Effect of Urolithin A on Bovine Sperm Capacitation and In Vitro Fertilization.

Reactive oxygen species (ROS) play a critical role in the functional competence of sperm cells. Conversely, excessive generation of ROS can impair sperm function, including their fertilization ability. Urolithin A (UA), a gut bacteria-derived metabolite produced from the transformation of ellagitannins, with anti-aging and antioxidant properties, was investigated for the first time in bovine sperm cells in the present study. Firstly, different doses of UA (0, 1, and 10 µM; 8-16 sessions) were used during the capacitation process of frozen-thawed bovine sperm. Sperm motility was assessed using optical microscopy and CASA. Sperm vitality (eosin-nigrosin), ROS, and ATP levels, as well as mitochondrial membrane potential (JC1) and oxygen consumption were evaluated. A second experiment to test the effect of different doses of UA (0, 1, and 10 µM; 9 sessions) in both the capacitation medium, as above, and the fertilization medium, was also implemented. The embryonic development and quality were evaluated. UA, at a concentration of 1 µM, significantly improved sperm movement quality (p < 0.03). There was a trend towards an increase in the oxygen consumption rate (OCR) of capacitated sperm with 1 µM and 10 µM UA supplementation. Moreover, an increase in ATP levels (p < 0.01) was observed, accompanied by a reduction in ROS levels at the higher UA concentration. These results suggest that UA may enhance spermatozoa mitochondrial function, modifying their metabolic activity while reducing the oxidative stress. Also, the number of produced embryos appears to be positively affected by UA supplementation, although differences between the bulls may have mitigated this effect. In conclusion, presented results further support previous findings indicating the potential therapeutic value of UA for addressing reproductive sub/infertility problems and improving ART outcomes. In addition, our results also reinforce the important bull effect on ART and that male sperm bioenergetic parameters should be used to predict spermatozoa functionality and developmental potential.

Moderate water stress causes different stomatal and non-stomatal changes in the photosynthetic functioning of Phaseolus vulgaris L. genotypes.

The impact of moderate water deficit on the photosynthetic apparatus of three Phaseolus vulgaris L. cultivars, Plovdiv 10 (P10), Dobrudjanski Ran (DR) and Prelom (Prel), was investigated. Water shortage had less impact on leaf hydration, RWC (predawn and midday) and predawn water potential in Prel. RWC and Ψ(p) were more reduced in P10, while there was no osmotic adjustment in any cultivar. Although drought drastically reduced stomatal opening in P10 and DR, reduced A(max) indicated non-stomatal limitations that contributed to the negligible P(n). These limitations were on potential thylakoid electron transport rates of PSI and II, pointing to photosystem functioning as a major limiting step in photosynthesis. This agrees with decreases in actual photochemical efficiency of PSII (F(v)'/F(m)'), quantum yield of photosynthetic non-cyclic electron transport (ϕ(e)) and energy-driven photochemical events (q(P)), although the impact on these parameters would also include down-regulation processes. When compared to DR, Prel retained a higher functional state of the photosynthetic machinery, justifying reduced need for photoprotective mechanisms (non-photochemical quenching, zeaxanthin, lutein, ß-carotene) and maintenance of the balance between energy capture and dissipative pigments. The highest increases in fructose, glucose, arabinose and sorbitol in Prel might be related to tolerance to a lower oxidative state. All cultivars had reduced A(max) due to daytime stomatal closure in well-watered conditions. Under moderate drought, Prel had highest tolerance, higher leaf hydration and maintenance of important photochemical use of energy. However, water shortage caused appreciable non-stomatal limitations to photosynthesis linked to regulation/imbalance at the metabolic level (and growth) in all cultivars. This included damage, as reflected in decreased potential photosystem functioning, pointing to higher sensitivity of photosynthesis to drought than is commonly assumed.

Impact of UV-B irradiation on photosynthetic performance and chloroplast membrane components in Oryza sativa L.

The impact of UV-B radiation on photosynthetic related parameters was studied in Oryza sativa L. cv. Safari plants, after an UV-B irradiation performed 1h per day for 7days (between 8 and 14days after germination) with a ten narrow-band (λ 311nm) that resulted in a total biological effective UV-B (UVB(BE)) of 2.975kJm(-2)day(-1) and a total of 20.825kJm(-2). Gas exchange measurements were severely affected, showing reductions higher than 80% in net photosynthesis (P(n)), stomatal conductance and photosynthetic capacity (A(max)), 1day after the end of the 7-days UV-B treatment. Similarly, several fluorescence parameters (F(o), F(v)/F(m), Fv'/Fm', ϕ(e), q(P) and q(E)) and thylakoid electron transport (involving both photosystems) were also severely reduced. Concomitantly, a decline of xanthophylls, carotenes, Chl a, Chl (a+b) and Chl (a/b) values was accompanied by the increase of the lipoperoxidation level in chloroplast membranes, altogether reflecting a loss of protection against oxidative stress. Seven days after of the end of UV-B treatment, most fluorescence parameters recovered, but in P(n), A(max), thylakoid electron transport rates, Chl a and lipid classes, as well as the level of lipoperoxidation, the impacts were even stronger than immediately after the end of stress, denoting a clear loss of performance of photosynthetic structures. However, only a moderate impact on total lipids was observed, accompanied by some changes in the relative weight of the major chloroplast membrane lipid classes, with emphasis on the decrease of MGDG and the increase of phospholipids. That suggested an ability to de novo lipid synthesis allowing qualitative changes in the lipid matrix. Notably, the leaves developed after the end of UV-B irradiation showed a much lower impact, with significantly decreased values only in P(n) and g(s), rises in several fluorescence parameters, thylakoid electron transport, photosynthetic pigments (xanthophylls and chls) and DEPS, while lipid classes presented values close to control. The results showed a global impact of UV-B in the photosynthetic structures and performance in irradiated leaves, but revealed also a low impairment extent in the leaves entirely developed after the end of the irradiation, reflecting a remarkable recovery of the plant after the end of stress, what could constitute an advantage under occasional UV-B exposure events in this vital worldwide staple food crop.

The impact of cold on photosynthesis in genotypes of Coffea spp.--photosystem sensitivity, photoprotective mechanisms and gene expression.

Environmental constraints disturb plant metabolism and are often associated with photosynthetic impairments and yield reductions. Among them, low positive temperatures are of up most importance in tropical plant species, namely in Coffea spp. in which some acclimation ability has been reported. To further explain cold tolerance, the impacts on photosynthetic functioning and the expression of photosynthetic-related genes were analyzed. The experiments were carried out along a period of slow cold imposition (to allow acclimation), after chilling (4°C) exposure and in the following rewarming period, using 1.5-year-old coffee seedlings of 5 genotypes with different cold sensitivity: Coffea canephora cv. Apoatã, Coffea arabica cv. Catuaí, Coffea dewevrei and 2 hybrids, Icatu (C. arabica×C. canephora) and Piatã (C. dewevrei×C. arabica). All genotypes suffered a significant leaf area loss only after chilling exposure, with Icatu showing the lowest impact, a first indication of a higher cold tolerance, contrasting with Apoatã and C. dewevrei. During cold exposure, net photosynthesis and Chl a fluorescence parameters were strongly affected in all genotypes, but stomatal limitations were not detected. However, the extent of mesophyll limitation, reflecting regulatory mechanisms and/or damage, was genotype dependent. Overnight retention of zeaxanthin was common to Coffea genotypes, but the accumulation of photoprotective pigments was highest in Icatu. That down-regulated photochemical events but efficiently protected the photosynthetic structures, as shown, e.g., by the lowest impacts on A(max) and PSI activity and the strongest reinforcement of PSII activity, the latter possibly reflecting the presence of a photoprotective cycle around PSII in Icatu (and Catuaí). Concomitant to these protection mechanisms, Icatu was the sole genotype to present simultaneous upregulation of caCP22, caPI and caCytf, related to, respectively, PSII, PSI and to the complex Cytb(6)/f, which could promote better repair ability, contributing to the maintenance of efficient thylakoid functioning. We conclude that Icatu showed the best acclimation ability among the studied genotypes, mostly due to a better upregulation of photoprotection and repair mechanisms. We confirmed the presence of important variability in Coffea spp. that could be exploited in breeding programs, which should be assisted by useful markers of cold tolerance, namely the upregulation of antioxidative molecules, the expression of selected genes and PSI sensitivity.

Dark stained tissues of the epicarp of encore mandarin: interactions with the production of hydroxyl radicals.

The interactions between hydroxyl radical production and the composition of the epicarp cells associated with the dark stained tissues of mature fruits of Encore mandarin were investigated. Phosphatidylinositol content of the cells associated with dark stains was lower in unpitted tissues, whereas the concentration of phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine did not vary significantly. In these pitted cells, protein content also showed a 1.59-fold increase. Additionally, the relative proportions of gln, thr, asp, glu and gly increased sharply, while ala and tyr decreased. The polypeptide patterns showed quantitative changes when samples of stained and unstained tissues were compared by SDS-PAGE electrophoresis. Qualitatively, the cells associated with pitted tissues revealed a new polypeptide band with an apparent mol. wt. of 50.4 kDa as well as the disappearance of another one of 10 kDa. Ethylene production in the dark stained tissues was lower than in unpitted tissues and these cells showed significant increases in membrane permeability, hydroxyl radical production, and lipid peroxidation. Furthermore, in these tissues the levels of the carotenoids increased significantly but levels of chlorophyll decreased. It was concluded that in the pitted tissues the significant changes of the membrane composition are closely associated with an increasing acyl lipid peroxidation mediated by hydroxyl radical production. The modulation of this metabolism further indicates an incomplete degradation of fatty acids. The implications of the increasing accumulation of carotenoids in the synthesis of oxy radicals are also discussed.