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An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided. © 1998 Chapman & Hall

This study focused on the intracellular signal transduction system and microtubule-associated proteins (MAPs), such as MAP-2 and Tau protein. The modulation of these proteins and their correlation with ultrastructural changes were investigated in rat pituitary prolactin (PRL) cells. Adult female Wistar rats were treated with estrogen and bromocriptine and their pituitary glands were removed for analysis of the expression of tubulin, MAP-2, Tau protein, protein kinase C (PKC), and calcium calmodulin (CaM) kinase. Western blot analysis showed that estrogen increased and bromocriptine decreased the expression of PKC alpha, beta 1, beta 2, CaM kinase alpha, beta, MAP-2, and Tau protein. MAP-2 and Tau protein, which are cytosolic proteins, being translated on free ribosomes, were associated with the membrane of whirling rough endoplasmic reticulum (RER) in estrogen-treated cells and dissociated with vesiculated RER induced by bromocriptine. These results suggested that the modulation of MAP-2 and Tau protein may reflect changes of PKC and CaM kinase, and that the quantitative changes and intracellular modulation of MAPs induced by estrogen and bromocriptine, i.e., estrogen-induced association and bromocriptine-induced dissociation of MAP-2 and Tau protein with membrane of RER, may reflect the dynamics of microtubules and are associated with structural changes in the RER and changes in the synthesis and intracellular transport of PRL.

The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopical in situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.

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.

There have been many reports on radioisotopic in situ hybridization (ISH) studies for the demonstration of pituitary hormone mRNAs in normal pituitary gland and pituitary adenomas. Recent studies have revealed that non-radioisotopic ISH has several advantages over the radioisotopic method. Using ISH with biotinylated oligonucleotide probes, we have been able to localize various pituitary hormone mRNAs in paraffin wax or frozen sections of rat normal pituitary gland and human pituitary adenomas. For control studies we used ISH with sense probes, ISH without probes, pretreatment with ribonuclease, ISH with a probe for β-actin and Northern blot hybridization. Using biotinylated probes, gene transcripts of rat growth hormone and prolactin were detected by Northern blot hybridization. The same biotinylated probes were used not only for light microscope ISH but also for the electron microscopical demonstration of rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticula. It is emphasized that biotinylated oligonucleotide probes are useful for the analysis of pituitary endocrine function because they are applicable to the three hybridization methods, namely, Northern blot hybridization and ISH at the light and electron microscope levels.

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. Three different approaches have been applied by the investigators in this EM-ISH study: preembedding method; non-embedding method using ultrathin frozen sections; and postembedding method. In order to obtain satisfactory morphological preservation and retain the messages, we routinely utilized 6 pmthick frozen sections fixed in 4% paraformaldehyde for the preembedding method and tissues embedded in LR White resin for the postembedding method. The hybridization signal intensity by the postembedding method was lower, and non-specific signals were relatively 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 applicability and preservation of mRNA, although quantitative analysis of the expression of mRNA is rather difficult in the preembedding method. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double-staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.

The expression and distribution of prolactin (PRL) mRNA and their alterations induced by estrogen and bromocriptine were investigated using non-radioisotopic in situ hybridization (ISH) at the electron microscopic (EM) level. Our EM-ISH studies using biotinylated oligonucleotide probes showed that estrogen induced whirling changes of the rough endoplasmic reticulum (RER) of female rat PRL cells and increased transcription of PRL genes located on the polysomes of the whirling RER. The presence of mammosomatotroph cells in the rat pituitary gland was also verified in our EM-ISH studies. After bromocriptine administration, PRL cells contained many secretory granules due to the inhibition of secretion. Pre- and post-embedding EM-ISH and northern hybridization studies revealed that bromocriptine induced the distorted, vesiculated, and dilated RER, and also the suppressed PRL mRNA expression. The activity of protein kinase C (PKC), which mediates PRL gene expression, tended to be elevated by estrogen and suppressed by bromocriptine. Therefore, it is considered that the ultrastructural and quantitative changes in PRL mRNA expression evoked by estrogen and bromocriptine may be mediated by the intracellular signal transduction system, including PKC.

We have previously reported that B cell abnormality in NZB/W F1 mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin-6 (IL-6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL-6, an increased production of both IgM and IgG, including anti-DNA antibody, was evident in NZB/W F1 mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL-6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL-6 was neutralized by treatment with rabbit anti-recombinant human IL-6 antibody. As B cells from athymic NZB/W F1 nude mice also responded to IL-6, it was suggested that B cells in NZB/W F1 mice differentiated into the IL-6-responding state in a thymus-independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W F1 mice.

Autoimmune MRL/Mp-lpr/lpr (MRL/l) mice were treated with the immunostimulating anti-cancer drug OK-432 (a streptococcal preparation), a potent inducer of tumour necrosis factor. Treatment was initiated at 8 weeks of age, before the onset of the autoimmune disease. OK-432 prevented the development of immune complex-mediated glomerulonephritis in a dose-dependent manner, and prolonged the life in this strain of mice. At 36 weeks of age, the incidence of proteinuria was 90% in the controls, 60% in the 0.5-KE(1 KE = 0.1 mg) treatment group, and 33% in the 2.0-KE group. The 50% survival time was 23 weeks for the controls; 32 weeks for the 0.5-KE group; and greater than 36 weeks for the 2.0-KE group. Immune complex deposition in glomeruli was significantly reduced in the treated groups. The IgM class of serum autoantibody levels was significantly increased by OK-432 treatment but the IgG class was almost unchanged. Furthermore, lymphadenopathy and splenomegaly were not suppressed. The results indicate that OK-432 may be useful in the treatment of autoimmune disease in humans.