Deoxynucleotidyl Transferase-mediated dUDP Nick-end Labelling Research Articles

Effect of pesticide exposure on human sperm characteristics, genome integrity, and methylation profile analysis.

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group.

Electroacupuncture of the Baihui and Shenting acupoints for vascular dementia in rats through the miR-81/IL-16/PSD-95 pathway

BackgroundThere is currently no effective treatment for vascular dementia (VaD). Scalp electroacupuncture (EA) has served clinically as an alternative treatment for VaD, but its mechanism is still unclear. In this study, we investigated the effect of EA at the Baihui (GV 20) and Shenting (GV 24) acupoints on spatial learning and memory ability, and the expression level of microRNA-81 (miR-81), interleukin-16 (IL-16), and postsynaptic density protein-95 (PSD-95) in the frontal cortex of VaD rats.MethodsMale Sprague-Dawley rats were randomly divided into four groups, sham, VaD, non-acupuncture (non-AP) and EA group. The VaD model was established by permanent bilateral occlusion of the common carotid arteries. Morris Water Maze was used to assess the rats’ spatial learning and memory. Immunochemistry (IHC), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blot analysis were performed to detect the expression level of miR-81, IL-16, and PSD-95. Finally, luciferase assay was used to determine the effect of miR-81 on IL-16 expression in PC12 cells.ResultsThe space exploration experiment of MWM showed the time and distance of the rat’s activities around the platform were decreased in the EA group. Compared to the VaD and non-AP group, the number of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-positive frontal cortical neurons was significantly decreased in EA group. The number of the PSD-95-positive cells and the miR-81 expression level in the frontal cortical in the EA group was dramatically increased in comparison with the other groups. In the PC12 cell validation experiment, IL-16 expression level was reduced under the condition of the miR-81 mimic treatment, while increased in the miR-81 inhibitor group. The PSD-95 protein level was up-regulated in the small interfering (si)RNA-IL16 group compared to the NC-IL16 groups with or without oxygen/glucose deprivation/reperfusion (OGD/R) conditions (P<0.05). However, this was abolished by miR-81 mimic.ConclusionsIn VaD rats, EA may improve spatial learning and memory through miR-81/IL-16/PSD-95 pathway.

Long-time low-temperature transportation of human ovarian tissue before cryopreservation

Research questionCan the low-temperature transport time of removed human ovarian tissue be prolonged until cryopreservation? DesignFresh ovarian cortex from nine premenopausal patients was either slow-frozen immediately or stored at 4°C for 24 or 48 h before slow-freezing. The fresh and frozen–thawed biopsies were evaluated by follicle counting via calcein staining, histologic analyses via haematoxylin and eosin staining, and apoptosis via terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL). The fresh cortex was assessed by reactive oxygen species (ROS) and total antioxidant capacity (TAC) assay to detect oxidative stress. The frozen–thawed cortex biopsies were also evaluated by quantitative PCR for messenger RNA (mRNA) expression of BCL-2, BAX, TNFa, HIF-1a, BMP15 and GDF9, and Western blot for detection of BCL-2, BMP15, GDF9 and CASPASE-3. The frozen–thawed cortex was cultured in vitro for 4 days, anti-Müllerian hormone and glucose were assessed in the supernatant, and ROS and TAC assay detected any oxidative stress in the cortex. ResultsIn the fresh cortex, there were no significant differences between the three groups. In the frozen–thawed cortex, there were no significant differences between the three groups regarding follicle viability, TUNEL, mRNA expression of TNFa, HIF-1a or BMP15. GDF9 mRNA and BAX/BCL-2 were lower and higher at 48 h than at 0 h, respectively. However, the protein expression of BCL-2, CASPASE-3, GDF9 and BMP15 were no different. In the cultured cortex, ROS, TAC and glucose uptake were no different across the three groups. ConclusionOvarian tissue transportation was validated for 24 h in the procedure used in clinical practice. This study showed that 4–8°C transportation for 24 or 48 h does not seem to damage the ovarian tissue. However, ovarian tissue transportation beyond 48 h needs to be further studied for conclusions to be made.

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.

DNA double strand breaks in human spermatozoa can be predictive for assisted reproductive outcome

Sperm DNA status has been reported to predict fertility outcomes in infertile men. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test (TUNEL) is the most widely used method to evaluate this; however, its prognostic value is still debated. One hundred infertile men undergoing intracytoplasmic sperm injection (ICSI) and 61 fertile men were tested for sperm parameters, sex hormones and sperm DNA status by chromatin tests (acridine orange, aniline blue, decondensation) and by direct assays (TUNEL and phosphorylated histone H2AX-γH2AX). In both groups, the prognostic value of each parameter to predict assisted clinical pregnancy was compared. Sperm parameters (P < 0.05 or P < 0.01), FSH levels (P < 0.05) and DNA status (P < 0.05 to P < 0.001) were significantly different in participants compared with controls. Among infertile men, 47 had positive and 53 had ICSI outcome. Both chromatin analysis and TUNEL test were unable to distinguish individuals who had successful outcomes from those who failed ICSI treatments. γH2AX percentage and γH2AX fragmentation index were significantly higher in sperm from non-pregnant compared with pregnant couples (P < 0.05 and P < 0.01). γH2AX assay is more predictive of ICSI outcome than TUNEL in infertile couples with male factor infertility.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 Mediates Apoptosis during Lung Vascular Permeability by Regulating Movement of Cleaved Caspase 3

Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p<0.001), but no correlations were found with fertilization or embryo development rates (p>0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p<0.05) and clinical pregnancy among IVF couples (p<0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p=0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p=0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available.

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30min and 24h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue.This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30min and 24h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.

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.

Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing

Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown. The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential. Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.

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.

Improvement in sperm DNA quality using an oral antioxidant therapy

Oxidative stress is now recognized as a common pathology that affects up to half of all infertile men. One of the principal mechanisms by which oxidative stress produces infertility is by damage to sperm DNA, either through direct oxidation of the DNA by reactive oxygen species (ROS) or by the initiation of apoptosis. The objective of this study was to determine if an oral antioxidant/mineral supplement could improve sperm DNA integrity in men with known oxidative stress. A total of 50 infertile men identified as exhibiting oxidative stress were administered oral antioxidant therapy for a period of 3 months. All participants were assessed at entry and exit for sperm DNA integrity with terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling, apoptosis with annexin V, protamination with chromomycin A(3) and ROS production with nitro blue tetrazolium assay. Sperm concentration, motility and morphology, together with assessment of serum male reproductive hormones (LH, FSH, testosterone, anti-Mullerian hormone), were also monitored. The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in sperm DNA integrity (P = 0.002) and protamine packaging (P < 0.001), accompanied by a reduction in seminal ROS production (P = 0.027) and apoptosis (P = 0.004). No significant changes in routine sperm parameters (concentration, motility, morphology) or male reproductive hormones were observed.

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.

MAPK Regulation of Aquaporin (AQP) Expression and Embryonic Apoptosis Following Exposure to Hyperosmotic Conditions

Preimplantation development is the "free-living" period of mammalian development that extends from insemination in the fallopian tube, to embryo implantation in the uterus. Blastocyst formation is required for implantation, establishment of pregnancy, and is a principal determinant of embryo quality prior to embryo transfer. Studies from our laboratory have revealed that the responses of preimplantation embryos to culture medium hyperosmolarity include increased p38 mitogen activated protein kinase (MAPK) activation and increased expression of Osmosensing scaffold for MeKK3. We have also reported that fluid movement across the trophectoderm is facilitated by the presence of Aquaporins (AQP) including AQP 3 and 9. The best current preimplantation embryo culture protocols employ media with osmolarities around 260 mOsm in contrast to the up to 350 mOsm reported for reproductive tract fluid. The rationale for this practice lies in that preimplantation embryo do not survive in media with osmolarities greater than 300 mOsm unless osmolytes are present when placed into culture. It is important to investigate and define the mechanisms and pathways that embryos employ to offset the deleterious effects that culture environments impose on preimplantation embryos. We have investigated the influences of hyperosmotic culture environments on preimplantation embryos and explored mechanisms and pathways embryos employ to offset these effects, all of which are crucial for a better understanding of embryo development and culture media optimization. We hypothesized that MAPK pathways mediate embryonic responses to hyperosmotic stress, by regulating AQP 3 and 9 expression as well as embryonic apoptosis. Real-Time PCR and whole-mount immunofluorescence were used to investigate AQP3 and 9 mRNA levels and protein distribution in embryos cultured in hyperosmotic media at 3h, 6h, and 24h. AQP3 and 9 mRNA did not vary significantly following 3 hours of exposure to sucrose hyperosmotic medium (300-460 mOsm) compared to control and glycerol hyperosmotic media. Following 6 hours we detected a significant increase in AQP3 and 9 mRNA in the sucrose 350mOsm treatment group. After 24 hours in sucrose hyperosmotic medium AQP3 and 9 mRNA levels remained significantly elevated compared to controls. When cultured in the presence of sucrose hyperosmotic medium and a p38 inhibitor, the increase in AQP3 and 9 mRNA levels were not observed. Whole-mount immunofluorescence revealed a dramatic shift in AQP9 protein from its predominantly cortical distribution observed in control embryos to become primarily localized to cytoplasmic/peri-nuclear regions in embryos cultured for 6 hours under hyperosmotic conditions (300mOsm-350mOsm). Apoptosis was investigated at 6h by using terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) and Caspase-3 assays. Following hyperosmotic treatment in 350mOsm sucrose, we observed a significant increase in apoptosis levels while embryos exposed to 350mOsm glycerol did not vary from control apoptosis levels. In the presence of JNK inhibitor, but not p38 or ERK inhibitors, the 350mOsm sucrose-treated embryos displayed a significant decrease in apoptosis. Our results demonstrate that AQP 3 and 9 mRNA levels and protein localization along with apoptosis are responsive to hyperosmotic culture stress. Furthermore, the use of MAPK inhibitors has determined that regulation of AQP expression and localization is primarily regulated by the p38 MAPK pathway while embryonic apoptosis is primarily regulated by the JNK/SAPK pathway. Research supported by operating funds from the Canadian Institute of Health Research.

Gonadotrophins regulate germ cell survival, not proliferation, in normal adult men

Gonadotrophins support spermatogenesis via poorly understood mechanisms. We aimed to determine the effect of FSH/LH suppression in regulating germ cell apoptosis and proliferation in normal fertile men. Testicular tissues were obtained after gonadotrophin suppression induced by testosterone alone or combined with depot medroxyprogesterone acetate for 2 or 6 weeks and an untreated group of men (referred to as 'normal men') served as controls (n = 5 or 10 men per group). Apoptosis and proliferation were identified by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) labelling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry using the pathway-specific proteins: activated caspase (aCaspase) 9 and 8 and quantified using stereological techniques. By 2 and 6 weeks, the proportion of TUNEL-labelled spermatogonia increased to 354% and 268% respectively, compared with normal men (P < 0.001), with increased caspase 9 [223 and 166% compared with normal men (P < 0.001)], but no increase in caspase 8, immunoreactivity. At 6 weeks, the proportions of TUNEL-labelled spermatocytes and round spermatids tended to increase (303 and 180% compared with normal men, NS), as did caspase 9 (199 and 147% compared with normal men, NS) and caspase 8 immunoreactivities (286 and 243% compared with normal men, NS and P = 0.06), respectively. The proportion of TUNEL-labelled elongating/elongated spermatids tended to increase (144 and 138% compared with normal men, NS) at 2 and 6 weeks, respectively, with no change in either caspase immunoreactivities. Even though the number of PCNA-labelled cells did not change with gonadotrophin suppression, the balance between proliferation and apoptosis was lower in spermatogonia (P = 0.01) and spermatocytes (P = 0.3) between treated and untreated normal men. We demonstrated that gonadotrophins act as spermatogonial survival factors via the regulation of intrinsic apoptotic pathway, whereas having no effect of cellular proliferation in normal men.

HIV-1 viral DNA is present in ejaculated abnormal spermatozoa of seropositive subjects

Semen is the major vehicle for HIV-1 infection as it contains free and cell-associated virions and infected cells. However, the presence of HIV-1 in spermatozoa has been a matter of debate, since the sperm cell fraction may contain somatic infected cells that jeopardize the attribution of the detected virus to the spermatozoa. Spermatozoa from 12 HIV-1 seropositive subjects were purified by multilayered Percoll gradient followed by osmotic shock. Residual presence of non-seminal cells (NCS) in purified spermatozoa, was then evaluated by cytometric and molecular analysis. HIV-1 DNA was revealed by nested PCR and in situ PCR after sperm chromatin decondensation. DNA-fragmented ejaculated spermatozoa in semen of infected subjects were detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) analysis. Purification procedure adopted allowed complete removal of NCS. On purified sperm cells, HIV-1 DNA was detected in 5 out of 12 subjects by nested-PCR. On crude semen of 10 out of 12 subjects, HIV-1 DNA was in situ detected in a small percentage of abnormal spermatozoa with a wide range of structural alterations. TUNEL analysis revealed an increased percentage of DNA-fragmented ejaculated spermatozoa in semen of infected subjects. We report molecular evidence demonstrating that HIV-1 infected subjects can ejaculate small amounts of HIV-1 DNA-positive abnormal spermatozoa. Their possible role in HIV-1 sexual transmission remains to be clarified.

Kidneys of Mice With Hereditary Tyrosinemia Type I Are Extremely Sensitive to Cytotoxicity

Children with hereditary tyrosinemia type 1 (HT1) suffer from liver failure, renal tubular dysfunction, and rickets. The disease is caused by deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of tyrosine catabolism, and leads to accumulation of the toxic substrate fumarylacetoacetate (FAA) in hepatocytes and renal proximal tubular cells. Patients are treated with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC), which prevents accumulation of FAA by blocking an enzyme upstream of FAH. Liver transplantation is performed when patients do not respond to NTBC or develop hepatocellular carcinoma. This reduces the tyrosine load for the kidney but does not abolish renal exposure to locally produced FAA. To investigate the pathogenesis of liver and kidney damage induced by tyrosine metabolites, we challenged FAH-deficient mice with various doses of homogentisic acid (HGA), a precursor of FAA. Injecting NTBC-treated Fah-/- mice with low doses of HGA caused renal damage and death of renal tubular cells, as was shown by histologic analyses and deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay but did not lead to liver damage. In addition, kidney function, but not liver function, was affected after exposure to low doses of HGA. Administration of high doses of HGA led to massive cell death in both the liver and kidneys. Resistance to HGA-induced cell death was seen after withdrawing NTBC from Fah-/- mice. The finding that the kidneys of Fah-/- mice are especially sensitive to damage induced by low doses of HGA underscores the need to perform careful monitoring of the kidney function of tyrosinemia patients undergoing any form of treatment.

Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells

Human endothelial EA.hy926 cells were incubated with BaP1, a hemorrhagic metalloproteinase purified from Bothrops asper snake venom. Since the first hour of incubation with the proteinase, cells started showing DNA fragmentation, detected by a terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-based photometric enzyme-linked immunosorbent assay (ELISA). At later times, DNA fragments were predominantly located outside the cells, evidencing plasma membrane rupture. DNA fragmentation was completely abolished by Batimastat, a potent inhibitor of metalloproteinase enzymatic activity. Apoptosis induced by BaP1 on endothelial cells was independent of two Bcl-2 family members (anti-apototic Bcl-xL and pro-apoptotic Bax), that did not show any changes in their expression during a 24 h-treatment period. Interestingly, IkappaBalpha, an inhibitor of NFkappaB, decreased after 24 h of treatment, suggesting further activation of the transcription factor. When some elements of the apoptotic extrinsic pathway were assessed, it was observed that procaspase-8 completely disappeared after 24 h of treatment with BaP1, probably indicating its activation by a death receptor, whereas caspase-8 inhibitor, cellular FLICE-inhibitory protein (cFLIP(L)), increased its expression since the first hours of BaP1 incubation. In conclusion, treatment of human endothelial cells with BaP1 induces apoptosis/anoikis, independently of Bcl-2 family members Bax and Bcl-xL and associated with caspase-8 activation and cFLIP(L) up-regulation. Apoptosis was completely dependent on BaP1 enzymatic activity. Similarities between this and other endothelial cell anoikis-related systems suggest that BaP1 and other snake venom metalloproteinases may be useful experimental tools in the study of death-related events that occur when adherent cells loose contact with extracellular matrix.

Effect of folic acid on homocysteine-induced trophoblast apoptosis.

In trophoblast cells exposed to homocysteine (Hcy) we observed cellular apoptosis and the inhibition of trophoblast functions. Because folate and Hcy, linked in the same metabolic pathway, are inversely related, we investigated the role of folic acid in reversing the Hcy effect in human placenta. In primary trophoblast cells we examined the cytosolic release of cytochrome c, both M30 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and DNA laddering. Hcy (20 micromol/l) treatment resulted in cytochrome c release from mitochondria to the cytosol, and an increased number of M30-positive trophoblast cells and TUNEL positive nuclei. Furthermore, DNA cleavage in agarose gel and the determination of histone-associated DNA fragments have been investigated. Homocysteine induced DNA fragmentation and significantly reduced hCG secretion. The addition of folic acid (20 nmol/l) resulted in inhibition of the effects of Hcy on human trophoblast. These results suggest a protective role of folic acid in the prevention of trophoblast apoptosis linked to Hcy.

Influence of deoxyribonucleic acid damage on fertilization and pregnancy

Objective To investigate sperm DNA damage in relation to fertilization and pregnancy. Design Prospective study. Setting The Institute of Reproductive Medicine, Giessen, Germany. Patient(s) Semen collected from 249 patients attending the IVF program. Main outcome measure(s) The percentage of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling- (TUNEL-), Fas-, and annexin-V-positive sperm and the proportion of green-fluorescing sperm in the acridine orange stain was determined and correlated with sperm concentration, motility, fertilization, and pregnancy. Result(s) Significant correlations with the concentration of motile sperm were only found for the acridine orange stain (before and after sperm separation) and for the TUNEL assay (after sperm separation). Moreover, patients whose sperm had a high percentage of DNA fragmentations showed significantly lower pregnancy rates (TUNEL assay: 19.05% vs. 34.65%; acridine orange stain: 24.58% vs. 37.93%). The apoptosis parameters (annexin V binding and Fas expression) showed no statistically significant differences. Conclusions Our data clearly demonstrate that DNA fragmentation, as determined by the TUNEL assay, is predictive for pregnancy in IVF. This implies that spermatozoa with DNA fragmentation can still fertilize an oocyte but that when paternal genes are “switched on,” further embryonic development stops, resulting in failed pregnancy. It seems that, at least in the patients we analyzed, apoptosis in the sperm does not play a role for fertilization. This would imply that DNA fragmentation in human spermatozoa is caused by external factors, such as reactive oxygen species, rather than by apoptosis.