Ultrastructural distribution of growth hormone and prolactin mRNAs in normal rat pituitary cells: a comparison between preembedding and postembedding methods

Abstract

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.

Application of confocal laser scanning microscopy (CLSM) to visualize prolactin (PRL) and PRLmRNA in the normal and estrogen-treated rat pituitary glands using non-fluorescentprobes

“…For EM observation of mRNA, the subsequent procedure was similar to that describe for pre-embedding immunoelectron microcopy (Matsuno et al, 1994a(Matsuno et al, ,b, 1995. …”

Application of Ultrastructural In Situ Hybridization Combined with Immunohistochemistry to Pathophysiological Studies of Pituitary Cell: Technical Review.

“…Ultrastructural ISH for mRNA, which was first described by Jacob et al [5], has further been developed by several investigators [4, 6-9, 11-13, 18-25] and has been carried out in the three different approaches: preembedding method [4, 12-14, 20, 23, 25], ultrathin frozen sections [9,18,19], postembedding method [6-9, 12, 14]. In recent years, we have developed a non-radioisotopic EM-ISH method using biotinylated synthesized oligonucleotide probes for the ultrastructural visualization of growth hormone (GH) and prolactin (PRL) mRNAs in rat pituitary cells, and have applied this method to pathophysiological studies of pituitary cells [12][13][14].…”

“…As described in our previous reports [12], compared with the preembedding method, the postembedding method has several difficulties: 1) Difficulty of message preservation during polymerization of LR White resin at high temperature for an extended period of time, which leads to mRNA degradation.…”

Applications of Plastic Embedding to Electron Microscopic Immunocytochemistry and In Situ Hybridization in Observations of Production and Secretion of Peptide Hormones

“…Postembedding EM-ISH studies have been reported by several investigators (Le Guellec et al 1990Guellec et al ,1991Guellec et al , 1992Matsuno et al 1994aMatsuno et al ,1995Matsuno et al ,1998c. Le Guellec and co-workers stated that no hybridization signals were observed in an EM-ISH study using Epon-embedded sections (Le Guellec et al 1992).…”

“…In these postembedding EM-ISH studies, relatively frequent nonspecific reactions of colloidal gold particles used for the detection of mRNA were observed in the cisternae of the RER. We experienced the decreased message preservation in the postembedding EM-ISH studies using LR White-embedded tissues (Matsuno et al 1994a(Matsuno et al ,1995(Matsuno et al ,1998c. To obtain specific hybridization signal detection and well-preserved messages, we have employed pre-embedding EM-ISH and subsequent immunoreaction for simultaneous ultrastructural identification of mRNA and encoded protein (Matsuno et al 1996(Matsuno et al ,1998a.…”