Tetrabromobisphenol Exposure Impairs Bovine Oocyte Maturation by Inducing Mitochondrial Dysfunction

Tetrabromobisphenol (TBBPA) is the most widely used brominated flame retardant in the world and displays toxicity to humans and animals. However, few studies have focused on its impact on oocyte maturation. Here, TBBPA was added to the culture medium of bovine cumulus–oocyte complexes (COCs) to examine its effect on oocytes. We found that TBBPA exposure displayed an adverse influence on oocyte maturation and subsequent embryonic development. The results of this study showed that TBBPA exposure induced oocyte meiotic failure by disturbing the polar-body extrusion of oocytes and the expansion of cumulus cells. We further found that TBBPA exposure led to defective spindle assembly and chromosome alignment. Meanwhile, TBBPA induced oxidative stress and early apoptosis by mediating the expression of superoxide dismutase 2 (SOD2). TBBPA exposure also caused mitochondrial dysfunction, displaying a decrease in mitochondrial membrane potential, mitochondrial content, mtDNA copy number, and ATP levels, which are regulated by the expression of pyruvate dehydrogenase kinase 3 (PDK3). In addition, the developmental competence of oocytes and the quality of blastocysts were also reduced after TBBPA treatment. These results demonstrated that TBBPA exposure impaired oocyte maturation and developmental competence by disrupting both nuclear and cytoplasmic maturation of the oocyte, which might have been caused by oxidative stress induced by mitochondrial dysfunction.     

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.     

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H₂O₂) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H₂O) and oxygen (O₂) in order to reduce the harm caused by H₂O₂. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H₂O₂ accumulates excessively, it produces radical-(HO-) through Fe^2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H₂O₂ by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.     

Altered mitochondrial structure and motion dynamics in living cells with energy metabolism defects revealed by real time microscope imaging.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 m wide and up to 10 m long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.     

Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.     

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.     

The role of microfilaments in early meiotic maturation of mouse oocytes.

Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes (gamma-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical "organizing pole" is hypothesized.     

Tamoxifen subcellular localization; observation of cell-specific cytotoxicity enhancement by inhibition of mitochondrial ETC complexes I and III

Recently, a nongenomic cytotoxic component of the chemotherapeutic agent tamoxifen (TAM) has been identified that predominantly triggers mitochondrial events. The present study delineates the intracellular fate of TAM and studies its interaction with a spectrum of cell homeostasis modulators primarily relevant to mitochondria. The subcellular localization of TAM was assessed by confocal fluorescence microscopy. The effect of the modulators on TAM cytotoxicity was assessed by standard MTT assays. Our findings show that in estrogen receptor positive MCF7 breast adenocarcinoma cells and DU145 human prostate cancer cells, TAM largely accumulates in the mitochondria and endoplasmic reticulum, but not lysosomes. Our results further demonstrate that in MCF7, but not in DU145 cells, mitochondrial electron transport chain complex I and III inhibitors exacerbate TAM toxicity with an order of potency of myxothiazol ≥ stigmatellin > rotenone > antimycin A, suggesting a cell-specific cytotoxic interplay between mitochondrial complex I and III function and TAM action.     

Noninvasive mitochondrial imaging in live cell culture

The observed distribution of mitochondria in a cell can vary with environmental influence, degree of differentiation and disease. Differences in the distribution of mitochondrial autofluorescence may be used to distinguish these different cellular states.     

Photoactivation of pheophorbide a induces a mitochondrial-mediated apoptosis in Jurkat leukaemia cells

The mechanism of cell death by pheophorbide a (Pba) which has been established to be a potential photosensitizer was examined in experimental photodynamic therapy (PDT) on Jurkat cells, a human lymphoid tumor cell line. In 30-60 min after irradiation, Pba treated cells exhibited apoptotic features including membrane blebbing and DNA fragmentation. Pba/PDT caused a rapid release of cytochrome c from mitochondria into the cytosol. Sequentially, activation of caspase-3 and the cleavage of poly ADP-ribose polymerase (PARP) were followed. Meanwhile, no evidence of activation of caspase-8 was indicated in the cells. In experiments with caspase inhibitors, it was found that caspase-3 alone was sufficient initiator for the Pba-induced apoptosis of the cells. Pba specific emission spectra were confirmed in the mitochondrial fraction and the light irradiation caused a rapid change in its membrane potential. Thus, mitochondria were entailed as the crucial targets for Pba as well as a responsible component for the cytochrome c release to initiate apoptotic pathways. Taken together, it was concluded that the mode of Jurkat cell death by Pba/PDT is an apoptosis, which is initiated by mitochondrial cytochrome c release and caspase-3-pathways.     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

A FACTOR FROM GRANULOSA CELLS CAN STIMULATE OOCYTE TISSUE PLASMINOGEN ACTIVATOR ACTIVITY

Several studies indicate that substances synthesized by granulosa cells are capableof regulating oocyte activity. We have studied the effect of factors synthesized by gran-ulosa cells on tPA activity of denuded oocytes using a co- culture system. The resultsshow that an FSH- dependent factor(s) synthesized by granulosa cells (but not by theca-interstitial cells) is capable of stimulating tPA activity of denuded oocytes. This findingis important for understanding hormonal regulation of oocyte tPA activity by mediatorssynthesized in granulosa cells.     

An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.     

A factor from granulosa cells can stimulate oocyte tissue plasminogen activator activity

Several studies indicate that substances synthesized by granulosa cells are capable of regulating oocyte activity. We have studied the effect of factors synthesized by granulosa cells on tPA activity of denuded oocytes using a co-culture system. The results show that an FSH-dependent factor(s) synthesized by granulosa cells (but not by theca-interstitial cells) is capable of stimulating tPA activity of denuded oocytes. This finding is important for understanding hormonal regulation of oocyte tPA activity by mediators synthesized in granulosa cells.     

Fluorescence imaging mitochondrial copper(Ⅱ) via photocontrollable fluorogenic probe in live cells

Monitoring mitochondrial derived copper(Ⅱ) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper(Ⅱ). Herein, we have reported the design,synthesis and characterization of photocontrollable fluorogenic probe, M_(Cu)~(-3), which is equipped with a photo-labile group(nitrobenzyl group) and mitochondria targeting unit(triphenylphosphonium salt).This novel probe showed an intense fluorescence enhancement in response to copper(Ⅱ) without interference from other metal cations in the biological condition(p H 6–9). The detection limit is 1.7 ×10~(-7) mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated M_(Cu)~(-3) can visualize mitochondrial copper(Ⅱ) in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper(Ⅱ) during mitochondria specific detection.     

Protective Effects of Liquiritigenin against Cisplatin-Induced Nephrotoxicity via NRF2/SIRT3-Mediated Improvement of Mitochondrial Function

Acute kidney injury (AKI) induced by cisplatin (CP), a first-line anticancer drug for chemotherapy, is common. To date, there is an urgent need to find effective treatments to reduce the nephrotoxicity caused by CP. Meanwhile, the restoration of mitochondrial dysfunction shows potential to be used as an adjunct to conventional therapeutic strategies. This study found that liquiritigenin can ameliorate mitochondrial dysfunction and acute kidney injury induced by CP in mice. The intraperitoneal injection of 15 mg/kg body weight liquiritigenin for 2 days markedly protected against CP-induced mitochondrial dysfunction, restored renal tubule and mitochondrial morphology, decreased blood Scr and BUN levels, and decreased cell apoptosis. Furthermore, the elevated expression of SIRT3 induced by liquiritigenin, which can be upregulated by NRF2, was confirmed in vivo and in vitro. The underlying protective mechanisms of liquiritigenin in CP-induced nephrotoxicity were then investigated. Molecular docking results showed that liquiritigenin has potent binding activities to KEAP1, GSK-3β and HRD1. Further results showed that liquiritigenin induced the nuclear translocation of NRF2 and increased the levels of mitochondrial bioenergetics-related protein such as PGC-1α, and TFAM, which are related to NRF2 activity and mitochondrial biogenesis. In addition, liquiritigenin was found to possibly reverse the decrease in BCL2/BAX ratio induced by CP in live cultured renal tubule epithelial cells. Collectively, these results indicated that liquiritigenin could be used as a potential nephroprotective agent to protect against cisplatin-induced acute kidney injury in a NRF2-dependent manner by improving mitochondria function.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

On the biological properties of alkynyl phosphine gold(i) complexes

Golden times for metal-based drugs? Alkynyl triphenylphosphine gold(I) complexes display interesting biological properties and show high potential for future drug development. They are strong inhibitors of the enzyme thioredoxin reductase, trigger antiproliferative effects in tumor cells, and influence tumor cell metabolism, mitochondrial respiration, and angiogenesis in zebrafish embryos.