Alterations of Spindle and Microfilament Assembly in Aged Cat Oocytes
ABSTRACT To obtain insights into the cytoplasmic maturation status of cat oocytes recovered from cat ovaries following hormone treatment, we first examined microtubule and microfilament assembly in cat oocytes recovered from hormone-treated ovaries at various stages of maturation. Additionally, we determined the alteration of spindle and microfilament assembly, as well as mitogen-activated protein kinase (MAPK) activity, in cat oocytes at 0, 6, 12 and 18 h of further maturation in vitro. We then looked at pronuclear formation and cleavage of these oocytes following parthenogenetic activation. Similar to other species, microtubules are present in germinal vesicle (GV) stage cat oocytes, and following GV breakdown, microtubules encompassed condensed chromatin particles to form the meiotic metaphase spindle. Microfilaments were located in the cortex and around the GV. A microfilament-rich area, in which the chromatin is located, was observed in the oocytes during meiotic maturation. Maturation rates in aged oocytes (cultured for 18 h) were increased when compared with that in relatively fresh oocytes (<12 h culture), and the number of oocytes with abnormal spindle shapes was also increased in aged oocytes. Furthermore, in aged oocytes, the incidence of the metaphase plate observed outside the thick microfilament domain was higher compared with that of young oocytes, and this seemed to result in an increase in the number of oocytes with two pronuclei and one polar body following activation. Western blot analysis revealed a decrease in MAPK activity in aged cat oocytes. Taken collectively, these results suggest that the optimum time for improved cytoplasmic maturation is <12 h in cat oocytes recovered from hormone-treated ovaries.
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ABSTRACT: 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 post-translational modifications and v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33 h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, 5 g-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription–polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of g-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively and (3) a 10 significant reduction in phosphorylation levels of serine 10 on histone 3. At 29 h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein expression levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.Reproduction Fertility and Development 04/2013; DOI:10.1071/RD13010