Ultrastructural distribution of growth hormone and prolactin mRNAs in normal rat pituitary cells: a comparison between preembedding and postembedding methods.
In situ hybridization (ISH) at the electron microscopic level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our electron microscopic ISH method using biotinylated oligonucleotide probes for rat growth hormone and prolactin mRNAs and compare the preembedding method with the postembedding method. Preembedding electron microscopic ISH localized rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticulum (RER). Rat growth hormone mRNA was distributed diffusely on the RER, whereas rat prolactin mRNA was scattered and distributed focally. Thus there might be a specific translational site for prolactin mRNA on the RER. Rat growth hormone mRNA signals were also recognized on the polysomes of the RER, using the postembedding method with streptavidin gold conjugate. The hybridization signal intensity using the postembedding method was lower, and non-specific signals were more frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of utility and preservation of mRNA. Electron microscopic ISH is considered to be an important tool for evaluating the intracellular localization of mRNA and the site of specific hormone synthesis on the RER.
Matsuno
A
A
Department of Neurosurgery, University of Tokyo Hospital, Japan.
Ohsugi
Y
Y
Utsunomiya
H
H
Takekoshi
S
S
Osamura
R Y
RY
Watanabe
K
K
Teramoto
A
A
Kirino
T
T
D000818:Animals; D001483:Base Sequence; D005260:Female; D013006:Growth Hormone / Q000302:isolation & purification*; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000166:cytology / Q000648:ultrastructure*; D011388:Prolactin / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D016610:Tissue Embedding / Q000379:methods
D012333:RNA, Messenger; D011388:Prolactin; D013006:Growth Hormone