1,721,060 research outputs found
Mechanisms underlying age-related defects in the mammalian oocyte: potential deleterious effects of methylglyoxal, a reactive dicarbonyl compound
No full textTatone C., Carbone M., Heizenrieder T., Gualtieri R., Eichenlaub-Ritter U. Mechanisms underlying age-related defects in the mammalian oocyte: potential deleterious effects of methylglyoxal, a reactive dicarbonyl compound University of L’Aquila, Dept of Health Sciences, L’Aquila (Italy) University of Bielefeld, Faculty of Biology, Gene Technolo/Microbiol, Bielefeld (Germany) University of Naples “Federico II”, Dept of Structural and Functional Biology, Naples (Italy) Methylglyoxal (MG) is a reactive dicarbonyl compound physiologically produced by various metabolic pathways. It causes inhibition of proliferation, and mitochondrial respiration,and increases reactive oxygen species, apoptosis and formation of advanced glycation end-products. Recent findings revealed a reduced MG scavenging efficiency in aged mouse ovaries. Therefore, we have investigated potential deleterious effects of MG on female gametes by exposing denuded (DO) and cumulus enclosed (COC) mouse oocytes from adult outbred mice (MF1,CD1) to 50-300M MG during in vitro maturation (IVM) for 16h or 19h. MG negatively affected the rate of polar body formation in both CEO and DO at 16h, with a more pronounced effect in DO. Oocytes with normal metaphase-II spindles decreased from about 73% (control) to 18% after 16h maturation in 75M MG. Moreover, oocytes in ana/telophase-I or with unaligned chromosomes were about three-to four-fold more abundant in the MG-exposed than in the control group. When maturation was prolonged to 19h MG-exposed oocytes exhibited normal appearing spindles and aligned chromosomes and increased PB rate. Time lapse analysis by polarisation microscopy confirmed that MG induced a pronounced meiotic delay. There was no increase in hyperploidy in the MG-exposed oocytes after 19h-IVM, while about 70% of meiosis I arrested oocytes had unaligned chromosomes at 16h-IVM suggesting that young oocytes are capable of dealing with a disturbance by MG by prolonging the spindle assembly checkpoint and progressing to meiosis II only when chromosomes are properly aligned and attached. CEO staining with JC-1 dye showed that MG was capable to induce changes in cytoplasmic localization of high polarized mitochondria. Furthermore, the number of foci of histone gamma H2AX increased in the nuclei of GV-arrested oocytes exposed for 5h to 75M MG, consistent with induction of DNA damage by MG. In accordance, TUNEL assay revealed about 30% apoptosis rate in CEO exposed to 300M MG. In conclusion, the present results indicate that MG may be one of the factors, which act synergistically to cause age-related changes in the ovarian and/or follicle microenvironment resulting in high susceptibility to meiotic errors and reduced developmental potential of aged oocytes
Effects of methylglyoxal-induced carbonyl stress on mitochondrial distribution and GSH-dependent redox potential in mouse oocytes
No full textTatone C, Heizenrieder T, Di Emidio G, Treffon P, Seidel T, Eichenlaub-Ritter U. Effects of methylglyoxal-induced carbonyl stress on mitochondrial distribution and GSH-dependent redox potential in mouse oocytes. Human Reproduction. 2011;26(Suppl.):I165
Biochemical and biological effects of KN-93, a selective inhibitor of calmodulin-dependent protein kinase II, on mouse egg activation induced by ethanol
No full textCalmodulin-dependent protein kinase ii (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mu mol KN-93 l(-1) produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mu mol KN-93 l(-1) was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation
La Procreazione Medicalmente assistita ed il SSN, la nostra esperienza 1999-2003
No full textSi descrive la nostra esperienza dal 1999 al 2003 nel campo della PMA in rapporto al SS
Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells.
No full textThe role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte
Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation.
No full textProtein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogenetically by specific PKC stimulators such as 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of 1) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited parthenogenones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence
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