Malcolm Purdey

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone.

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte‐secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs—namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15‐induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose‐6‐phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15‐treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione‐reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH‐stimulation alone decreases the oocytes’ ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence. Mol. Reprod. Dev. 82: 281–294, 2015.

A Dual Sensor for pH and Hydrogen Peroxide Using Polymer-Coated Optical Fibre Tips

This paper demonstrates the first single optical fibre tip probe for concurrent detection of both hydrogen peroxide (H2O2) concentration and pH of a solution. The sensor is constructed by embedding two fluorophores: carboxyperoxyfluor-1 (CPF1) and seminaphtharhodafluor-2 (SNARF2) within a polymer matrix located on the tip of the optical fibre. The functionalised fibre probe reproducibly measures pH, and is able to accurately detect H2O2 over a biologically relevant concentration range. This sensor offers potential for non-invasive detection of pH and H2O2 in biological environments using a single optical fibre.

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence

ABSTRACT Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Cancer Detection in Human Tissue Samples Using a Fiber-Tip pH Probe

Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis, and are restricted to excised tissue. In this work, we report the development of an optical fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration, which could find use in the operating theater. Use of this probe in surgery either on excised or in vivo tissue has the potential to improve success rates for complete removal of cancers. Cancer Res; 76(23); 6795-801. ©2016 AACR.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%)

STUDY QUESTION Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? SUMMARY ANSWER Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. WHAT IS KNOWN ALREADY Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. STUDY DESIGN, SIZE, DURATION The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. PARTICIPANTS/MATERIALS, SETTING, METHODS In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. MAIN RESULTS AND THE ROLE OF CHANCE Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. LIMITATIONS, REASONS FOR CAUTION Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. WIDER IMPLICATIONS OF THE FINDINGS Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premiers Science and Research Fund. The authors declare there are no conflict of interest.

Localised hydrogen peroxide sensing for reproductive health

The production of reactive oxygen species (ROS) is known to affect the developmental competence of embryos. Hydrogen peroxide (H2O2) an important reactive oxygen species, is also known to causes DNA damage and defective sperm function. Current techniques require incubating a developing embryo with an organic fluorophore which is potentially hazardous for the embryo. What we need is a localised ROS sensor which does not require fluorophores in solution and hence will allow continuous monitoring of H2O2 production without adversely affect the development of the embryo. Here we report studies on such a fibre-based sensor for the detection of H2O2 that uses a surface-bound aryl boronate fluorophore carboxyperoxyfluor-1(CPF1). Optical fibres present a unique platform due to desirable characteristics as dip sensors in biological solutions. Attempts to functionalise the fibre tips using polyelectrolyte layers and (3-aminopropyl)triethoxysilane (APTES) coatings resulted in a limited signal and poor fluorescent response to H2O2 due to a low tip surface density of the fluorophore. To increase the surface density, CPF1 was integrated into a polymer matrix formed on the fibre tip by a UV-catalysed polymerisation process of acrylamide onto a methacrylate silane layer. The polyacrylamide containing CPF1 gave a much higher surface density than previous surface attachment methods and the sensor was found to effectively detect H2O2. Using this method, biologically relevant concentrations of H2O2 were detected, enabling remote sensing studies into ROS releases from embryos throughout early development.

A portable device for cancer margin assessment using a pH sensitive optical fibre probe

We report the development of an optical fibre probe which could find use as an aid for margin detection during surgery. A fluorophore doped polymer is coated on an optical fibre tip, allowing for pH values of unknown tissue to be measured and compared to healthy tissue, to discriminate between healthy and cancerous tissue. This probe is integrated within a portable system, requiring no alignment or access to specialised vibration damped optical tables. A miniature spectrometer allows for measurements to be performed on-site, opening up the potential for use of these probes within the operating theatre for in-vivo measurements.

An organic fluorophore-nanodiamond hybrid sensor for photostable imaging and orthogonal, on-demand biosensing

Organic fluorescent probes are widely used to detect key biomolecules; however, they often lack the photostability required for extended intracellular imaging. Here we report a new hybrid nanomaterial (peroxynanosensor, PNS), consisting of an organic fluorescent probe bound to a nanodiamond, that overcomes this limitation to allow concurrent and extended cell-based imaging of the nanodiamond and ratiometric detection of hydrogen peroxide. Far-red fluorescence of the nanodiamond offers continuous monitoring without photobleaching, while the green fluorescence of the organic fluorescent probe attached to the nanodiamond surface detects hydrogen peroxide on demand. PNS detects basal production of hydrogen peroxide within M1 polarised macrophages and does not affect macrophage growth during prolonged co-incubation. This nanosensor can be used for extended bio-imaging not previously possible with an organic fluorescent probe, and is spectrally compatible with both Hoechst 33342 and MitoTracker Orange stains for hyperspectral imaging.

Biosensors for detecting stress in developing embryos

Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.

A simple optical fibre probe for differentiation between healthy and tumorous tissue

Incomplete removal of malignant tumours continues to be a significant issue in cancer surgery. It increases the risk of local recurrence and impaired survival, and results in the need for additional surgery with associated attendant costs and morbidity. While pathological methods exist to determine tissue type during surgery, these methods can compromise post-operative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis and are restricted to excised tissue. In this work we report the development of an optical fibre probe which could find use as an aid for margin detection during surgery. A fluorophore doped polymer coating is deposited on the tip of an optical fibre, which can then be used to record the pH by monitoring the emission spectra from the embedded indicator. The pH values of unknown tissue are measured and compared to healthy tissue, allowing for discrimination between healthy and cancerous tissue. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration which could find use in the operating theatre. Use of this probe in surgery either on excised or in-vivo tissue has the potential to improve success rates for complete removal of cancers.