Detection of proopiomelanocortin mRNA by in situ hybridization,using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

Summary A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (MSH/ACTH[4-11]), ws synthesized and labelled in the 3-end by use of terminal transferase. Probes tailed with either [³H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment,hybridization and probe labelling conditions

Summary Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40–45° C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4° C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9° C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1×SSC, 45° C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4–11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length. Image analyses of stainings obtained with individual probe fractions, varying in tail length, proved that probes extended to 27-mer size produced the best results. Finally, similar experiments using a 24-mer probe complementary to the dynorphin B[10–18]-coding region of prodynorphin mRNA revealed best hybridization results with a tail length of 4 nucleotides. Purification and testing of labelled probes can result in major improvements in hybridization detection efficiency. Abbreviations used: AL Anterior (pituitary) lobe; APAAP Alkaline phosphatase-antialkaline phosphatase; BCIP-NBT Beta-chloroindolyl phosphate-nitroblue tetrazolium; BSA Bovine serum albumin, CAP Cell area percentage; CCD Charge-coupled device; CPV Corrected pixel values; F Formalin; HPLC High performance liquid chromatography; IA Image analysis; IL Intermediate (pituitary) lobe; MPV Mean pixel values; MSH Melanocyte-stimulating hormone; OD Optical density; PB 0.1 M sodium phosphate buffer, pH 7.4; PBS Phosphate-buffered saline; PF Paraformaldehyde; PG Paraformaldehyde:glutaraldehyde; POMC Proopiomelanocortin; RAM Random access memory; SD Standard deviation; SON Synthetic oligodeoxynucleotide; SSC Sodium chloride:sodium citrate; Tm Melting point; VE Visual evaluation     

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in rat pituitary POMC mRNA after exposure to cold or a novel environment, detected by in situ hybridization.

Plasma adrenocorticotropin (ACTH) levels increase after acute cold exposure. The purpose of this study was to determine if there were parallel changes in pituitary proopiomelanocortin (POMC) mRNA. Male rats were exposed to cold (3-5 degrees C) or a novel environment for 15 or 30 min. Others were unstressed. POMC mRNAs in frozen sections or dissociated cells were hybridized with a photobiotinylated oligonucleotide probe which was detected in situ by streptavidin-alkaline phosphatase. The percentage of area labeled for POMC mRNA was quantified by the Cue-3 color image analysis system. In frozen sections, 24-fold increases in the percentage of area labeled for POMC mRNA were evident in intermediate lobes (IL) 30 min after stress. No change was seen in anterior lobes (AL). If the ALs were dissociated, a 66-99% increase in percentage of labeled cells was detected 2-3 hr after the cold exposure. Fifteen min of cold stress (CS) also caused a 117% increase in the area of label for POMC mRNA per corticotrope. No change was seen after 30 min. Exposure to a novel environment caused a 73% increase in the percentage of area labeled for POMC mRNA per AL corticotrope and an 11-fold increase in the IL. These results indicate that both AL corticotropes and IL melanotropes are stimulated by acute exposure to cold and novel environments.     

Proopiomelanocortin gene is expressed in many normal human tissues and in tumors not associated with ectopic adrenocorticotropin syndrome

Immunoreactive (IR) POMC peptides have been detected in several human nonpituitary tissues and most pheochromocytomas and lung cancers, including those not associated with ectopic ACTH syndrome. We found IR-ACTH, IR-gamma MSH, IR-beta-endorphin (beta END), and IR-lipotropin in extracts from the following 10 normal human tissues, listed in order of decreasing POMC peptide concentrations: adrenal, testis, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum. IR-ACTH, IR-gamma MSH, and IR-beta END were detected in all six pheochromocytomas and all 12 lung tumors (six squamous cell carcinomas, five adenocarcinomas, and one small cell carcinoma) we examined, as well as in a squamous cell carcinoma of the larynx. None of the patients had clinical evidence of ectopic ACTH syndrome. To determine whether these nonpituitary tissues and tumors actually synthesize POMC, rather than simply absorb POMC peptides from plasma, we examined poly(A) RNA prepared from these tissues and total RNA from pituitary by Northern blot hybridization for the presence of POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1150 bases. A short POMC-like mRNA of about 900 bases was found in all normal nonpituitary tissues, three of five pheochromocytomas, eight of nine lung cancers, and the laryngeal squamous cell tumor. In addition, larger POMC-like mRNA species between 1200 to 1500 bases were detected in adrenal, testis, ovary, placenta, two pheochromocytomas, and three squamous cell lung tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.     

Immunocytochemistry of 5-bromo-2'-deoxyuridine labelled oligonucleotide probes. A novel technique for in situ hybridization

A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2'-deoxyuridine (5-BrdU) and with [gamma-32P]-ATP. The labelled probes were used for in situ hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [32P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-brdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.     

In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs.

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.     

In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression

We performed in situ hybridization of myosin heavy-chain (MHC) mRNA on rabbit muscle using a biotin-labeled complementary RNA probe. An 1107-nucleotide fragment from an alpha-cardiac MHC cDNA was used to transcribe an RNA probe 97% similar to slow-twitch and 75% similar to fast-twitch sequences. Serial sections were used to identify slow-twitch fibers in medial gastrocnemius, soleus, and tibialis anterior by immunofluorescence of slow MHC and oxidative capacity by histochemistry. Slow-twitch fibers hybridized by the RNA probe stained heavily after detection with streptavidin-alkaline phosphatase (89% dark and 11% medium density). Fast-oxidative fibers stained intermediately (26% dark, 58% medium, and 16% light) and fast-glycolytic fibers stained lightly (12% medium and 88% light). Biotin-labeled probe and enzymatic detection allowed greater resolution of the subcellular location of the MHC mRNA, a distinct advantage over isotope labeling and autoradiography. A non-uniform distribution of MHC mRNA was recognized within an adult skeletal muscle fiber. High concentrations of MHC mRNA were found under the sarcolemma and between the myofibrils, suggesting the existence of a distribution mechanism. The combination of in situ hybridization and immunocytochemistry allows rapid subcellular localization of both MHC mRNA and its translated protein.     

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?

We hypothesized that urocortin might be produced in the pituitary of the late-gestation ovine fetus in a manner that could contribute to the regulation of ACTH output. We used in situ hybridization and immunohistochemistry to identify urocortin mRNA and protein in late-gestation fetal pituitary tissue. Levels of urocortin mRNA rose during late gestation and were associated temporally with rising concentrations of pituitary proopiomelanocortin (POMC) mRNA. Urocortin was localized both to cells expressing ACTH and to non-ACTH cells by use of dual immunofluorescence histochemistry. Transfection of pituitary cultures with urocortin antisense probe reduced ACTH output, whereas added urocortin stimulated ACTH output from cultured pituitary cells. Cortisol infusion for 96 h in chronically catheterized late-gestation fetal sheep significantly stimulated levels of pituitary urocortin mRNA. We conclude that urocortin is expressed in the ovine fetal pituitary and localizes with, and can stimulate output of, ACTH. Regulation of urocortin by cortisol suggests a mechanism to override negative feedback and sustain feedforward of fetal hypothalamic-pituitary-adrenal function, leading to birth.     

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the -s ubunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions. © Chapman & Hall     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.