Genetic Association in the Maintenance of the Mitochondrial Microenvironment and Sperm Capacity.

Sperm motility is one of the major determinants of male fertility. Since sperm need a great deal of energy to support their fast movement by active metabolism, they are thus extremely vulnerable to oxidative damage by the reactive oxygen species (ROS) and other free radicals generated as byproducts in the electron transport chain. The present study is aimed at understanding the impact of a mitochondrial oxidizing/reducing microenvironment in the etiopathology of male infertility. We detected the mitochondrial DNA (mtDNA) 4,977 bp deletion in human sperm. We examined the gene mutation of ATP synthase 6 (ATPase6 m.T8993G) in ATP generation, the gene polymorphisms of uncoupling protein 2 (UCP2, G-866A) in the uncoupling of oxidative phosphorylation, the role of genes such as manganese superoxide dismutase (MnSOD, C47T) and catalase (CAT, C-262T) in the scavenging system in neutralizing reactive oxygen species, and the role of human 8-oxoguanine DNA glycosylase (hOGG1, C1245G) in 8-hydroxy-2'-deoxyguanosine (8-OHdG) repair. We found that the sperm with higher motility were found to have a higher mitochondrial membrane potential and mitochondrial bioenergetics. The genotype frequencies of UCP2 G-866A, MnSOD C47T, and CAT C-262T were found to be significantly different among the fertile subjects, the infertile subjects with more than 50% motility, and the infertile subjects with less than 50% motility. A higher prevalence of the mtDNA 4,977 bp deletion was found in the subjects with impaired sperm motility and fertility. Furthermore, we found that there were significant differences between the occurrences of the mtDNA 4,977 bp deletion and MnSOD (C47T) and hOGG1 (C1245G). In conclusion, the maintenance of the mitochondrial redox microenvironment and genome integrity is an important issue in sperm motility and fertility.

Mitochondrial translocation of estrogen receptor ß affords resistance to oxidative insult-induced apoptosis and contributes to the pathogenesis of endometriosis.

Endometriosis is the major cause of female infertility and has been linked to the action of estrogen and estrogen receptor (ER). A new pool of ERß locates within mitochondria, which regulates the endometriotic cell withstanding external insults, but its effect remains controversial. We hypothesize that mitochondrial estrogen receptor ERß (mtERß) is a pivotal regulator in estradiol-mediated cell protection leading to the endometriotic progression. We observed elevated levels of ERß in the endometriotic tissues. A dramatic increase of ERß in mitochondria (mtERß) was found in the ectopic endometriotic tissues, or the estradiol-primed primary endometriotic cells. We analyzed the mtERß-specific overexpressing clone (mtsERß), which exhibited higher mitochondrial bioenergetics and lower reactive oxygen species (ROS) generation. The mtsERß-overexpressed endometriotic cells displayed an enhanced migration phenotype, whereas significantly attenuated migration by mitochondrial respiratory inhibitor (oligomycin) or ERß deficiency by shERß. Further investigations revealed that ERß directly modulated mitochondrial DNA (mtDNA) gene expression by interacting with mtDNA D-loop and polymerase γ. The mtsERß afforded a resistance to oxidative insult-induced apoptosis through the induction of the ROS scavenger enzyme Mn-superoxide dismutase and anti-apoptotic protein Bcl-2. Collectively, the demonstration of mtERß responses in restoration of mitochondrial bioenergetics and inhibition of mitochondria-dependent apoptotic events provides insight into the pathogenesis of endometriosis, suggesting ERß-selective estrogen receptor modulator may serve as novel therapeutics of endometriosis in the future.

Pleural fluid osteopontin, vascular endothelial growth factor, and urokinase-type plasminogen activator levels as predictors of pleurodesis outcome and prognosticators in patients with malignant pleural effusion: a prospective cohort study.

BACKGROUND: Rapidly growing cancer cells secrete growth-promoting polypeptides and have increased proteolytic activity, contributing to tumor progression and metastasis. Their presentation in malignant pleural effusion (MPE) and their predictive value for the outcome of pleurodesis and survival were studied. METHODS: Between February 2011 and March 2012, MPE samples were prospectively collected from 61 patients. Twenty-five patients with non-malignant pleural effusion in the same period were included as controls. Pleural fluid osteopontin (OPN), vascular endothelial growth factor (VEGF), and urokinase-type plasminogen activator (uPA) concentrations were measured. RESULTS: Patients with MPE had higher pleural fluid OPN, VEGF, and uPA concentrations than those with non-malignant pleural effusion, but only differences in VEGF were statistically significant (p = 0.045). Patients with distant metastases had significantly elevated pleural fluid VEGF concentrations than those without (p = 0.004). Pleural fluid OPN, VEGF, and uPA concentrations were positively correlated in most patients. However, there was no significant difference in pleural fluid OPN, VEGF, and uPA concentrations between patients with successful pleurodesis and those without. There was also no significant difference in cancer-specific survival between sub-groups with higher and lower pleural fluid OPN, VEGF, or uPA concentrations. Patients with successful pleurodesis had significantly longer cancer-specific survival than those without (p = 0.015). CONCLUSIONS: Pleural fluid OPN, VEGF, and uPA concentrations are elevated in MPE but are not satisfactory predictors of pleurodesis outcome or survival. Patients with higher pleural fluid VEGF concentration have higher risk of distant metastasis. Evaluating the benefits of therapy targeting the VEGF pathway in these patients warrants further studies.

Estrogen receptor-ß in mitochondria: implications for mitochondrial bioenergetics and tumorigenesis.

Estrogen enhances mitochondrial function by enhancing mitochondrial biogenesis and sustaining mitochondrial energy-transducing capacity. Shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation to glycolysis have been hypothesized to be involved in estrogen-induced tumorigenesis. Studies have shown that mitochondria are an important target of estrogen. Estrogen receptor-ß (ERß) has been shown to localize to mitochondria in a ligand-dependent or -independent manner and can affect mitochondrial bioenergetics and anti-apoptotic signaling. However, the functional role of mitochondrial ERß in tumorigenesis remains unclear. Clinical studies of ERß-related tumorigenesis have shown that ERß stimulates mitochondrial metabolism to meet the high energy demands of processes such as cell proliferation, cell survival, and transformation. Thus, in elucidating the precise role of mitochondrial ERß in cell transformation and tumorigenesis, it will be particularly valuable to explore new approaches for the development of medical treatments targeting mitochondrial ERß-mediated mitochondrial function and preventing apoptosis.

TCDD induces the hypoxia-inducible factor (HIF)-1α regulatory pathway in human trophoblastic JAR cells.

The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α) stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS) and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or N-acetylcysteine (a ROS scavenger). The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

High-dialysate-glucose-induced oxidative stress and mitochondrial-mediated apoptosis in human peritoneal mesothelial cells.

Human peritoneal mesothelial cells (HPMCs) are a critical component of the peritoneal membrane and play a pivotal role in dialysis adequacy. Loss of HPMCs can contribute to complications in peritoneal dialysis. Compelling evidence has shown that high-dialysate glucose is a key factor causing functional changes and cell death in HPMCs. We investigated the mechanism of HPMC apoptosis induced by high-dialysate glucose, particularly the role of mitochondria in the maintenance of HPMCs. HPMCs were incubated at glucose concentrations of 5 mM, 84 mM, 138 mM, and 236 mM. Additionally, N-acetylcysteine (NAC) was used as an antioxidant to clarify the mechanism of high-dialysate-glucose-induced apoptosis. Exposing HPMCs to high-dialysate glucose resulted in substantial apoptosis with cytochrome c release, followed by caspase activation and poly(ADP-ribose) polymerase cleavage. High-dialysate glucose induced excessive reactive oxygen species production and lipid peroxidation as well as oxidative damage to DNA. Mitochondrial fragmentation, multiple mitochondrial DNA deletions, and dissipation of the mitochondrial membrane potential were also observed. The mitochondrial dysfunction and cell death were suppressed using NAC. These results indicated that mitochondrial dysfunction is one of the main causes of high-dialysate-glucose-induced HPMC apoptosis.

Low-dose testosterone treatment decreases oxidative damage in TM3 Leydig cells.

Testosterone replacement therapy has benefits for aging men and those with hypogonadism. However, the effects of exogenous testosterone on Leydig cells are still unclear and need to be clarified. In this report, we demonstrate that testosterone supplementation can reduce oxidative damage in Leydig cells. The TM3 Leydig cell line was used as an in vitro cell model in this study. Cytoprotective effects were identified with 100-nmol l⁻¹ testosterone treatment, but cytotoxic effects were found with ≥ 500-nmol l⁻¹ testosterone supplementation. Significantly reduced reactive oxygen species (ROS) generation, lipid peroxide contents and hypoxia induction factor (HIF)-1α stabilization and activation were found with 100-nmol l⁻¹ testosterone treatment. There was a 1.72-fold increase in ROS generation in the 500-nmol l⁻¹ compared to the 100-nmol l⁻¹ testosterone treatment. A 1.58-fold increase in steroidogenic acute regulatory protein (StAR) expression was found in 50-nmol l⁻¹ testosterone-treated cells (P < 0.01). Chemically induced hypoxia was attenuated by testosterone supplementation. Leydig cells treated with low-dose testosterone supplementation showed cytoprotection by decreasing ROS and lipid peroxides, increasing StAR expression and relieving hypoxia stress as demonstrated by HIF-1α stabilization. Increased oxidative damage was found with ≥ 500-nmol l⁻¹ testosterone manipulation. The mechanism governing the differential dose effects of testosterone on Leydig cells needs further investigation in order to shed light on testosterone replacement therapy.

Endocrine disruptor, dioxin (TCDD)-induced mitochondrial dysfunction and apoptosis in human trophoblast-like JAR cells.

The endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to disrupt hormone signalling, reduce fertility, interfere with embryo development and cause spontaneous miscarriage in humans. The precise mechanisms of its effects on early implantation in humans are still unclear. In this study, we examined the relationship between mitochondrial function and dioxin-induced toxicity in JAR cells, a human trophoblast-like cell line. Several experiments were performed to address the effects of TCDD on cell viability, reactive oxygen species (ROS) generation, oxidative damage (indicated by the presence of lipoperoxides and oxidized DNA bases), mitochondrial DNA (mtDNA) copy number, ATP content, mtDNA mutations and the protein levels of p53, Bax, Bcl2, cytochrome c and caspase 3. Increased oxidative damage and mitochondrial dysfunction in TCDD-treated trophoblast-like cells was demonstrated. A 2.58-fold increase in lipid peroxides was detected in cells treated with 2 nM TCDD for 4 h. The oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine was significantly increased by TCDD treatment in a time-dependent manner. Meanwhile, reductions in mtDNA copy number and ATP content and an increase in mtDNA deletions were found. Furthermore, we observed increased apoptosis, p53 accumulation, Bax overexpression, cytochrome c release and sequential caspase 3 activation after TCDD exposure. These results indicate that oxidative damage and mitochondrial dysfunction may be responsible for the apoptotic effects of TCDD.

The cytotoxicity to leukemia cells and antiviral effects of Isatis indigotica extracts on pseudorabies virus.

AIM OF THE STUDY: Isatis indigotica (I. indigotica), Cruciferae, has been used in Chinese medicine for anti-leukemia and anti-severe acute respiratory syndrome (SARS). The aim of this study was to evaluate the cytotoxicity of Isatis indigotica extracts on human leukemia cell line (HL-60) and the antiviral activity on swine pseudorabies virus (PrV) in in vitro assays. MATERIALS AND METHODS: Extracts and derived fractions of Isatis indigotica were prepared from root (R) and leaf (L) using methanol (M), ethyl acetate (E) and distilled water (D). The cytotoxic effect of extracts on swine peripheral blood mononuclear cells (PBMCs) and HL-60 was assessed by MTT method. The cytopathic effect (CPE) reduction, plaque reduction and inhibition assays on viral replication, and virucidal activity were further conducted to investigate the anti-PrV activity. RESULTS: Indirubin, one of the biological active compounds of Isatis indigotica, had the most significant cytotoxicity on HL-60 cells and inhibitory effect on PrV replication. Extracts from roots and leaves of Isatis indigotica also presented CPE inhibition either before or after infection of PrV on porcine kidney (PK-15) cells. Leaf extracts had better virucidal activity than roots, and ethyl acetate extracts exhibited the highest efficacy among extracts tested. CONCLUSION: Isatis indigotica posses a valuable virucidal effect in disease control of pseudorabies virus infection in swine.

Increase of oxidative stress in human sperm with lower motility.

OBJECTIVE: To investigate the causal role of oxidative-stress status on human sperm motility. DESIGN: To demonstrate that sperm with higher oxidative damage have a lower antioxidant capacity. SETTING: University hospital infertility center. PATIENT(S): Seventy-eight semen samples were obtained from 35 healthy donors who had normal semen characteristics and from 43 infertile or subfertile males. INTERVENTION(S): The levels of oxidative damage (8-hydroxy-2'-deoxyguanosine [8-OHdG] and lipid peroxides) and antioxidants (retinol, alpha-tocopherol, ascorbate, and protein thiols) in the spermatozoa and/or seminal plasma were measured. MAIN OUTCOME MEASURE(S): We analyzed the specific content of 8-OHdG and lipid peroxides by using high-performance liquid chromatography (HPLC)-electrochemical detection and HPLC-fluorescence analysis, respectively. Retinol and alpha-tocopherol were analyzed by using an HPLC system, whereas ascorbate and protein thiols were determined by using spectrophotometry. 8-Hydroxy-2'-deoxyguanosine was visualized by immunofluorescent staining with an anti-8-OHdG antibody that was conjugated with fluorescein isothiocyanate conjugate. Lipid peroxides in spermatozoa were stained with a fluorescent dye, C11-BODIPY(581/591). RESULT(S): Statistically significant negative correlations were revealed between sperm motility and 8-OHdG and between motility and lipid peroxides. Statistically significant positive correlations were found between sperm motility and the levels of retinol, alpha-tocopherol, ascorbate, and protein thiols of seminal plasma. 8-Hydroxy-2'-deoxyguanosine and lipid peroxides in spermatozoa were found to be present mostly in mitochondria. CONCLUSION(S): Oxidative stress and oxidative damage were increased significantly in spermatozoa with declined motility, and the antioxidant capacities in the spermatozoa and seminal plasma were lower in males who had infertility or subfertility.

Repeated ovarian stimulations induce oxidative damage and mitochondrial DNA mutations in mouse ovaries.

Superovulation by injection of exogenous gonadotropin is the elementary method to produce in vivo-derived embryos for embryo transfer in women. Increased oocyte aneuploidy, embryo mortality, fetal growth retardation, and congenital abnormalities have been studied at higher-dose stimulations. Ovarian and oocyte biological aging possibly may have adverse implications for human oocyte competence with repeated hyperstimulation. In this study, we found that reduced competence for the human oocyte has been associated with degenerative embryo upsurge during embryo culture and failure to develop into the blastocyst stage in the three, four, five, and six stimulation cycles. On the other hand, the numbers of ovulated oocytes were decreased in the groups with more ovarian stimulation. More aggregated mitochondria were found in the cytoplasm of the repetitively stimulated embryos. Higher amounts of oxidative damage including 8-OH-dG, lipoperoxides, and carbonyl proteins were also revealed in the ovaries with more cycle numbers of ovarian stimulation. Higher proportions of mtDNA mutations were also found. The detected molecular size of the mutated band was approximately 675 bp. Increased amounts of carbonyl proteins were also revealed after repeated stimulation. An understanding of the relationship between oocyte competence and ovarian responses to stimulation in the mouse may provide insights into the origin of oocyte defects and the biology of ooplasmic aging that could be of clinical relevance in the diagnosis and treatment of human infertility.