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.

In vivo and in vitro postovulatory aging: when time works against oocyte quality?

“…Furthermore, studies in mouse POAoocytes detected a significant aging-dependent increase in pericentromeric ATP-dependent helicase (ATRX) [105], together with abundance in histone H4K12 acetylation and pericentromeric histone H3K9 demethylation [93,105]. These findings are in accordance with studies in mice and pigs reporting increased histone acetylation also in H3K14 and H4K8 [64,65,[106][107][108]. On the contrary, Huang and coworkers showed an accelerated aging process when mouse oocytes treated for 5 h with trichostatin A, due to raising levels of histone acetylation [106].…”

“…Mimicking MPF trend, MAPK1/3 reduced activity is demonstrated in both mouse [39] and cat oocytes [64]. In in vivo mouse POA-oocytes, kinase levels undergo a significant decrease, affecting the phosphorylation of the myosin-II regulatory light chain and increasing the rates of parthenogenetic activation [44].…”

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model

“…Ovarian sections were incubated overnight at 4 °C with a mixture of mouse monoclonal anti α-tubulin (1:1000, Sigma Chemical CO, St. Louis, MO, USA; Cat n° T5168) and mouse monoclonal anti β-tubulin (1:100; Sigma Chemical CO, Cat n° T-7941) antibodies then labeled with donkey anti-mouse Alexa Fluor 488 (1:100, Cat n° A21202, Life Technologies, Invitrogen, Carlsbad, California, USA) for 1 h at room temperature. These specific primary antibodies were chosen for immunofluorescence due to their high affinity to α and β tubulin as indicated by the manufacturer and other Authors [ 41 – 44 ].…”