Apoptosis plays an essential role in normal spermatogenesis, but deregulations of this biological process, which is closely associated with male infertility, have been found. Whereas calcium homeostasis is a key regulator of cell survival, sustained elevation of intracellular calcium plays a role in apoptosis. The aim of this research was to determine the role of two different calcium mobilizing agents, hydrogen peroxide (H(2)O(2)) and the physiological agonist progesterone, on the apoptosis process of human ejaculated spermatozoa. Translocation of membrane phosphatidylserine was examined with an annexin V binding assay, DNA damage was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and caspase-3 activity was assessed using a fluorometric assay. After incubation of spermatozoa for 1 h with either 10 microM H(2)O(2) or 20 microM of progesterone, there was a significant increase in both caspase-3 activity and the percentage of annexin V-positive cells. Similarly, the TUNEL results were significantly higher 1 h after incubation with either 10 microM H(2)O(2) or 20 microM of progesterone. In fact, progesterone-treated cells showed a three-fold increase (from 17.6 to 52.9%) of TUNEL-positive cells compared to untreated cells, while H(2)O(2)-treated cells exhibited a two-fold increase (from 17.6 to 37.9%). In sum, our results suggest that spermatozoa treated with calcium mobilizing agents, such as H(2)O(2) and progesterone, seem to undergo an apoptosis process that is dependent on caspase-3 activation.

Red grapes contain elevated amounts of antioxidant compounds (polyphenols) that may potentially prevent cell aging, cardiovascular disease and oxidation-related disorders. Since functional drinks are presently one of the most dynamic sectors of the market, the present work was aimed at evaluating the possible antioxidant effect of an experimental grape juice in terms of urinary 6-sulfatoxymelatonin (aMT6-s) and total antioxidant capacity in young (20 ± 10 yr-old), middle-aged (45 ± 10 yr-old) and elderly (75 ± 10 yr-old) individuals. Grapes (Vitis vinifera cv. Tempranillo) were de-stemmed, racked and pressed. The juice was subsequently stabilized by high hydrostatic pressure (HHP). Participants consumed 200 mL of grape juice twice a day (as the lunch and dinner desserts) for 5 days. First-void morning urines were collected before treatment (basal values), the day immediately after the last ingestion of juice (assay), and one day afterwards (post-assay). aMT6-s and total antioxidant capacity were quantified using commercial ELISA and colorimetric assay kits, respectively. The intake of grape juice cv. Tempranillo induced a significant increase of urinary aMT6-s and total antioxidant capacity in the three groups of age analyzed as compared to their corresponding basal and post-assay values. These functional/nutraceutical properties may be of interest for a prospective commercialization of the grape juice. The novel technology used for juice stabilization may be suitable for introducing into the market a product with high sensory and nutritional quality, as it has been shown in this study.

Abstract:  Ageing is associated with an increased production of free radicals and alterations in the mechanisms of adaptation to oxidative stress. In fact, the free radical theory of ageing proposes that deleterious actions of free radicals are responsible for the functional deterioration associated with ageing. Moreover, a close relationship exists between calcium homeostasis and oxidative stress. The current work was aimed at proving that intracellular calcium overload induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and/or thapsigargin leads to oxidative stress. We additionally examined the effect of melatonin on the levels of reactive oxygen species (ROS) and cell viability in human leucocytes collected from young (20–30‐year‐old) and elderly (65–75‐year‐old) individuals under both basal and oxidative stress‐induced conditions. Treatments with 10 nM FMLP and/or 1 μM thapsigargin induced a transient increase in cytosolic free‐calcium concentration ([Ca^2 +]_c) in human leucocytes due to calcium release from internal stores, and led in turn to oxidative stress, as assessed by intracellular ROS measurement. Non‐treated leucocytes from aged individuals exhibited higher ROS levels and lower rates of cell survival when compared to leucocytes from young individuals. Similar results were obtained in FMLP and/or thapsigargin‐treated leucocytes from elderly individuals when compared to those from the young individuals. Melatonin treatment significantly reduced both hydrogen peroxide (H₂O₂) and superoxide anion levels, likely due to its free‐radical scavenging properties, and enhanced leucocyte viability in both age groups. Therefore, melatonin may be a useful tool for the treatment of disease states and processes where an excessive production of oxidative damage occurs.