Detection of receptor mRNAs using nonradioactive in situ hybridization

To understand better the regulatory mechanism of cell function through bioactive substance such as hormones and cytokines, analysis of the gene expression of their receptors at the individual cell level is required. Since the presence of specific mRNA is a good index of gene expression, in situ hybridization (ISH) has been recognized as a powerful tool. Especially, nonradioactive ISH with synthetic oligodeoxynucleotide probe is a safe, quick and highly sensitive method. We have developed a unique method with thymine-thymine (T-T) dimer as a nonradioactive labeling, in which adjacent thymines are dimerized by ultraviolet light irradiation. The T-T dimers in hybrids are immunohistochemically recognized using an anti-T-T dimer antibody. In this article, we will present an example of ISH protocol, which works very well both T-T dimer and digoxigenin-labeled probes.     

Opioid regulation of gonadotropin-releasing hormone gene expression in the male rat brain as studied by in situ hybridization

It is well documented that gonadotropin-releasing hormone (GnRH) release is negatively regulated by opiates. In order to investigate the role of opiates in the regulation of GnRH gene expression in the rat brain, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on GnRH mRNA levels as measured by in situ hybridization. Four-day treatment with morphine (40 mg kg day-2) produced a 44% decrease in the number of silver grains overlying GnRH neurones. Conversely, naloxone (4 mg kg day-2) also administered during 4 days increased by hybridization signal by 22%. The concomitant administration of morphine and naloxone completely prevented the effect of morphine on GnRH gene expression indicating the inhibitory influence of morphine is likely to be mediated by opioid receptors. The present data clearly indicate that opiates can inhibit not only the release of GnRH and consequently LH secretion but also the biosynthesis of the neuropeptide as evaluated by mRNA level measurements.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.     

Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes

Fluorescence in situ hybridization using two or three probes was utilized to estimate the incidence of diploidy, the incidence of disomy for the sex chromosomes and chromosomes 16 and 18, and the proportion of Y- and X-chromosome bearing sperm, in a series of normal males. Our results demonstrate the importance of using an approach capable of distinguishing disomy from diploidy, as most donors had levels of diploidy higher than the disomy levels of individual chromosomes. Our analyses suggest the existence of chromosome-specific mechanisms of paternal non-disjunction, as sex chromosome disomy was approximately 1.5 times as common as disomy 16, and over two times as common as disomy 18. In studies of gametic sex ratio, we found little evidence for marked deviation from an expected 1:1 ratio.     

Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect alpha 2 adrenoceptor subtype mRNAs in adult and developing rat brain

The mRNA expression of highly homologous alpha 2 adrenoceptor subtypes was determined using a highly sensitive in situ hybridization protocol that allowed the detection of low abundance mRNA. Full-length 35S-labeled riboprobes specific for alpha 2A, alpha 2B and alpha 2C adrenoceptors were used for maximal sensitivity. The probes were hydrolyzed to an average length of 600 bp which, in combination with proteinase K digestion, resulted in optimal probe penetration in developmental and adult tissue. The expression intensity could be quantified and the ontogeny of receptor mRNA expression determined. At the same time receptor binding sites or functional proteins could be detected simultaneously in adjacent sections, because fresh frozen and post-fixed tissue was used.     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

Estrogen facilitation of progesterone-induced incubation behavior in castrated male ring doves.

Previous studies have shown that incubation is shared by both male and female ring doves and is induced, without prior participation in courtship and nest building, by progesterone (100 mg/day) treatment. In the present experiment with 52 mature male ring doves, it was shown that the effectiveness of progesterone is markedly reduced by castration and restored after estradiol benzoate (200 mg/day * 14) pretreatment, thus simulating the endocrine events which precede incubation in the female. Estrogen also stimulated the appearance of nest cooing (another isomorphic behavior) but not bow cooing (a male-specific display). Results are discussed in terms of hormonal specificity underlying reproductive behavior and the possible physiological roles of estrogen and progesterone in the ring dove. (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cytogenetic analysis using simultaneous and sequential fluorescence in situ hybridization

BACKGROUND: Trichosporon beigelii, causal agent of white piedra can cause disseminated infection in immunodepressed subjects. Systemic infections due to this pathogen have been reported mainly in neutropenic patients and rarely in AIDS patients. CASE REPORT: A 36-year-old HIV+ man from Senegal was hospitalized for fever and meningoencephalitis associated with skin lesions. T. beigelii was isolated from skin biopsies and cerebrospinal fluid cultures. The patients was treated with amphotericin B with regression of the skin lesions. The diagnosis of disseminated T. beigelii infection was retained. DISCUSSION: Disseminated T. beigelii infections are known to occur in immunodepressed subjects, especially in case of neutropenia. In our patient, the presence of two proven localizations (meninges and skin) and the favorable outcome with amphotericin B favored disseminated infection. The good response to treatment can probably be explained by the absence of neutropenia. Skin lesions are frequent, usually occurring as disseminated papulae or purpural nodules. Pathology examination and skin biopsy culture can provide rapid diagnosis allowing appropriate treatment.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Gonadal hormone activation of male courtship ultrasonic vocalizations and male copulatory behavior in castrated male deer mice (Peromyscus maniculatus bairdi).

Examined the influence of testosterone (T), a 5-alpha-reduced metabolite of T (dihydrotestosterone), and an aromatized metabolite of T (estradiol) on 35-kHz ultrasonic calling and copulatory behavior by 72 male deer mice. Daily treatment with T propionate (TP), dihydrotestosterone propionate (DHTP), or estradiol benzoate (EB) restored ultrasonic calling in long-term castrated Ss. Both TP and DHTP restored copulatory behavior, but EB was ineffective. Synergism of EB and DHTP action was observed; when subthreshold doses of EB (1 μg/day) and DHTP (50 μg/day) were administered in combination, ultrasonic calling and copulatory behavior were activated. In relation to other comparative findings, results indicate that the degree to which male sexual behavior is facilitated by 5-alpha-reduced androgens and/or estrogens is influenced by the species and the particular pattern of masculine behavior under consideration. (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Direct solution hybridization of guanidine thiocyanate-solubilized cells for quantitation of mRNAs in hepatocytes

The sensitivity of direct solution hybridization of hepatocytes solubilized in guanidium thiocyanate (GuSCN) for detecting alpha 1-acid glycoprotein and albumin mRNAs was studied. The sensitivity of detection was inversely correlated with the DNA concentration. Raising the hybridization temperature from 20 to 37 or 50 degrees C (with formamide) increased the hybridization efficiency three- to fourfold in cell lysates with a high DNA concentration (1 microgram/microliter), whereas the hybridization efficiency was already maximal at 20 degrees C in diluted samples. It was most important to normalize all hybridization reactions with an internal standard, such as sense mRNA, because of the great variation in hybridization efficiency from one cell preparation to another depending on the DNA concentration. Direct hybridization of GuSCN cell lysates labeled in vivo with [6-14C]orotic acid was more efficient than hybridizing equivalent amounts of purified [6-14C]-labeled RNA, perhaps because of greater mRNA integrity and/or better recoveries of mRNA in GuSCN cell lysates. Therefore, direct solution hybridization of GuSCN-solubilized hepatocytes, which avoids the problem of RNA purification, appears to be a rapid, sensitive, and reliable method for quantifying mRNA in hepatocytes.     

Decline of sexual behavior in castrated male rats: effects of female precopulatory behavior

Ventricle cells from neonatal rats were cultured in a medium preventing cell proliferation on a modified Roller apparatus with a defined pericellular oxygen partial pressure (pO2) of 38 or 0.6 mm Hg for about one week. The cells were harvested after the second and eighth day of culture (corresponding to one or seven days of culture under a defined pO2) and prepared for electron microscopy. Electron micrographs of this material were examined by stereologic techniques. The obtained results showed some deviations of mitochondrial fine structure of heart muscle cells depending on oxygen supply. During cultivation with a pO2 of 0.6 mm Hg (hypoxic conditions) we observed a proportional increase in mitochondria without cristae and an increase in size accompanied by a more irregular shape. On the contrary, the mitochondria of cells cultured with a pericellular pO2 of 28 mm Hg, which we consider to be a normoxic condition, did not show any deviation of inner membrane arrangement, but an increase in number and a decrease of size during cultivation was apparent. The mitochondria-myofibrils ratio decreased during cultivation in both conditions of oxygen supply. The ratio between sarcoplasmatic reticulum and myofibrils decreased markedly at a pO2 of 0.6 mm Hg.