9 research outputs found
Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration
Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described
Design and Synthesis of CNS-targeted Flavones and Analogues with Neuroprotective Potential Against H; 2; O; 2; - and Aβ; 1-42; -Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells
With the lack of available drugs able to prevent the progression of Alzheimer's disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their; C; -glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analysis, log; D; 7.4; was determined. From all screened compounds, the; p; -morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H; 2; O; 2; - and Aβ; 1-42; -induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of; C; -glucosylflavones as neuroprotective agents, and presents the; p; -morpholinyl; C; -glucosylflavone; 37; , which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 μM, as a new lead structure for further development against AD