The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

L-cysteine's effect on modulated rat brain enzymes with forced swimming.

BACKGROUND: Forced exercise is associated with oxidative stress, and L-cysteine (L-cys) administration reduces free-radical production. AIM: To investigate whether L-cys (5 mg/kg) intraperitoneal administration can ameliorate modulated total antioxidant status (TAS), protein concentration, and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase, and Mg2+-ATPase in rat brain after 2 and 3 hr of forced swimming. METHODS: TAS, protein, and enzyme activities were measured spectrophotometrically before and after 2 and 3 hr of exercise without or with L-cys administration. RESULTS: TAS concentration (55.6 +/- 1.5 vs. 42.1 +/- 1.0 vs. 37.4 +/- 1.2 micromol/L, p < .001), protein concentration (5.68 +/- 0.36 vs. 5.40 +/- 0.18 vs. 4.01 +/- 0.16 mg/ml, p < .01), and AChE activity (0.89 +/- 0.05 vs. 0.61 +/- 0.04 vs. 0.48 +/- 0.03 DeltaOD/min x mg protein, p < .001) were significantly reduced, whereas Na+,K+-ATPase (6.00 +/- 0.36 vs. 10.44 +/- 1.04 vs. 11.90 +/- 1.21 micromol phosphorus inorganic/hr x mg protein, p < .001) and Mg2+-ATPase activity (7.20 +/- 0.65 vs. 10.88 +/- 1.08 vs. 11.55 +/- 1.22 mmol phosphorus inorganic/hr x mg protein, p < .001) were statistically significantly increased after 2 and 3 hr of forced exercise. Post-L-cys administration, AChE activity was decreased (0.90 +/- 0.04 vs. 0.47 +/- 0.02 DeltaOD/min x mg protein, p < .001) and remained unaltered (0.64 +/- 0.04 vs. 0.67 +/- 0.04 DeltaOD/min x mg protein, p > .05) 2 and 3 hr postexercise (0.47 +/- 0.02 vs. 0.54 +/- 0.02 DeltaOD/min x mg protein, p > .05). Na+,K+-ATPase was decreased and remained unchanged (1.85 +/- 0.17 vs. 1.77 +/- 0.19 mumol phosphorus inorganic/hr x mg protein, p > .05) 2 and 3 hr postswimming (1.91 +/- 0.19 vs. 2.06 +/- 0.17 mumol phosphorus inorganic/hr x mg protein, p > .05). Mg2+-ATPase activity was similar with L-cys supplementation pre- vs. postswimming. CONCLUSIONS: L-cys administration might ameliorate modulated rat brain enzyme activities induced by free-radical production during forced swimming.

The in vivo and in vitro effects of L-carnitine supplementation on the erythrocyte membrane acetylcholinesterase, Na+, K+-ATPase and Mg2+-ATPase activities in basketball players.

BACKGROUND: We investigated whether the activities of erythrocyte membrane acetylcholinesterase (AChE), Na+, K+-ATPase and Mg2+-ATPase are modulated in basketball players pre- vs. post-forced training with or without L-carnitine (L-C) supplementation. METHODS: Blood was obtained from 10 male players pre-game (group A) and post-game (group B) and after 1 month L-C supplementation (2 g/24 h orally) pre-training (group C) and post-training (group D). Lactate, pyruvate and total antioxidant status (TAS) were measured with commercial kits, catecholamines with HPLC and the enzyme activities spectrophotometrically. RESULTS: Lactate, pyruvate, AChE, Na+, K+-ATPase and catecholamines were increased (p<0.001) and TAS was decreased (p<0.001) in group B. In contrast, TAS remained unaltered and the all enzyme activities were reduced (p<0.001) in group D at the same time of study. Mg2+-ATPase activity remained unchanged. In vitro incubation of the modulated AChE and Na+, K+-ATPase with L-C (25 microM) from group B and group D resulted in a non-significant reduction of the enzymes in group B and complete restoration of their activities in group D. CONCLUSIONS: The increase of AChE and Na+, K+-ATPase activities may be due to the elevation of catecholamines in group B. Carnitine utilization by the muscles during training may result in a reduction of the enzyme activities (group D). The latter is supported by the recovery of the enzyme activities after incubation of the membranes from group D with L-C.

The effect of aspartame metabolites on the suckling rat frontal cortex acetylcholinesterase. An in vitro study.

UNLABELLED: Aspartame (ASP) consumption is suggested to be implicated with muscarinic dysfunction. The aim of this work was to evaluate the effect of ASP and its metabolites on acetylcholinesterase (AChE) activity in rat frontal cortex and pure enzyme. Rat frontal cortex homogenate or pure enzyme AChE (eel E. Electricus) were incubated with ASP and each of ASP components, phenylalanine (Phe), aspartic acid (asp), and methanol (MeOH) for 1 h at 37 degrees C. AChE was measured spectrophotometrically. The results showed that incubation of rat tissue or pure enzyme with the sum of ASP metabolites, as reported to be found in the CSF after 150 or 200 mg/kg ASP consumption, inhibited frontal cortex and pure AChE about -11% to -29% (p<0.001). Asp, Phe or MeOH concentrations related to their CSF levels after ingestion of abuse or toxic ASP doses, when separately incubated with frontal cortex or pure AChE, resulted in a significant decrease of the enzyme activities. IN CONCLUSION: ASP compounds may directly and/or indirectly act on the frontal cortex AChE. High or toxic doses of the sweetener remarkably decreased the enzyme activity. If this in vitro finding comes into human reality, it may be suggested that cholinergic symptoms are related to the consumption of the above ASP doses.

L-cysteine supplementation prevents exercise-induced alterations in human erythrocyte membrane acetylcholinesterase and Na+,K+-ATPase activities.

BACKGROUND: L-Cysteine (L-Cys) is implicated in the reduction of free radical production. The aim of this study was to investigate whether L-Cys supplementation prevents modulation of the activities of erythrocyte membrane acetylcholinesterase (AChE), Na(+),K(+)-ATPase and Mg(2+)-ATPase induced by free radicals in basketball players during training. METHODS: Blood was obtained from 10 basketball male players before (group A) and after a game (group B) and after 1 week of L-Cys (0.5 g/24 h orally) supplementation before (group C) and after training (group D). Lactate, pyruvate and total antioxidant status (TAS) were measured using commercial kits and the enzyme activities were determined spectrophotometrically. RESULTS: Both lactate and pyruvate levels remarkably increased after exercise. In contrast, TAS levels significantly decreased in group B, increased in group C and then declined (group D), reaching those of group A. AChE activity was statistically increased post-exercise (3.98+/-0.04 Delta OD/min x mg protein) compared with pre-training (2.90+/-0.05 Delta OD/min x mg protein, p<0.01). Na(+),K(+)-ATPase activity was also higher post-exercise (1.27+/-0.05 micromol Pi/h x mg protein) than that pre-exercise (0.58+/-0.04 micromol Pi/h x mg protein, p<0.001). When the players were supplemented with L-Cys, both AChE and Na(+),K(+)-ATPase activities remained unaltered post-exercise. Mg(2+)-ATPase activities were unchanged in all groups studied. CONCLUSIONS: L-Cys supplementation may protect the enzyme activities studied against stimulation induced by free radical production during training in athletes by ameliorating their total antioxidant capacity.