistry to fluorescein imaging.
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.
authors.affiliation: Department of Neurosurgery, Teikyo University Chiba Medical Center, Chiba 299-0111, Japan. akirakun@med.teikyo-u.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Matsuno
authors.forename: Akiko
authors.initials: A
authors.lastname: Mizutani
authors.forename: Hiroko
authors.initials: H
authors.lastname: Okinaga
authors.forename: Koji
authors.initials: K
authors.lastname: Takano
authors.forename: So
authors.initials: S
authors.lastname: Yamada
authors.forename: Shoko M
authors.initials: SM
authors.lastname: Yamada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakaguchi
authors.forename: Katsumi
authors.initials: K
authors.lastname: Hoya
authors.forename: Mineko
authors.initials: M
authors.lastname: Murakami
authors.forename: Masato
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Sugaya
authors.forename: Johbu
authors.initials: J
authors.lastname: Itoh
authors.forename: Susumu
authors.initials: S
authors.lastname: Takekoshi
authors.forename: R Yoshiyuki
authors.initials: RY
authors.lastname: Osamura
mesh_terms: D000818:Animals; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods; D008854:Microscopy, Electron; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism*
chemical_list: D013006:Growth Hormone