Quantification of In Situ Hybridization Signals in Rat Testes

Kimura, Touji; Kosaka, Jun; Nomura, Takako; Yamada, Teruo; Miki, Yukari; Takagi, Koji; Kogami, Takashi; Sasaki, Junzo

doi:10.1369/jhc.4a6249.2004

Cited by 8 publications

(9 citation statements)

References 23 publications

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“…All corrected signal intensities from the same slide were then Z-transformed to overcome the problem of their non-linearity and absence of normal distribution, which finally enabled us to compare various brain regions on each slide. It is, however, important to keep in mind that ISH signals only reflect relative changes in copy numbers and thus only allow a relative assessment of the signal intensity in each section [42]. Our study would thus not be suited to detect an absolute overall increase in mitochondrial gene transcription after birth, as described by other scientists [31]–[33].…”

Section: Discussionmentioning

confidence: 89%

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Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Wirtz

Schuelke

2011

PLoS ONE

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Mutations in the nuclear encoded subunits of mitochondrial complex I (NADH:ubiquinone oxidoreductase) may cause circumscribed cerebral lesions ranging from degeneration of the striatal and brainstem gray matter (Leigh syndrome) to leukodystrophy. We hypothesized that such pattern of regional pathology might be due to local differences in the dependence on complex I function. Using in situ hybridization we investigated the relative expression of 33 nuclear encoded complex I subunits in different brain regions of the mouse at E11.5, E17.5, P1, P11, P28 and adult (12 weeks). With respect to timing and relative intensity of complex I gene expression we found a highly variant pattern in different regions during development. High average expression levels were detected in periods of intense neurogenesis. In cerebellar Purkinje and in hippocampal CA1/CA3 pyramidal neurons we found a second even higher peak during the period of synaptogenesis and maturation. The extraordinary dependence of these structures on complex I gene expression during synaptogenesis is in accord with our recent findings that gamma oscillations – known to be associated with higher cognitive functions of the mammalian brain – strongly depend on the complex I activity. However, with the exception of the mesencephalon, we detected only average complex I expression levels in the striatum and basal ganglia, which does not explain the exquisite vulnerability of these structures in mitochondrial disorders.

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Section: Discussionmentioning

confidence: 89%

“…In contrast to mRNA array analysis, ISH signals are notoriously difficult to quantify because numerous confounding factors might interfere [42]. On the other hand, ISH offers the advantage to assess gene expression at the cellular level.…”

Section: Discussionmentioning

confidence: 99%

Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Wirtz

Schuelke

2011

PLoS ONE

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“…We used rat testes for basic research to quantify ISH signals because spermatogenic cells in this organ undergo synchronized development and show stage-specific gene expression, allowing the differences in signal intensities among spermatogenic cell groups to be clearly demonstrated [14]. In that experimental model, we selected a well-preserved segment of rRNA [39] as the hybridizable RNA in paraffin sections and concluded that ISH rRNA staining and the posterization of the signal intensity were useful parameters for the quantitative analysis of mRNA; in other words, for the detection of the total transcriptional activity in the tissue sections.…”

Section: Discussionmentioning

confidence: 99%

“…In a previous paper, we described several problems that had been overcome in the quantification of mRNA in tissue sections [14]. These problems were 1) whether or not there was linearity between the probe concentration and the ISH signals, 2) whether or not the number of probe molecules per unit area or per cell cross-section accurately reflected the cellular mRNA content [17], 3) that there were differences in the accessibility of the probes to mRNA among the mRNAs, 4) that the variance in the degree of fixation reflected the degree of accessibility of probes among different tissue sections [5], and 5) that proteolytic permeabilization depended on the degree of fixation and was critical [16, 33], while different cell types often had different optima for permeabilization.…”

Section: Discussionmentioning

confidence: 99%

“…We therefore tried to estimate the amount of each transcript in certain cell types; however, such estimates tended to be subjective. Next, we performed basic research to quantify the ISH signals in rat testes [14], a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this experimental model, a well preserved segment of rRNA [39] was selected as the hybridizable RNA in paraffin sections.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

Kogami

Miki

Yamada

et al. 2006

Acta Histochem. Cytochem

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We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.

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Pre‐ and postnatal calorie restriction perturbs early hypothalamic neuropeptide and energy balance

Shin

Dai

Thamotharan

et al. 2012

J of Neuroscience Research

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Energy balance is regulated by circulating leptin concentrations and hypothalamic leptin receptor (ObRb) signaling via STAT3 but inhibited by SOCS3 & PTP1B. Leptin signaling enhances anorexigenic neuropeptides and receptor (POMC, MC3-R, MC4-R) activation while suppressing orexigenic neuropeptides (NPY, AgRP). We investigated in a sex-specific manner the early (PN2) and late (PN21) postnatal hypothalamic mechanisms in response to intrauterine (IUGR), postnatal (PNGR) and combined (IPGR) calorie and growth restriction. At PN2, while both male and female IUGR were hypoleptinemic, hypothalamic leptin signaling in females was activated as seen by enhanced STAT3. In addition, increased SOCS3 and PTP1B supported early initiation of leptin resistance in females that led to elevated AgRP but diminished MC3-R and MC4-R. In contrast, males demonstrated leptin sensitivity seen as a reduction in PTP1B and MC3-R and MC4-R with no effect on neuropeptide expression. At PN21 with adequate postnatal caloric intake, a sex-specific dichotomy in leptin concentrations was seen in IUGR with euleptinemia in males indicative of persisting leptin sensitivity and hyperleptinemia in females consistent with leptin resistance, both with normal hypothalamic ObRb signaling, neuropeptides and energy balance. In contrast, superimposition of PNGR upon IUGR (IPGR) led to diminished leptin concentrations with enhanced PTP1B and an imbalance in arcuate nuclear NPY/AgRP and POMC expression that favored exponential hyperphagia and diminished energy expenditure post-weaning. We conclude that IUGR results in sex-specific leptin resistance mainly observed in females while PNGR and IPGR abolish this sex-specificity setting the stage for acquiring obesity after weaning.

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Quantification of In Situ Hybridization Signals in Rat Testes

Cited by 8 publications

References 23 publications

Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

Pre‐ and postnatal calorie restriction perturbs early hypothalamic neuropeptide and energy balance

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