Masashi Shin

Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling.

To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the tissues first. However, the effects of decalcifying reagents on the integrity of DNA are largely unknown. In the present study, we evaluated the usefulness of various decalcifying reagents including 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), 5% formic acid, 5% HCl, 10% nitric acid, Plank–Rychlos solution, Morses solution and K-CX solution in TUNEL staining. Mouse maxilla was selected as the experimental system. Apoptotic cells naturally occurring in the epithelium were analysed. Tissues were assessed by soft X-ray imaging to confirm complete decalcification. The time required for decalcification of the tissue was 7 days with 10% EDTA and 2 days with other decalcifiers. Decalcified tissues were stained with Methyl/Green–Pyronine Y or 4′, 6-diamidino-2-phenylindole for assessment of DNA integrity. Nuclei of epithelial cells were strongly positive for both dyes after decalcification with 10% EDTA, 5% TCA, Morses solution and 5% formic acid. The other reagents failed to retain DNA. Our results demonstrated good TUNEL staining of the maxilla treated with 10% EDTA or 5% TCA . Based on the required time for processing and the signal-noise ratio, we recommend 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.

Fas‐Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation‐arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that “maturation arrest” is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.

Southwestern histochemistry as a molecular histochemical tool for analysis of expression of transcription factors: application to paraffin-embedded tissue sections.

 Southwestern histochemistry is used to localize transcription regulatory factors that bind to specific sequences of DNA and regulate the transcriptional activity of the genes, using a haptenized double-stranded DNA. This method has been developed in fresh frozen sections, but it was difficult to detect a specific signal in paraffin sections. In this review, we focus on the use of Southwestern histochemistry in paraffin sections. To detect a reliable signal in paraffin sections, we autoclaved or microwaved the sample before incubation with probe DNA. As a model system, we assessed estrogen receptor (ER) in paraffin sections of mouse ovary. ER was detected by Southwestern histochemistry in autoclaved or microwaved paraffin sections, and no signal was detected without such pretreatment. The ER staining pattern detected by Southwestern histochemistry was well consistent with that of ER α and ER β detected by immunohistochemistry. Next, we analyzed the expression of cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) in regenerating livers of rats and mice. CREB was detected in hepatocyte nuclei in autoclaved paraffin sections by Southwestern histochemistry. Immunohistochemistry of CREB displayed homogeneous staining in hepatocyte nuclei, while the signal detected by Southwestern histochemistry showed a heterogeneous speckled pattern in the nuclei. CREB expression increased prior to the induction of proliferating cell nuclear antigen (PCNA). Because the PCNA gene has a cAMP-responsive element in its promoter, it can be suggested that CREB may activate the PCNA gene and lead to the induction of hepatocyte DNA synthesis. Our results indicate that autoclave or microwave treatment is very useful for Southwestern histochemistry in paraffin-embedded sections.

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( n -GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of n -GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.

Enhanced expression of transcription factor E2F in Helicobacter pylori-infected gastric mucosa.

Objective. Helicobacter pylori is implicated in gastric carcinogenesis through increased gastric epithelial cell turnover. In fact, high proportions of proliferating and apoptotic epithelial cells are found in H. pylori‐infected gastric mucosa. E2F, a transcription factor, induces coordinated transactivation of a set of genes involved in cell cycle progression. The aim of this study was to investigate the expression of E2F in H. pylori‐infected gastric mucosa and examine the correlation between such expression and gastric epithelial cell proliferation and apoptosis.