Microoxygraph Device for Biosensoristic Applications

Oxygen consumption rate (OCR) is a significant parameter helpful to determine in vitro respiratory efficiency of living cells. Oxygen is an excellent oxidant and its electrocatalytic reduction on a noble metal allows accurately detecting it. By means of microfabrication technologies, handy, low-cost, and disposable chip can be attained, minimizing working volumes and improving sensitivity and response time. In this respect, here is presented a microoxygraph device (MOD), based on Clark’s electrode principle, displaying many advantageous features in comparison to other systems. This lab-on-chip platform is composed of a three-microelectrode detector equipped with a microgrooved electrochemical cell, sealed with a polymeric reaction chamber. Au working/counter electrodes and Ag/AgCl reference electrode were fabricated on a glass slide. A microchannel was realized by photoresist lift-off technique and a polydimethylsiloxane (PDMS) nanoporous film was integrated as oxygen permeable membrane (OPM) between the probe and the microreaction chamber. Electrochemical measurements showed good reproducibility and average response time, assessed by periodic injection and suction of a reducing agent. OCR measurements on 3T3 cells, subjected, in real time, to chemical stress on the respiratory chain, were able to show that this chip allows performing consistent metabolic analysis

A Krill Oil Supplemented Diet Suppresses Hepatic Steatosis in High-Fat Fed Rats

Krill oil (KO) is a dietary source of n-3 polyunsaturated fatty acids, mainly represented by eicosapentaenoic acid and docosahexaenoic acid bound to phospholipids. The supplementation of a high-fat diet with 2.5% KO efficiently prevented triglyceride and cholesterol accumulation in liver of treated rats. This effect was accompanied by a parallel reduction of the plasma levels of triglycerides and glucose and by the prevention of a plasma insulin increase. The investigation of the molecular mechanisms of KO action in high-fat fed animals revealed a strong decrease in the activities of the mitochondrial citrate carrier and of the cytosolic acetyl-CoA carboxylase and fatty acid synthetase, which are both involved in hepatic de novo lipogenesis. In these animals a significant increase in the activity of carnitine palmitoyl-transferase I and in the levels of carnitine was also observed, suggesting a concomitant stimulation of hepatic fatty acid oxidation. The KO supplemented animals also retained an efficient mitochondrial oxidative phosphorylation, most probably as a consequence of a KO-induced arrest of the uncoupling effects of a high-fat diet. Lastly, the KO supplementation prevented an increase in body weight, as well as oxidative damage of lipids and proteins, which is often found in high-fat fed animals

Stallion semen quality depends on major histocompatibility complex matching to teaser mare.

The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm numbers as compared to exposure to an MHC-similar mare. Sperm velocity seemed not affected by the treatment. Overall genetic similarity between stallions and mares (determined from polymorphic microsatellites on 20 different chromosomes) played no significant role here. The MHC type of the teaser mare also affected characteristics of cold-stored sperm after 24 and 48 hr. As expected from ejaculate economics, sperm viability was elevated after exposure to an MHC-dissimilar mare. However, oxidative stress and the percentage of sperm with a high DNA fragmentation were mostly increased after exposure to an MHC-dissimilar mare, depending also on whether the teaser mare was in oestrous or not. We conclude that males can quickly adjust ejaculate quality relative to a female's MHC, and that this male reaction to the social environment can also affect important characteristics of cold-stored semen

Oxygen uptake by mitochondria in demembranated human spermatozoa: a reliable tool for the evaluation of sperm respiratory efficiency.

In this work we report a relatively simple and fast method for analyzing oxygen consumption and therefore mitochondrial functionality, in individual human ejaculates. This oxygraphic method requires a low number of cells, is highly reproducible and linearly correlates with sperm concentration. Our results showed that oxygen uptake by mitochondria of demembranated sperm cells is significantly stimulated by respiratory substrates and ADP. The RCR values indicate a good coupling between respiration and phosphorylation by sperm mitochondria and thus a well preserved integrity of the mitochondria themselves. Interestingly, whereas the rates of oxygen uptake (V3 and V4), as expected, changed with different sperm concentrations, the RCR values remained constant thus demonstrating a linear response of the assay. The results obtained with the use of a panel of specific inhibitors of the respiratory chain suggested that this method, besides its potential clinical application, can also be useful for a deeper understanding of the biochemical properties of sperm mitochondria and their role in ATP production in human spermatozoa

The role of mitochondria in energy production for human sperm motility

Mitochondria of spermatozoa are different from the corresponding organelles of somatic cells, in both their morphology and biochemistry. The biochemical differences are essentially related to the existence of specific enzyme isoforms, which are characterized by peculiar kinetic and regulatory properties. As mitochondrial energy metabolism is a key factor supporting several sperm functions, these organelles host critical metabolic pathways during germ cell development and fertilization. Furthermore, spermatozoa can use different substrates, and therefore activate different metabolic pathways, depending on the available substrates and the physico-chemical conditions in which they operate. This versatility is critical to ensure fertilization success. However, the most valuable aspect of mitochondria function in all types of cells is the production of chemical energy in the form of ATP which can be used, in the case of spermatozoa, for sustaining sperm motility. The latter, on the other hand, represents one of the major determinants of male fertility. Accordingly, the presence of structural and functional alterations in mitochondria from asthenozoospermic subjects confirms the important role played by these organelles in energy maintenance of sperm motility. The present study gives an overview of the current knowledge on the energy-producing metabolic pathways operating inside human sperm mitochondria and critically analyse the differences with respect to somatic mitochondria. Such a comparison has also been carried out between the functional characteristics of human sperm mitochondria and those of other mammalian species. A deeper understanding of mitochondrial energy metabolism could open up new avenues of investigation in bioenergetics of human sperm mitochondria, both in physiological and pathological condition

The role of mitochondria in energy production for human sperm motility

Mitochondria of spermatozoa are different from the corresponding organelles of somatic cells, in both their morphology and biochemistry. The biochemical differences are essentially related to the existence of specific enzyme isoforms, which are characterized by peculiar kinetic and regulatory properties. As mitochondrial energy metabolism is a key factor supporting several sperm functions, these organelles host critical metabolic pathways during germ cell development and fertilization. Furthermore, spermatozoa can use different substrates, and therefore activate different metabolic pathways, depending on the available substrates and the physico-chemical conditions in which they operate. This versatility is critical to ensure fertilization success. However, the most valuable aspect of mitochondria function in all types of cells is the production of chemical energy in the form of ATP which can be used, in the case of spermatozoa, for sustaining sperm motility. The latter, on the other hand, represents one of the major determinants of male fertility. Accordingly, the presence of structural and functional alterations in mitochondria from asthenozoospermic subjects confirms the important role played by these organelles in energy maintenance of sperm motility. The present study gives an overview of the current knowledge on the energy-producing metabolic pathways operating inside human sperm mitochondria and critically analyse the differences with respect to somatic mitochondria. Such a comparison has also been carried out between the functional characteristics of human sperm mitochondria and those of other mammalian species. A deeper understanding of mitochondrial energy metabolism could open up new avenues of investigation in bioenergetics of human sperm mitochondria, both in physiological and pathological conditions