Transferase-mediated dUTP-digoxigenin Nick End-labelling Staining Research Articles

Toxicological effects of 3-methyl-4-nitrophenol on mouse ovarian and testicular cell proliferation, apoptosis and oocyte maturation

The aim of the present study was to reveal the effects of 3-methyl-4-nitrophenol (PNMC) on mouse gonadal cell proliferation and apoptosis. Immature female and male mice were intramuscularly injected with 100 mg/kg PNMC or vehicle every five days. One-month later, ovarian rather than testicular weights were significantly decreased. The positive terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling staining was enhanced in both testicular seminiferous epithelium and ovarian corpus luteum. Further study in ovaries showed that PNMC treatment increased the protein expressions of PCNA, p21 and p27, and decreased the expression of Cyclin D2. Whereas PNMC had no significantly influence on those protein expressions in testes. In addition, we demonstrated that 100 nM PNMC significantly suppressed mouse oocyte meiotic resumption and cumulus cell expansion in vitro. These findings suggest that the reproductive toxicities of PNMC involve affecting the processes of gonadal cell apoptosis and proliferation.

Proliferation and apoptosis processes in the seasonal testicular development of the wild Daurian ground squirrel (Citellus dauricus Brandt, 1844).

The aim of the present study was to elucidate the regulatory role of cell proliferation and apoptosis in testicular development of wild Daurian ground squirrels during the breeding season (April), the non-breeding season (June) and before hibernation (September). Gross mass and hormonal analysis showed that the testis:body mass ratio and plasma testosterone concentration fluctuated seasonally, with a peak in April and lowest values in June. Similarly, spermatogenesis was fully developed in April but suppressed in June and September. Testicular decellularisation and vacuolisation was seen during the transition from the breeding to the non-breeding season. Furthermore, testicular levels of proliferating cell nuclear antigen, cyclin D2 and caspase-3 protein were significantly increased in June and September. Intriguingly, positive terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling staining and nuclear translocation of caspase-3 in testicular germ cells appeared only during the prehibernation period, whereas accumulation of cyclin D2 in spermatocyte nuclei occurred in September. These findings demonstrate, for the first time, that both cell proliferation and apoptosis are stimulated during the prehibernation period, indicating that a hormonal-regulated balance of testicular germ cell proliferation and apoptosis may play a pivotal role in preparing for testicular recrudescence of wild Daurian ground squirrels.

Downregulation of survival signalling pathways and increased apoptosis in the transition of pressure overload‐induced cardiac hypertrophy to heart failure

1. Transition from compensated left ventricular (LV) hypertrophy to decompensated heart failure was characterized using a pressure-overload induced model to elucidate the temporal relationship between cardiomyocyte apoptosis and survival signalling in this transition. 2. Mice were subjected to transverse aortic constriction (TAC) or sham operation for 1-16 weeks and were studied by echocardiography, catheterization and histology. Relevant gene expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, Akt and glycogen synthase kinase (GSK)-3beta were determined. 3. Transverse aortic constriction resulted in myocyte hypertrophy and fibrosis from Week 4 and a progressive increase in left ventricular (LV) dimensions and wall thicknesses with maintained contractile function by Week 12. However, a sharp decline in contractile function and elevated LV end-diastolic pressure from 12 to 16 weeks were observed after TAC, indicating functional decompensation. 4. Following TAC, mRNA levels of atrial natriuretic peptide, B-type natriuretic peptide, beta-myosin heavy chain (MHC) and transforming growth factor-beta1 were increased time dependently, whereas mRNA expression of alpha-MHC, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a and Bcl-2 were decreased. The ratio of Bcl-2/Bax was decreased and this was consistent with progressively increased myocyte apoptosis demonstrated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling staining. Phosphorylation of ERK1/2 was increased by Week 4, but decreased thereafter. Levels of phosphorylated Akt declined from Week 8, whereas GSK3beta phosphorylation increased from 1 to 8 weeks, then decreased from Week 12 after TAC. 5. In conclusion, TAC resulted in early concentric and late eccentric hypertrophy with eventual development of LV dysfunction. This transition was temporally associated with a progressive increase in cell size, fibrosis and myocyte apoptosis. Downregulation of ERK1/2, Akt and GSK3beta and enhanced cardiomyocyte apoptosis are implicated as important mechanisms in the transition from compensated hypertrophy to heart failure.

Transglutaminase Participates in UVB-Induced Cell Death Pathways in Human Corneal Epithelial Cells

Ultraviolet light (UVB) is known to cause apoptosis in human corneal epithelial cells. This study evaluates the role of transglutaminase in regulating tumor necrosis factor (TNF) receptor clustering as well as caspase activation in UVB-induced apoptosis in human corneal epithelial cells. A human corneal epithelial cell line was used. A single dose of UVB (20 mJ/cm2) was used as a stimulus. Cell viability and cell death were investigated by MTT, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL), and caspase-3 assays. Immunofluorescent staining was used to investigate TNF receptor-I clustering at various time intervals after UVB. Short interfering RNA was used to knock down transglutaminase-2 expression. Fluorescein-cadaverine uptake was used to assess transglutaminase activity. A noncovalent peptide delivery system was used to transfect guinea pig liver transglutaminase into corneal epithelial cells. UVB increased transglutaminase activity, reduced cell viability, and increased TUNEL staining. UVB or TNF-alpha promoted TNF-receptor-I clustering, a process inhibited by the transglutaminase inhibitor, mono-dansyl cadaverine. UVB also increased activated caspase-3, in a manner suppressible by mono-dansyl cadaverine. Intracellular delivery of exogenous transglutaminase markedly increase caspase-3 activation compared with the vehicle control. Transglutaminase enzymatic activity is involved in corneal epithelial cell death after UVB and appears to participate in two steps regulating this process, clustering of TNF receptor-I and caspase-3 activation.

Hypoxia–reoxygenation‐induced apoptosis in cultured neonatal rat cardiomyocyets and the protective effect of prostaglandin E1

The aim of the present study was to investigate the effect of prostaglandin (PG) E1 on hypoxia/re-oxygenation (H/R) apoptosis and the expression of bcl-2 and bax in cultured neonatal rat cardiomyocytes. The H/R model was made using the first generation of cultured neonatal rat cardiomyocytes. Hypoxia/re-oxygenation apoptosis was studied by electron microscopy and agarose gel electrophoresis. The percentage of apoptotic cells was measured by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL). The expression of bcl-2 and bax was detected by in situ hybridization and immunohistochemical staining. Most cells of the H/R group tested by electron microscopy showed cytoplasmic concentration, nuclear chromatin condensation and margination. Prostaglandin E1 (5, 15 and 45 microg/L) relieved the injury. The results of DNA electrophoresis in the H/R group showed a typical DNA ladder and the DNA ladder decreased gradually corresponding with increasing doses of PGE1. The TUNEL staining showed that the total number of apoptotic cells in the H/R group was much more than that in the PGE1 (45 microg/L) group. The results of in situ hybridization and immunohistochemical staining showed that the bcl-2 content in the H/R group was lower than that in the control group; bax content showed the reverse. Compared with the H/R group, bcl-2 content was significantly higher in the PGE1 (5, 15 and 45 microg/L) groups. However, bax content in the PGE1 (5, 15 and 45 microg/L) groups was significantly lower than that in the H/R group. 6. In conclusion, H/R injury can induce cardiomyocyte apoptosis. Prostaglandin E1 obviously has anti-apoptotic effects on cardiomyocytes and the mechanisms probably involve the inhibition of bax expression and increased expression of bcl-2.

Trichosanthin‐induced apoptosis in gastric cancer is related to the downexpression of bcl‐2

OBJECTIVE: To study: (i) the induction of apoptosis in gastric cancer cells by trichosanthin; and (ii) the relationship between apoptosis and the expression of bcl‐2. METHODS: During in vitro experiments, morphological studies and the terminal deoxynucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labeling (TUNEL) method were used to detect apoptosis in gastric adenocarcinoma cell line SGC‐7901 before and after trichosanthin treatment. An immunohistochemical staining method and northern blot hybridization were used to detect the expression of the apoptosis‐related gene bcl‐2 before and after trichosanthin treatment. RESULTS: When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 36 h), they presented some typical apoptotic morphological changes that were observed by fluorescent staining. These morphological changes included nuclear condensation and nucleosomal fragments forming a lunate body under the nuclear membrane. When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL) for 36, 42 or 48 h, TUNEL staining revealed a significant increase in the apoptotic index (AI), from 3.78 ± 1.11%, 3.98 ± 1.12% and 3.85 ± 1.08%, to 11.30 ± 2.33%, 10.22 ± 2.00% and 11.18 ± 1.85% (P < 0.01), respectively. When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 32 h), immunohistochemical staining revealed a decreased expression of the bcl‐2 protein product: the staining density decreased from ++/+++ to –/+ (P < 0.01). When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 24 h), northern blot hybridization showed a decreased expression of bcl‐2 RNA: hybridization decreased from 35.19 ± 2.34 to 22.27 ± 3.90 (P < 0.01). CONCLUSIONS: Trichosanthin is able to induce apoptosis in gastric cancer. The apoptosis may be mediated by the downexpression of the apoptosis‐related gene bcl‐2.

Silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice.

To examine whether induction of apoptosis plays a role in the pathogenesis of street rabies, we compared the distribution of viral antigens, histopathology, and the induction of apoptosis in the brain of mice infected with a street rabies virus (silver-haired bat rabies virus, SHBRV) and with a mouse-adapted laboratory rabies virus strain (challenge virus standard, CVS-24). Inflammation was identified in the meninges, but not in the parenchyma of the brain of mice infected with either CVS-24 or SHBRV. Necrosis was present in numerous cortical, hippocampal, and Purkinje neurons in CVS-24-infected mice, but only minimal necrosis was identified in mice infected with SHBRV. Likewise, extensive terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining was observed in the brain of mice infected with CVS-24 but little or none in the brain of mice infected with SHBRV. Rabies virus antigens were distributed similarly in the CNS infected with either virus. However, the expression of the glycoprotein (G) is more widespread and the staining of G is generally stronger in CVS- than SHBRV-infected mice, whereas the expression of rabies virus nucleoprotein (N) is similar in mice infected with either CVS or SHBRV. The positive TUNEL staining thus correlates with the high level of G expression in CVS-infected mouse brain. Northern blot hybridization revealed that the ratio between the N and G transcripts is similar in brains infected with either virus, indicating that the reduced expression of G protein is not caused by reduced transcription in SHBRV-infected animals. Taken together, these observations suggest that apoptosis is not an essential pathogenic mechanism for the outcome of a street rabies virus infection and that other pathologic processes may contribute to the profound neuronal dysfunction characteristic of street rabies.

Apoptotic cell death is an important cause of neuronal injury in experimental Venezuelan equine encephalitis virus infection of mice.

Mice develop a fatal encephalomyelitis after infection with the Trinidad donkey strain of Venezuelan equine encephalitis (VEE) virus. Adult mice were inoculated intraperitoneally with VEE virus and the brains were examined at different time points. Morphological changes were assessed by histological staining. VEE virus antigen was detected with immunoperoxidase staining, and DNA fragmentation was evaluated in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. VEE antigen was found in many areas of the brain and it was prominent in neurons. There were mild associated inflammatory changes. DNA fragmentation was demonstrated in many of these areas using TUNEL. In areas with TUNEL staining, morphological neuronal changes ranged from nuclear chromatin condensations to nuclear and cellular fragmentation, which are characteristic of apoptosis. There is strong morphological and biochemical evidence of apoptotic cell death in this experimental model of VEE virus infection.