Detrimental Effects of Non-Functional Spermatozoa on the Freezability of Functional Spermatozoa from Boar Ejaculate

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0 –native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H2DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa

Recent Advancements in the LC- and GC-Based Analysis of Malondialdehyde (MDA): A Brief Overview

Malondialdehyde (MDA) is an end-product of lipid peroxidation and a side product of thromboxane A2 synthesis. Moreover, it is not only a frequently measured biomarker of oxidative stress, but its high reactivity and toxicity underline the fact that this molecule is more than “just” a biomarker. Additionally, MDA was proven to be a mutagenic substance. Having said this, it is evident that there is a major interest in the highly selective and sensitive analysis of this molecule in various matrices. In this review, we will provide a brief overview of the most recent developments and techniques for the liquid chromatography (LC) and gas chromatography (GC)-based analysis of MDA in different matrices. While the 2-thiobarbituric acid assay still is the most prominent methodology for determining MDA, several advanced techniques have evolved, including GC–MS(MS), LC–MS(MS) as well as several derivatization-based strategies

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Background Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). Results The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. Conclusion The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of FBA to estimate intracellular fluxes also provides flux values that can be used as a starting point for rational engineering of C. reinhardtii. From these initial estimates, it is clear that aerobic heterotrophic growth on acetate has a low yield on carbon, while mixotrophically and autotrophically grown cells are significantly more carbon efficient

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with 13C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes

Application of medical and analytical methods in Lyme borreliosis monitoring

Lyme borreliosis (LB) is one of the most common tick-borne diseases in the northern hemisphere. It is a chronic inflammatory disease caused by the spirochaete Borrelia burgdorferi. In its early stages, pathological skin lesions, namely erythema chronicum migrans, appear. The lesions, usually localised at the site of the bite, may become visible from a few weeks up to 3 months after the infection. Predominant clinical symptoms of the disease also involve joint malfunctions and neurological or cardiac disorders. Lyme disease, in all its stages, may be successfully treated with antibiotics. The best results, however, are obtained in its early stages. In order to diagnose the disease, numerous medical or laboratory techniques have been developed. They are applied to confirm the presence of intact spirochaetes or spirochaete components such as DNA or proteins in tick vectors, reservoir hosts or patients. The methods used for the determination of LB biomarkers have also been reviewed. These biomarkers are formed during the lipid peroxidation process. The formation of peroxidation products generated by human organisms is directly associated with oxidative stress. Apart from aldehydes (malondialdehyde and 4-hydroxy-2-nonenal), many other unsaturated components such as isoprostenes and neuroprostane are obtained. The fast determination of these compounds in encephalic fluid, urine or plasma, especially in early stages of the disease, enables its treatment. Various analytical techniques which allow the determination of the aforementioned biomarkers have been reported. These include spectrophotometry as well as liquid and gas chromatography. The analytical procedure also requires the application of a derivatization step by the use of selected reagents

Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle