dUDP Nick-end Labelling Assay Research Articles

Protective effects and mechanism of action of ruscogenin in a mouse model of ovalbumin-induced asthma

Objective Ruscogenin is a natural product exhibiting anti-inflammatory, antioxidant, and anti-apoptotic effects; however, its effectiveness for asthma management has not yet been reported. The aim of this study was to explore the role of ruscogenin in airway inflammation and apoptosis in asthma. Methods In vivo, female 6- to 8-week-old CL57 mice were sensitized to ovalbumin and challenged intranasally for 7 days. One group was gavaged with ruscogenin before ovalbumin challenge. At the end of the challenge period, airway hyperresponsiveness and airway inflammation were evaluated. Enzyme-linked immunosorbent assay was used to estimate the oxidative stress levels. A terminal deoxynucleotidyl transferase dUDP nick-end labeling assay was used to determine the extent of apoptosis. Immunohistochemistry and western blotting were performed to examine VDAC1 expression. In vitro, human bronchial epithelial (HBE) cells were treated with H2O2, ruscogenin, or disulfonate salt, and flow cytometry was used to calculate the apoptosis ratio and detect mitochondrial calcium levels Results In vivo, ruscogenin improved airway hyperresponsiveness and airway inflammation, while reducing oxidative stress, the apoptosis ratio and VDAC1 expression in asthmatic lungs. In vitro, ruscogenin attenuated apoptosis in HBE cells by decreasing the levels of VDAC1 expression and mitochondrial calcium. Conclusion Ruscogenin reduced oxidative stress and apoptosis in the airway epithelium by inhibiting VDAC1 expression and mitochondrial handling of calcium.

Advanced glycation end products present in the obese uterine environment compromise preimplantation embryo development

Research questionProinflammatory advanced glycation end products (AGE), highly elevated within the uterine cavity of obese women, compromise endometrial function. Do AGE also impact preimplantation embryo development and function? DesignMouse embryos were cultured in AGE equimolar to uterine fluid concentrations in lean (1–2 µmol/l) or obese (4–8 µmol/l) women. Differential nuclear staining identified cell allocation to inner cell mass (ICM) and trophectoderm (TE) (day 4 and 5 of culture). Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay (day 5). Day 4 embryos were placed on bovine serum albumin/fibronectin-coated plates and embryo outgrowth assessed 93 h later as a marker of implantation potential. AGE effects on cell lineage allocation were reassessed following pharmacological interventions: either 12.5 nmol/l AGE receptor (RAGE) antagonist; 0.1 nmol/l metformin; or combination of 10 µmol/l acetyl-l-carnitine, 10 µmol/l N-acetyl-l-cysteine, and 5 µmol/l alpha-lipoic acid. Results8 µmol/l AGE reduced: hatching rates (day 5, P < 0.01); total cell number (days 4, 5, P < 0.01); TE cell number (day 5, P < 0.01), and embryo outgrowth (P < 0.01). RAGE antagonism improved day 5 TE cell number. ConclusionsAGE equimolar with the obese uterine environment detrimentally impact preimplantation embryo development. In natural cycles, prolonged exposure to AGE may developmentally compromise embryos, whereas following assisted reproductive technology cycles, placement of a high-quality embryo into an adverse ‘high AGE’ environment may impede implantation success. The modest impact of short-term RAGE antagonism on improving embryo outcomes indicates preconception AGE reduction via pharmacological or dietary intervention may improve reproductive outcomes for overweight/obese women.

Survival and growth of human preantral follicles after cryopreservation of ovarian tissue, follicle isolation and short-term xenografting

In women, chemotherapy and radiotherapy can be harmful to the ovaries, causing loss of endocrine and reproductive functions. When gonadotoxic treatment cannot be delayed, ovarian tissue cryobanking is the only way of preserving fertility. This technique, however, is not advisable for patients with certain types of cancer, because of the risk of reintroducing malignant cells present in the cryopreserved tissue. Our objective is therefore to develop a transplantable artificial ovary. To this end, cryopreserved human preantral follicles were isolated and embedded in fibrin formulations prepared with 50 mg/ml fibrinogen and 10 IU/ml thrombin supplemented or not with 3% hyaluronic acid, and respectively xenografted to specially created right and left peritoneal pockets in eight nude mice. On days 0 and 7, the animals were killed and the matrices retrieved. On day 7, no difference was observed in the recovery rate of follicles embedded in fibrin alone (23.4%) or fibrin-hyaluronic acid (20.5%). Ki67 staining confirmed growth of the grafted follicles and terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay revealed 100% of the follicles to be viable in both groups on day 7. In conclusion, fibrin seems to be a promising material for creation of an artificial ovary, supporting follicle survival and development.

Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man

Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P < 0.001) and decrease in the pachytene spermatocytes (P < 0.001) were observed in the t(5;7;9;13) carrier compared with controls. It was further observed that 93% of octavalents were found partially asynapsed between homologous chromosomes. A significant decrease in the recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

Sohlh2 inhibits the apoptosis of mouse primordial follicle oocytes via C-kit/PI3K/Akt/Foxo3a signalling pathway

We previously reported that bone morphogenetic protein 4/drosophila mothers against decapentaplegic protein (BMP4/Smad) signalling pathway initiated primordial follicle growth and prevented oocyte apoptosis via up-regulation of Sohlh2 and receptor for kit ligand (c-kit). The mechanism underlying this process was not fully elucidated. In the present study, primary oocyte cultures were established from ovaries of 3-day-old female mouse pups by two-step enzyme digestion. Cultures were divided into Sohlh2 small interference RNA (SiRNA) group, negative SiRNA group, Sohlh2 overexpression plasmid group and pCAG-puro group. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay was carried out to detect the oocyte apoptosis; immunocytochemical staining and quantitative real time-polymerase chain reaction detected the expression of c-kit and Forkhead box O3a (Foxo3a); Western blot was performed to detect the expression of Sohlh2, C-kit, saerine/threonine kinases (Akt1) and Foxo3a. The results showed that Sohlh2 inhibited oocyte apoptosis and upregulated c-kit expression; Sohlh2 decreased the endonuclear Foxo3a via the upregulation of phosphorylated Akt1 (P-Akt1) and phosphorylated Foxo3a (P-Foxo3a) but not total Akt1 (T-Akt1) or total Foxo3a (T-Foxo3a); Sohlh2 increased P-Akt1 but not T-Akt1; the PI3K (phosphotidylinsitol-3-kinase) inhibitor LY294002 ameliorated the role of Sohlh2 on phosphorylation of Akt1 and Foxo3a. Sohlh2 may inhibit oocyte apoptosis via c-kit/PI3K/Akt/Foxo3a signalling pathway.

Gonadotrophin-releasing hormone antagonist induces apoptosis in human decidual stromal cells: effect on GADD45 and MAPK signaling

The impact of gonadotrophin-releasing hormone (GnRH) antagonists used in IVF protocols on endometrial tissue remodeling, embryo implantation and the programming of early pregnancy is still unclear. Pregnancy and infant outcomes after treatment with GnRH antagonist for IVF are particular causes of concern. The purpose of this study was to investigate the mechanisms of GnRH antagonist-induced apoptosis of human decidual stromal cells and the effects of GnRH antagonist on the activation of ERK1/2, JNK and GADD45α signaling. Human decidual stromal cells were isolated from decidual tissue. The expression of GnRH-I receptor (GnRH-IR) was examined by immunoblot analysis and immunohistochemistry. The cells were treated with the GnRH antagonist, Cetrorelix. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay was used to examine cell viability. Cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay were used as indicators for cell apoptosis. Mitogen-activated protein kinase function was tested for the elucidation of intracellular signalings through the pre-treatment of stromal cells with ERK1/2 (U0126) and JNK (SP600125) inhibitors prior to the Cetrorelix treatment. To characterize the signaling pathway of GnRH antagonist, the endogenous GnRH-IR and GADD45α were knocked down by specific small-interfering RNA (siRNA). The GnRH-IR is expressed in human decidual stromal cells. Treatment with GnRH antagonist decreased cell viability, induced apoptosis and increased the phosphorylation of ERK1/2 and JNK. Cells pre-treated with U0126 and SP600125 were rescued from the GnRH antagonist-mediated inhibition of cell growth and did not exhibit GnRH antagonist-induced apoptosis and downstream GADD45α signaling. GnRH antagonist-mediated cell growth inhibition and apoptosis were also abolished by the knockdown of the endogenous GnRH-IR or GADD45α with siRNAs. The GnRH antagonist suppresses the growth of decidual stromal cells by inducing apoptosis through the GnRH-IR and through the ERK1/2 and JNK phosphorylation-dependent induction of GADD45α signaling. These results indicate that ERK1/2, JNK and GADD45α are coordinately regulated by the GnRH antagonist through the GnRH-IR to induce apoptosis in human decidual stromal cells, suggesting that the GnRH antagonist may play a role in decidual programming in human pregnancy.

Impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes

A prospective study was performed to assess the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. The study population included 178 couples (62 cycles of IVF, 116 of intracytoplasmic sperm injection (ICSI)) with own ( n = 77) and donor ( n = 101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Correlation between DNA damage to oocyte fertilization, embryo quality and clinical pregnancy, implantation and miscarriage rates was evaluated. DNA fragmentation was not related to fertilization rates in either IVF ( r = 0.08) or ICSI ( r = −0.04) cycles. DNA fragmentation was similar in patients with <50% embryo utilization rate compared with ⩾50%, in cancelled and in embryo transfer cycles and in miscarriages and in successful deliveries. Moreover, DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the multivariable analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation threshold, results did not vary. It is concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. The study of sperm DNA damage is especially relevant in an era in which advanced forms of assisted reproductive technologies are frequently used and in which barriers to natural selection are bypassed. A prospective study was carried out with the aim of assessing the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. We studied 178 couples undergoing 62 cycles of IVF and 116 of intracytoplasmic sperm injection (ICSI) with own ( n = 77) and donor ( n = 101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Oocyte fertilization, embryo quality and pregnancy, implantation and miscarriage rates were correlated with DNA damage. DNA fragmentation was not related to fertilization rates in either IVF ( r = 0.08) or ICSI ( r = −0.04) cycles. DNA fragmentation was higher in patients with <50% of good-quality embryos than with ⩾50%, in cancelled than in embryo transfer cycles and in miscarriages than in successful deliveries. DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the logistic regression analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation cut-off point, results did not vary. According to these findings, it can be concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes.

Relationship between DNA damage and sperm head birefringence

Birefringence or double refraction is the decomposition of a ray of light into two rays when it passes through an anisotropic material such as quartz. Sperm cells have been demonstrated to be optically anisotropic. The objective of this study was to evaluate the relationship between the pattern of human sperm head birefringence (SHBF) and DNA damage. A total of 26 patients with normal semen were included. DNA damage (fragmentation and denaturation) was evaluated in the sperm head in the context of birefringence, both total (SHBF-T) and partial (SHBF-P), by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUDP nick-end labelling assay and acridine orange fluorescence, respectively. Positive DNA fragmentation in spermatozoa with SHBF-T (205/1053; 19.5%) was significantly higher ( P < 0.0001) than in spermatozoa that presented SHBF-P (60/820; 7.3%). However, the percentage of denatured DNA in spermatozoa with SHBF-T (824/1256; 65.6%) was not significantly different from the ones with SHBF-P (666/1009; 66.0%). In conclusion, the data support a positive relationship between spermatozoa with total SHBF in their head and increased DNA fragmentation.

Quality assessment of induced spermatogenesis in hypogonadotrophic hypogonadic men treated with gonadotrophins

Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenesis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared with sperm samples from normospermic control donors. Sperm cells were analysed by fluorescence in-situ hybridization, using probes specific for chromosomes 13, 21, 18, X and Y, and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients. Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenensis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve a paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared to sperm samples from normospermic control donors. Sperm cells were analysed by FISH, using probes specific for chromosomes 13, 21, 18, X and Y, and by TUNEL assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients.

Pyrrolidine dithiocarbamate inhibits nuclear factor- B pathway activation, and regulates adhesion, migration, invasion and apoptosis of endometriotic stromal cells

The activation of nuclear factor-κB (NF-κB) has been implicated in the development and progression of endometriosis. The aim of this study is to investigate the potential application of pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, in the treatment of endometriosis. NF-κB-DNA-binding activity, IκB phosphorylation and expression of nuclear p65 protein in endometriotic ectopic stromal cells (EcSCs), endometriotic eutopic stromal cells (EuSCs) and normal endometrial stromal cells (NESCs) were detected by electrophoretic mobility shift assay and western blot analysis. Adhesion, migration, invasion and apoptosis of EcSCs were observed by means of adhesion, migration, invasion and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay, respectively. Gene and protein expressions of CD44s, matrix metalloproteinase (MMP)-2, MMP-9 and survivin in EcSCs were measured by RT-PCR and western blot analysis. The results showed that PDTC in the absence or presence of interleukin (IL)-1β showed stronger inhibitory effects on NF-κB-DNA-binding activity, IκB phosphorylation and expression of nuclear p65 protein in EcSCs than those in EuSCs or NESCs. PDTC enhanced apoptosis, and suppressed IL-1β-induced cellular adhesion, migration and invasion of EcSCs. Pretreatment of EcSCs with PDTC attenuated IL-1β-induced expressions of CD44s, MMP-2, MMP-9 and survivin at gene and protein levels. All these findings suggest that PDTC induces apoptosis and down-regulates adhesion, migration and invasion of EcSCs through the suppression of various molecules. Therefore, PDTC could be used as a therapeutic agent for the treatment of endometriosis.

Polymorphisms of nucleotide-excision repair genes may contribute to sperm DNA fragmentation and male infertility

The nucleotide-excision repair (NER) system is crucial for the removal of bulky DNA adducts during spermatogenesis. Dysfunction of its repair capacity is likely related to the increased susceptibility to DNA damage. In this study, four polymorphisms in NER pathway (XPA(-4) G/A, ERCC1 C8092A, XPD Lys751Gln and XPF Ser835Ser) were selected to evaluate their potential impact on sperm DNA damage and male infertility. Genotypes were determined by PCR-restriction fragment length polymorphism. Sperm DNA damage was evaluated by TdT-mediated dUDP nick-end labelling assay. A case-only study of 620 infertile men found a significant association between XPA(-4) G/A polymorphism and sperm DNA damage. Individuals with the XPA(-4) A allele showed more sperm DNA damage and lower sperm concentration than G allele carriers. Further analysis, including 620 patients and 385 controls, revealed a 1.52-fold risk (95% CI 1.08-2.02) of developing male infertility in the XPA(-4) AA carriers compared with noncarriers. Luciferase assay verified that the promoter with the XPA(-4) A allele had a lower transcriptional activity than that with the G allele. These data provide the first evidence that -4 G/A polymorphism in XPA promoter alters its transcriptional activity and, thus, might contribute to sperm DNA damage and male infertility. Sperm DNA integrity is essential for the accurate transmission of genetic information. To our knowledge, few studies have elucidated the effect of DNA repair gene single-nucleotide polymorphisms on sperm DNA integrity, although the DNA repair system is indispensable in maintaining genetic stability and normal spermatogenesis. In this original study, we evaluated the potential impact of the polymorphisms in the nucleotide-excision repair pathway on the risk of sperm DNA damage based on 620 infertile patients and 385 controls, and provided the first evidence that -4 G/A polymorphism in the promoter for the xeroderma pigmentosum group A gene altered its transcriptional activity, which might contribute to sperm DNA damage and male infertility.

Evaluation of human sperm function after being cryopreserved within the zona pellucida

To investigate the fertilization ability, chromatin structure, and DNA integrity of spermatozoa after being cryopreserved within an empty zona pellucida (ZP). Prospective study. Reproductive research center in a university-affiliated hospital. Normozoospermic patients. Spermatozoa were cryopreserved within the ZP or with traditional methods. The sperm recovery rate, sperm motility, fertilization ability, chromatin structure, and DNA integrity were assessed before and after cryopreservation. Significantly higher sperm recovery rate was identified for the spermatozoa cryopreserved within the ZP than those cryopreserved with traditional methods, but the motility recovery was similar. Frozen-thawed samples showed increased damage to the sperm chromatin and DNA, which were assessed by acridine orange test (AO) and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay; however, no difference of chromatin and DNA integrity was observed for spermatozoa cryopreserved within the ZP or with traditional methods. In addition, the fertilization ability, as assessed by injecting spermatozoa into hamster oocytes, was similar for spermatozoa cryopreserved with different cryopreservation methods. Cryopreservation of spermatozoa within an empty ZP results in higher sperm recovery rate, and the post-thaw sperm functions of spermatozoa cryopreserved within the ZP are comparable with spermatozoa cryopreserved with traditional methods.

Increased apoptosis and decreased proliferation of colorectal cancer cells using insulin‐like growth factor binding protein‐4 gene delivered locally by gene transfer

Insulin-like growth factor (IGF)-I induces proliferation of transformed cells. Its binding proteins (IGFBP) are involved in local regulation of IGF. This study assessed the effects of overexpression of IGFBP-4 on the development of cancer in vivo. Nude mice were subcutaneously inoculated with HT-29 colorectal cancer cells (3 x 10(6)). When the tumour became visible (1 week after inoculation), animals received either 150 microg of mammalian expression vector containing IGFBP-4 cDNA or vector alone (n = 6 each) by peritumoural injection. Tumour size was measured during the growth. After 3 weeks of IGFBP-4 induction, animals were killed and tumour tissue samples were collected for examining the level of IGFBP-4 expression. Tumour mitotic activities were determined by counting numbers of mitotic cells on the tissue section. Apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUDP nick end labelling assay. Following IGFBP-4 treatment, tumour showed large necrotic areas, significantly increased numbers of apoptotic cells (36.67 +/- 7.36 vs 7.07 +/- 1.91, P < 0.01 vs control), decreased cells undergoing mitosis (2.31 +/- 0.32 vs 3.61 +/- 0.27, P < 0.01 vs control) and higher expression of IGFBP-4 (P < 0.05 vs control). IGFBP-4 gene transfer increased apoptosis and decreased mitosis, but tumour volume was not significantly altered possibly due to cellular debris filling the centre of tumours.

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Triiodothyronine is a survival factor for developing oligodendrocytes

Thyroid hormone plays an important role in oligodendrocyte development. The studies presented here suggest that thyroid hormone is required for oligodendrocyte survival during development. Oligodendrocyte precursor cells, astrocytes and microglia were cultured in a defined media. Oligodendrocyte precursor cell differentiation was induced by growth factor removal. Time course studies revealed that oligodendrocytes cultured in the presence or absence of triiodothyronine (T3) develop similarly during the first 3 days of development. Oligodendrocytes cultured in the absence of T3, however, die after developmental day 3. TdT-Mediated dUDP Nick End Labeling assay and Hoechst staining indicate that T3 rescues developing oligodendrocytes from death by apoptosis. Apoptosis is likely induced by the presence of the cytokines TNFα and IL-1β. However, expression of these cytokines is not altered by thyroid hormone administration. Thus, thyroid hormone has been demonstrated to effect proliferation, myelin gene expression and now the survival of developing oligodendrocytes.

Molecular Response to Ischemia-Reperfusion of Rat Skin: Study of Expression of p53, p21WAF-1, and Bax Proteins, and Apoptosis

The authors investigated the expression of p53, p21WAF-1, and Bax proteins, and apoptosis to elucidate the cellular response to ischemia-reperfusion of the skin. The rat left lower limb was dissected at the inguinal region retaining the bone and femoral vessels, and the vessels were clamped to produce an ischemic condition. After 6 hours the clamps were removed, and the plantar skin was resected at various times up to 72 hours after reperfusion. Five skin specimens were obtained at each time point from 5 rats. When a rat died during the study, additional rats were used until five specimens could be obtained from 5 rats at each time point. The expression of the three proteins was detected by Western blot analysis. The apoptotic cells were detected using the terminal deoxytransferase-mediated dUDP nick-end labeling assay. After reperfusion, the levels of p53 and p21WAF-1 were significantly higher in the ischemia-reperfusion rats compared with the sham-operated rats. However, the levels of Bax protein did not show a noticeable increase at any period. The apoptotic cells in both the epidermis and dermis were not evident compared with the sham skin, which were similar to those in the nontreated, normal skin. These results demonstrate that p53 and p21WAF-1 proteins accumulate after 6 hours of ischemia of the skin during reperfusion. Moreover, it is speculated that accumulation of these proteins plays an important role in the survival of the skin by inducing growth arrest of the cells, but not apoptosis.

Differential effects of repeated ovarian stimulation on cytoplasmic and spindle organization in metaphase II mouse oocytes matured in vivo and in vitro.

The effects of four rounds of ovarian stimulation spaced 1-6 weeks apart on the normality of metaphase II (MII) spindle formation, chromosomal alignment and cytoplasmic organization were examined in intact ovulated mouse oocytes and at MII for oocytes obtained at the germinal vesicle stage from the same ovaries and matured in vitro. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay was used to identify DNA strand breaks in chromosomes, and histological studies of ovaries between and at each round of ovarian stimulation were performed. The results demonstrate a progressive and significant increase in the frequency of spindle defects with each round of ovarian stimulation, including those spaced weeks apart. Oocytes with spindle defects were also characterized by the occurrence of detached chromosomes and cytoplasmic asters. In contrast, in-vitro matured oocytes derived from the same ovaries were normal. No evidence of DNA strand breaks with repeated rounds of ovarian stimulation was detected in ovulated or in-vitro matured oocytes. The development and persistence of nodules of hypertrophied granulosa in regions where follicular growth occurs suggest that a progressively increasing proportion of oocytes in the ovulatory pathway may experience an intrafollicular milieu that has negative consequences for competence. The results are discussed with respect to ovarian and oocyte biological ageing and possible adverse implications for human oocyte competence with repeated hyperstimulation.