Deoxynucleotide Transferase-mediated dUTP Nick-end Labeling Research Articles

Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) invitro. Experimental basic science study. Reproductive biology laboratory. ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. Invitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70by means of immunofluorescent assay. The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation invitro. The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.

Ulinastatin Alleviates Neurological Deficiencies Evoked by Transient Cerebral Ischemia via Improving Autophagy, Nrf-2-ARE and Apoptosis Signals in Hippocampus

Ulinastatin [or called as urinary trypsin inhibitor (UTI)] plays a role in regulating neurological deficits evoked by transient cerebral ischemia. However, the underlying mechanisms still need to be determined. The present study was to examine the effects of UTI on autophagy, Nrf2-ARE and apoptosis signal pathway in the hippocampus in the process of neurological functions after cerebral ischemia using a rat model of cardiac arrest (CA). CA was induced by asphyxia followed by cardiopulmonary resuscitation (CPR) in rats. Western blot analysis was employed to determine the expression of representative autophagy (namely, Atg5, LC3, Beclin 1), p62 protein (a maker of autophagic flux), and Nrf2-ARE pathways. Neuronal apoptosis was assessed by determining expression levels of Caspase-3 and Caspase-9, and by examining terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). The modified neurological severity score (mNSS) and spatial working memory performance were used to assess neurological deficiencies in CA rats. Our results show that CA amplified autophagy and apoptotic Caspase-3/Caspase-9, and downregulated Nrf2-ARE pathway in the hippocampus CA1 region. Systemic administration of UTI attenuated autophagy and apoptosis, and largely restored Nrf2-ARE signal pathway following cerebral ischemia and thereby alleviated neurological deficits with increasing survival of CA rats. Our data suggest that UTI improves the worsened protein expression of autophagy and apoptosis, and restores Nrf2-ARE signals in the hippocampus and this is linked to inhibition of neurological deficiencies in transient cerebral ischemia. UTI plays a beneficial role in modulating neurological deficits induced by transient cerebral ischemia via central autophagy, apoptosis and Nrf2-ARE mechanisms.

Alpha-lipoic acid reduces retinal cell death in diabetic mice

Oxidative stress plays an important role in the development of diabetic retinopathy. Here, we examined whether α-lipoic acid (α-LA), a natural antioxidant, attenuated retinal injury in diabetic mice. The α-LA was orally administered to control mice or mice with streptozotocin-induced diabetes. We found that α-LA reduced oxidative stress, decreased and increased retinal 4-hydroxy-2-nonenal and glutathione peroxidase, respectively, and inhibited retinal cell death. Concomitantly, α-LA reversed the decreased activation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and increased the levels of peroxisome proliferator-activated receptor delta and sirtuin3 in diabetic mouse retinas, similar to results shown after metformin treatment of retinal pigment epithelial cells (RPE) exposed to high glucose. Moreover, α-LA lowered the levels of O-linked β-N-acetylglucosamine transferase (OGT) and thioredoxin-interacting protein (TXNIP) in diabetic retinas that were more pronounced after metformin treatment of RPE cells. Importantly, α-LA lowered interactions between AMPK and OGT as shown by co-immunoprecipitation analyses, and this was accompanied by less cell death as measured by double immunofluorescence staining by terminal deoxynucleotide transferase-mediated dUTP nick-end labelling and OGT or TXNIP in retinal ganglion cells. Consistently, α-LA lowered the levels of cleaved poly(ADP-ribose) polymerase and pro-apoptotic marker cleaved caspase-3 in diabetic retinas. Our results indicated that α-LA reduced retinal cell death partly through AMPK activation or OGT inhibition in diabetic mice.

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis-inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber.

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. A characteristic feature of aestivation in this species is the degeneration of the intestine. In the current study, we hypothesized that energy conservation and cytoprotective strategies need to be coordinated in the intestine to ensure long-term survival during aestivation, and there was potential relationship between heat shock protein 70 (HSP70) and apoptosis-inducing factor mitochondrial 1 (AIFM1) during extreme environmental stress. AIFM1 is a bifunctional flavoprotein that is involved in the caspase-independent activation of apoptosis. The gene and protein expression profiles of AjAIFM1 and AjHSP70 in intestinal tissue during aestivation were analyzed and results showed an inverse correlation between them, AjAIFM1 being suppressed during aestivation whereas AjHSP70 was strongly upregulated. Comparable responses were also seen when intestinal cells were isolated and analyzed in vitro for responses to heat stress at 25°C (a water temperature typical during aestivation), compared with 15°C control cells. Combined with co-immunoprecipitation studies in vivo and in vitro, our results suggested that AjHSP70 protein may have potential interaction with AjAIFM1. To determine the influence of heat stress on apoptotic rate of intestinal cells, we also assessed the DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and results also supported a potential antiapoptotic response in sea cucumber during heat stress. This type of cytoprotective mechanism could be used to preserve the existing cellular components during long-term aestivation in sea cucumber.

The cardioprotective effect of interleukin-11 against ischemia-reperfusion injury in a heart donor model.

Previously, we have demonstrated the cardioprotective effect of interleukin (IL)-11 in animal models of acute coronary syndrome. In this study, we sought to evaluate its cardioprotective potential during prolonged hypothermic global ischemia and subsequent reperfusion using a rat heart donor model. IL-11 was administered intravenously 10 minutes before harvesting the rat heart. The hearts were preserved in cold (4 °C) Krebs-Henseleit buffer for 6 hours, and then attached to a Langendorff perfusion apparatus and reperfused with an oxygenated Krebs-Henseleit solution containing IL-11. Normal saline was used instead of IL-11 in the control group. Functional recovery of the reperfused heart was observed by using a left ventricular balloon. Myocardial cell injury was quantified by measuring the biomarkers collected from the coronary effluent. Apoptotic cells were identified and counted using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining technique. IL-11 administration improved myocardial function after 6 hours of cold ischemia. Although there were no significant differences in any of the baseline-measured values between the two groups, left ventricular developed pressure (LVDP) and changes in left ventricular pressures (dP/dt) were significantly higher in the IL-11 group at 120-minute reperfusion. The number of TUNEL-labeled cardiomyocytes was also significantly smaller in the IL-11 group. The administration of IL-11 showed a significant recovery of cardiac contractile function after 6 hours of cold ischemia. Our data suggest that it may have significant therapeutic potential for maintaining the functional viability of the heart exposed to prolonged hypothermic global ischemia.

Metformin protects against retinal cell death in diabetic mice

Retinal degeneration is an early feature of diabetic retinopathy, the major cause of blindness in the developed world. Here we investigated how the widely used antidiabetic drug metformin reduces retinal injury in diabetic mice. Metformin was orally administered to control mice or mice with streptozotocin-induced diabetes. Western blot analysis showed that levels of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and other related proteins such as carbohydrate-responsive element-binding protein (ChREBP) and thioredoxin-interacting protein (TXNIP) were significantly increased, and nuclear factor kappaB (NF-κB) and poly (ADP-ribose) polymerase (PARP) were activated in the diabetic retinas or retinal pigment epithelial (RPE) cells exposed to high glucose compared to controls. More importantly, RPE cells exposed to high glucose and treated with thiamet-G had higher levels of those proteins, demonstrating the role of elevated O-GlcNAcylation. Double immunofluorescence analysis revealed increased co-localization of terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL)-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB in diabetic retinas compared to control retinas. Co-immunoprecipitation analysis showed that interaction between OGT and ChREBP or NF-κB was increased in diabetic retinas compared to control retinas, and this was accompanied by more cell death. Notably, metformin attenuated the increases in protein levels; reduced co-localization of TUNEL-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB; and reduced interaction between OGT and ChREBP or NF-κB. Our results indicate that OGT inhibition might be one of the mechanisms by which metformin decreases retinal cell death.

Nafamostat Mesilate Attenuates Ischemia-Reperfusion-Induced Renal Injury.

It has been reported that nafamostat mesilate (NM) inhibits inflammatory injury via inhibition of complement activation in ischemic heart, liver, and intestine. However, it is unclear if NM also inhibits apoptosis in ischemia-reperfusion (IR)-injured kidney. We therefore investigated whether NM attenuates IR renal injury that involves inhibition of apoptosis. HK-2 cells and male C57BL/6 mice were used for this study. C57Bl/6 mice were divided into 4 groups: sham, NM (2mg/kg)+ sham, IR injury (IR injury; reperfusion 27 minutes after clamping of both the renal artery and vein), and NM+ IR injury. Kidneys were harvested 24 hours after IR injury, and functional and molecular parameters were evaluated. For invitro studies, HK-2 cells were incubated for 6 hours with mineral paraffin oil to induce hypoxic injury, and then treated with various doses of NM to evaluate the antiapoptotic effects. Blood urea nitrogen, serum creatinine levels, and renal tissue injury scores in NM+ IR-injured mice were significantly lower than those of control IR mice (all P< .01). NM significantly improved cell survival in hypoxic HK-2 cells (P< .01), significantly decreased renal Bax expression (P< .05), and increased renal Bcl-2 protein levels in IR kidneys and hypoxic HK-2 cells compared with those of the sham and control groups. The numbers of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling- and 8-oxo-2'-deoxyguanosine-positive cells were significantly lower in NM+ IR-injured kidneys compared with those in control IR-injured mice (P< .05); NM treatment decreased the expression of inducible and endothelial nitric oxide synthase in IR-injured mice (P< .05). NM ameliorates IR renal injury via inhibition of apoptosis by, at least in part, lowering nitric oxide overproduction, reducing Bax, and increasing Bcl-2.

DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters

To evaluate whether sperm DNA fragmentation (sDF), measured in brighter, dimmer, and total populations, predicts natural conception, and to evaluate the intra-individual variability of sDF. Prospective study. Outpatient clinic and diagnostic laboratory. A total of 348 unselected patients and 86 proven fertile men. None. sDF was revealed with the use of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)/propidium iodide (PI). Receiver operating characteristic (ROC) curves were built before and after matching fertile men to patients for age (76:152) or semen parameters (68:136) or both (49:98). Intra-individual variability of sDF was assessed over 2years. Brighter (area under ROC curve [AUC] 0.718 ± 0.54), dimmer (AUC 0.655 ± 0.63), and total (AUC 0.757 ± 0.54) sDF predict male fertility in unmatched and age- or semen parameters-matched subjects. After matching for both age and semen parameters, only brighter (AUC 0.711 ± 0.83) and total (AUC 0.675 ± 0.92) sDF predict male fertility. At high values of total sDF, brighter predicts natural conception better than total sDF. Intra-individual coefficients of variation of sDF were 9.2 ± 8.6% (n = 25), 12.9 ± 12.7% (n = 53), and 14.0 ± 12.6% (n = 70) over, respectively, 100-day and 1- and 2-year periods, appearing to be the most stable of the evaluated semen parameters. The predictive power of total sDF partially depends on age and semen parameters, whereas brighter sDF independently predicts natural conception. Therefore, brighter sDF is a fraction of sDF that adds new information to the routine semen analysis. At high levels of sDF, distinguishing the two sperm populations improves the predictive power of sDF. Overall, our results support the idea that TUNEL/PI can be of clinical usefulness in the male fertility workup.

Ethanol-Associated Cardiomyocyte Apoptosis and Left Ventricular Dilation Are Unrelated to Changes in Myocardial Telomere Length in Rats

AimThe aim of this work was to determine whether ethanol-associated myocardial apoptosis and cardiac dilation are related to myocardial telomere shortening in rats. Methods and ResultsSprague-Dawley (SD) rats received either drinking water with (ethanol: n = 19) or without (control: n = 19) 5% (v/v) ethanol ad libitum, for 4 months. Left ventricular (LV) dimensions and function (echocardiography and isolated perfused heart preparations), cardiomyocyte apoptosis (terminal deoxynucleotide transferase–mediated dUTP nick-end labeling), and leukocyte and myocardial telomere length (real-time polymerase chain reaction) were determined at the end of the study. Ethanol administration resulted in a marked increase in cardiomyocyte apoptosis (ethanol 0.85 ± 0.13% vs control 0.36 ± 0.06%; P = .0021) and LV dilation (LV end-diastolic diameter: ethanol 8.20 ± 0.14 mm vs control 7.56 ± 0.11 mm [P = .0014]; volume intercept at 0 mm Hg (V0) of the LV end-diastolic pressure-volume relationship: ethanol 0.40 ± 0.03 mL vs control 0.31 ± 0.02 mL [P = .020]). However, there were no changes in systolic chamber function as indexed by LV endocardial fractional shortening or the slope of the LV systolic pressure-volume relationship (end systolic elastance). The percentage of myocardial apoptosis was correlated with the degree of LV dilation (% apoptosis vs LV EDD: r = 0.39; n = 38; P = .021; vs V0: r = 0.44; n = 19; P = .046). No differences in leukocyte or cardiac telomere length were noted between the ethanol and control groups. Furthermore, cardiac telomere length was not associated with indexes of LV dilation (LVEDD and V0) or cardiomyocyte apoptosis. ConclusionsChronic ethanol-associated myocardial apoptosis and adverse remodeling occurs independently from changes in cardiac telomere length. Telomere shortening may not be a critical mechanism responsible for cardiomyocyte apoptosis and adverse cardiac remodeling.

In vivo mechanisms of uterine myoma volume reduction with ulipristal acetate treatment

To study the in vivo mechanisms of action of ulipristal acetate (UPA) on uterine myomas. Retrospective histologic and immunohistochemical (IHC) study of myomas. Academic research unit. Among 59 women with symptomatic myomas who underwent myomectomy, 42 were treated preoperatively with UPA, while 17 were not. Histology and IHC were analyzed on tissue microarrays obtained from surgical specimens. Proliferation, apoptosis, extracellular matrix (ECM) remodeling, and matrix metalloproteinase 2 (MMP-2) expression. Proliferation was low in all conditions, with no statistical difference between groups. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay showed an increase in cell death in UPA-treated myomas compared with untreated myomas, but only after short-term treatment; this was not associated with elevated levels of cleaved caspase-3. After long-term treatment, cell density was higher and the ECM volume fraction lower in UPA-treated myomas than in untreated myomas. MMP-2 expression was found to be increased after treatment, showing the highest level after long-term treatment, compared with untreated myomas. Regarding sustained clinical volume reduction of myomas, this study strongly points to multifactorial mechanisms of action of UPA, involving: 1) a persistently low cell proliferation rate; 2) a limited period of cell death; and 3) ECM remodeling concomitant with stimulation of MMP-2 expression.

Effects of Cryopreservation on the Molecular Integrity of Ovarian Grafts

INTRODUCTION: The objective of this study was to compare the effects of slow freezing and vitrification on the molecular integrity of ovarian grafts. METHODS: Fresh bovine ovaries were processed and divided into three groups (fresh control, slow freezing, and vitrification) before transplantation. For slow freezing and vitrification, 1.5 M dimethylsulfoxide-based cryoprotectant and a two-step vitrification method were used, respectively. Two pieces of ovarian tissue were transplanted onto the back muscle of the severe combined immunodeficient mouse (six animals for each group). Ovarian grafts were recovered on day 7 and day 28 after transplantation to assess DNA damage, apoptosis, and autophagy by histology, immunohistochemistry, and Western blot analysis. RESULTS: The slow freezing group showed significantly higher normal follicle count compared with the vitrification group. There was no significant difference in apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase 3) among three groups; however, autophagy (LC3B) was decreased in both slow freezing and vitrification groups. On day 28, a significant decrease in LC3B in the vitrification group was noted compared with the slow freezing group. There was no difference in expression of DNA damage and repair markers (γH2AX, Rad51, PCNA) and vascular density makers (CD31). CONCLUSION: The expression pattern of biochemical markers for DNA damage and repair and apoptosis in ovarian grafts was similar between three groups. There is not yet strong evidence that vitrification is better than slow freezing. Therefore, further improved vitrification protocols are needed.

C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress-Related Renal Apoptosis Is Involved in Rats With Brain Death

BackgroundC/EBP homologous protein (CHOP) is an important marker in endoplasmic reticulum stress (ERS)-associated cell apoptosis. The role of CHOP-induced renal apoptosis remains unclear in rats with brain death (BD). The present study aims to investigate the possible implication of CHOP-mediated, ERS-related BD-induced apoptosis in rats. Materials and MethodsForty male Sprague–Dawley rats were divided randomly into 4 experimental groups. We examined activation of ERS and apoptosis-related protein expression using Western blot and immunohistochemical staining. In addition, apoptosis is assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. ResultsKidneys harvested after BD show increased ERS- and apoptosis-related protein markers compared with kidneys of non-BD rats. Salubrinal (Sal) significantly increased levels of p-eIF2a and decreased the activity of CHOP at 6 hours after BD compared with vehicle-treated dimethylsulfoxide. ConclusionsTreating the BD donor with Sal influences renal apoptosis compared with vehicle-treated BD rats. Our results indicate that targeting the CHOP pathway provides a promising therapeutic approach for kidney injury associated with donor BD.

Antithymocyte globulin facilitates alloreactive T-cell apoptosis by means of caspase-3: potential implications for monitoring rejection-free outcomes.

Alloreactive T-cell apoptosis may explain reduced immunosuppression requirements with proapoptotic immunosuppression and among rejection-free recipients. This possibility remains unproven. Apoptotic (caspase-3+, cathepsin B+) and inflammatory (CD154+) T-cell subsets were evaluated before and after adding rabbit antithymocyte globulin (rATG) to mixed lymphocyte co-cultures between human leukocyte antigen-mismatched peripheral blood lymphocytes from healthy adults. In random samples from children with liver (LTx-20) and intestine (ITx-13) transplantation, apoptotic T cells were evaluated for association with rejection-free outcomes using the caspase-3 substrate, phiphilux. In mixed lymphocyte co-cultures between normal human peripheral blood lymphocytes, (1) frequencies of memory (M) and naive (N) Th and Tc, which expressed activated caspase-3, were enhanced most by the combination of allostimulation and rATG, than either stimulus alone. These findings were confirmed with antibody to activated caspase-3, phiphilux, and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay; (2) frequencies of Th subsets, which expressed activated cathepsin B, were similarly increased with combined stimulation. Tc seemed resistant to cathepsin B activation; (3) with increasing rATG concentrations, proportionately more allospecific CD154+T-cytotoxic memory cells (TcM) survived than TcM, resulting in relative enrichment of allospecific CD154+TcM. In random blood samples, phiphilux+T-cell subset frequencies were higher among 14 rejection-free LTx and ITx recipients and demonstrated a greater increase with ex vivo rATG pretreatment than 19 rejectors. In logistic regression analysis, phiphilux+TcM associated best with rejection-free outcomes with a sensitivity of 57% and a specificity of 89%. Rabbit antithymocyte globulin facilitates apoptosis of alloreactive T cells by means of caspase-3 activation, which may explain its steroid-sparing effect in pediatric liver and intestine recipients. Apoptotic susceptibility of T-cytotoxic memory cells, which resist cathepsin B activation, may distinguish rejection-free and rejection-prone liver recipients.

Endoplasmic Reticulum Stress Mediates Brain Death-Induced Apoptosis in Rat Liver.

Objectives The aim of this study was to assess whether Endoplasmic reticulum stress contribute to liver cell apoptosis and injury induced by brain death. Methods: Sixty rats were randomly divided into sham operation group (group A) and brain death group (group B). Activation of ER stress and apoptosis related protein expression were examined by the Western blot and immunohistochemical staining (IHC).In additional, apoptosis was assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and transmission electron microscopy(TEM). Results In the present study, we have shown that brain death induces a variety of signature ER stress markers including ER stress-specific X box-binding protein-1 (Xbp-1), and up-regulation of glucose-regulated protein 78 (Grp78). Furthermore, brain death causes up-regulation of C/EBP homologous protein (Chop) and Caspase12. Consistent with this, TUNEL and TEM confirmed apoptosis in the liver following brain death. Conclusion The present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain death-induced apoptosis and liver injury.

Apoptosis of Cardiomyocytes in Children with Right Ventricular Pressure Overload with and without Hypoxemia

Cardiomyocyte apoptosis has been implicated in ventricular remodeling and initiation of cardiac failure. We sought to determine the severity of right ventricular (RV) cardiomyocyte apoptosis in cyanotic and acyanotic children with RV pressure overload. Fourteen patients, seven with tetralogy of Fallot (group I) and seven with pulmonary stenosis and ventricular septal defect (group II), undergoing open-heart surgery were studied. Right ventricular biopsies were examined for cardiomyocyte apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. The magnitude of cardiomyocyte apoptosis was related to preoperative oxygen saturation and postoperative inotrope use and hospital stay. Compared with group I patients, group II patients were significantly older at operation (p = 0.002) and had a larger body size (p < 0.01) and higher preoperative oxygen saturation (p = 0.01). The prevalence of cardiomyocyte apoptosis in both group I and II patients as a whole was 0.24 ± 0.29% (range, 0% to 1.10%). The prevalence was similar between group I (median 0.30%, range 0% to 1.10%) and group II (median 0.20, range 0% to 0.40%, p = 0.65). The prevalence of cardiomyocyte apoptosis correlated positively with preoperative oxygen saturation on room air (r = -0.69, p < 0.005) and postoperative inotrope score (r = 0.67, p = 0.001). A higher postoperative inotrope score (r = 0.68, p = 0.001) was associated with a significant longer duration of postoperative stay in the hospital. The prevalence of cardiomyocyte apoptosis in the pressure-overloaded right ventricle is related to the severity of hypoxia and may have an impact on postoperative course in terms of early postoperative use of inotropes and duration of hospital stay.

A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold

To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. In vivo experimental study. Gynecology research unit in a university hospital. Six-week-old female NMRI mice. Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.

Gambogic acid synergistically potentiates cisplatin-induced apoptosis in non-small-cell lung cancer through suppressing NF-κB and MAPK/HO-1 signalling

Background:Gambogic acid (GA) has been reported to have potent anticancer activity and is authorised to be tested in phase II clinical trials for treatment of non-small-cell lung cancer (NSCLC). The present study aims to investigate whether GA would be synergistic with cisplatin (CDDP) against the NSCLC.Methods:1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI) isobologram, western blot, quantitative PCR, flow cytometry, electrophoretic mobility shift assay, xenograft tumour models and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling analysis were used in this study.Results:The cell viability results showed that sequential CDDP-GA treatment resulted in a strong synergistic action in A549, NCI-H460, and NCI-H1299 cell lines, whereas the reverse sequence and simultaneous treatments led to a slight synergistic or additive action. Increased sub-G1 phase cells and enhanced PARP cleavage demonstrated that the sequence of CDDP-GA treatment markedly increased apoptosis in comparison with other treatments. Furthermore, the sequential combination could enhance the activation of caspase-3, -8, and 9, increase the expression of Fas and Bax, and decrease the expression of Bcl-2, survivin and X-inhibitor of apoptosis protein (X-IAP) in A549 and NCI-H460 cell lines. In addition, increased apoptosis was correlated with enhanced reactive oxygen species generation. Importantly, it was found that, followed by CDDP treatment, GA could inhibit NF-κB and mitogen-activated protein kinase (MAPK)/heme oxygenase-1 (HO-1) signalling pathways, which have been validated to reduce ROS release and confer CDDP resistance. The roles of NF-κB and MAPK pathways were further confirmed by using specific inhibitors, which significantly increased ROS release and apoptosis induced by the sequential combination of CDDP and GA. Moreover, our results indicated that the combination of CDDP and GA exerted increased antitumour effects on A549 xenograft models through inhibiting NF-κB, HO-1, and subsequently inducing apoptosis.Conclusion:Gambogic acid sensitises lung cancer cells to CDDP in vitro and in vivo in NSCLC through inactivation of NF-κB and MAPK/HO-1 signalling pathways, providing a rationale for the combined use of CDDP and GA in lung cancer chemotherapy.

Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time

To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. Comparative prospective study. Center for reproductive medicine. Twenty-seven semen samples from infertile patients. None. Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (<20%); and B) Abnormal TUNEL (≥ 20%). TUNEL was performed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples and in TG2 samples after 0.5, 1.0, and 1.5 hours of incubation in PVP or HA. TUNEL levels were significantly reduced after gradient separation compared with neat values. In group A, TUNEL levels were significantly higher in the TG2 + 1.5 hours in PVP and HA samples but did not reach abnormal levels. In group B, TUNEL levels were significantly higher in the TG2 + 1 hour in PVP and HA samples. Sperm DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection.

English

This study involves the antitumor potential of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) to induce apoptosis and enhance antitumor activity of imatinib in&nbsp;K562 cells. Though PGG was reported to have antitumor activities in breast, prostate, kidney, liver cancers and HL-60 leukemia cells, there is no report on synergistic antitumor effect of PGG with imatinib until now. In the present study, PGG significantly enhanced the cytotoxicty and cleavage of poly (ADP-ribose) polymerase (PARP) in imatinib induced chronic myeloid leukemia K562 cells. Furthermore, oral administration of PGG or imatinib significantly inhibited the growth of K562 cells inoculated in Balb/c athymic nude mice and also immunohistochemistry revealed decreased expression of Ki67 (proliferation), CD34 (blood density) and&nbsp;death domain-associated protein&nbsp;(DAXX) and increased terminal deoxynucleotide transferasemediated dUTPnick end&nbsp;labeling&nbsp;(TUNEL)&nbsp;positive cells as one of the&nbsp;apoptotic feature&nbsp;in tumor sections of K562 mouse xenograft model&nbsp;comparable to imatinib treated group.&nbsp;Overall, our findings suggest the potency of PGG to induce apoptosis and enhance antitumor activity of imatinib inK562 cells. &nbsp; Key words:&nbsp;Chronic myeloid leukemia, apoptosis, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), imatinib.

Mystery of idiopathic male infertility: is oxidative stress an actual risk?

To study the role of oxidative stress in sperm dysfunction in Turkish idiopathic infertile men. Prospective study. Medical laboratory. Semen samples from 28 idiopathic infertile men and 14 fertile men. Sperm DNA fragmentation and reactive oxygen species (ROS) formation were assayed with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) test and 2',7'-dichlorodihydrofluorescein, respectively. Seminal plasma protein carbonyl groups (PC), nitrotyrosine (NT), malondialdehyde (MDA), and total thiol (SH) levels and ferric reducing antioxidant power (FRAP) were determined. Sperm DNA fragmentation in relation to ROS formation and seminal plasma oxidative parameters. The number of TUNEL-positive spermatozoa from idiopathic infertile men was higher than from fertile men, and ROS formation was increased as well in infertile males. A positive correlation was detected between TUNEL-positive cells and ROS content. Seminal plasma MDA, PC, and NT levels were elevated in idiopathic infertile males. No difference was observed in the total SH content and FRAP. Seminal plasma MDA levels correlated positively with both NT and PC levels. Positive correlations were detected between DNA fragmentation and MDA, NT, and PC of seminal plasma, and between sperm ROS content and MDA levels. The results of this study support the presence of oxidative stress in sperm dysfunction in Turkish idiopathic infertile men.