The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 posttranslational modifications and Akt expression levels. To this end, oocytes were retrived at 13, 29 and 33 h after hCG treatment. The presence/distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, Plk1 and Ser473/Thr308 pAkt as well as histone H3 and H4 acetylation/phosphorylation levels were assayed via immunofluorescence; Akt expression levels were determined via RT-PCR and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h post-hCG treatment showed similar levels of acetylated tubulin but ageing induced 1) translocation of γ-tubulin from spindle poles to microtubules; 2) absence of Thr308 and Ser473-pAkt in 76% and 30% of oocytes, respectively; 3) a significant reduction of H3S10 phosphorylation levels. At 29 h, a significant decrease of Akt mRNA, but not of pAKT/Akt protein expression levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability/fertilizability by altering the expression levels and spindle distributions of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.

Post-ovulatory ageing of mouse oocytes affects the distribution of specific spindle-associated proteins and Akt expression levels

CECCONI, Sandra;PATACCHIOLA, FELICE;CARTA, Gaspare;MACCHIARELLI, GUIDO;
2014

Abstract

The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 posttranslational modifications and Akt expression levels. To this end, oocytes were retrived at 13, 29 and 33 h after hCG treatment. The presence/distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, Plk1 and Ser473/Thr308 pAkt as well as histone H3 and H4 acetylation/phosphorylation levels were assayed via immunofluorescence; Akt expression levels were determined via RT-PCR and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h post-hCG treatment showed similar levels of acetylated tubulin but ageing induced 1) translocation of γ-tubulin from spindle poles to microtubules; 2) absence of Thr308 and Ser473-pAkt in 76% and 30% of oocytes, respectively; 3) a significant reduction of H3S10 phosphorylation levels. At 29 h, a significant decrease of Akt mRNA, but not of pAKT/Akt protein expression levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability/fertilizability by altering the expression levels and spindle distributions of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/9187
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