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Alterations and reversibility in the chromatin, cytoskeleton and development of pig oocytes treated with roscovitine
Author(s) -
Ju JyhCherng,
Tsay Chih,
Ruan ChiWai
Publication year - 2003
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.10234
Subject(s) - germinal vesicle , biology , oocyte , andrology , meiosis , maturation promoting factor , cytoskeleton , cyclin dependent kinase 1 , endocrinology , medicine , embryo , microbiology and biotechnology , genetics , cell cycle , cell , gene
Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus‐oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 μM in Experiment 1 and 0, 40, 60, 80, 100, 120 μM in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (∼20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10–50 μM (Experiment 1) of roscovitine were used (19–34%). When oocytes were released from the inhibitor, similar proportions (70–83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80–120 μM with 83–91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40–60 μM (27–43%) groups ( P < 0.05). Although 15–21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 μM) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups ( P < 0.05). This study demonstrated that roscovitine‐treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species. Mol. Reprod. Dev. 64: 482–491, 2003. © 2003 Wiley‐Liss, Inc.