Factors affecting the efficiency and reversibility of roscovitine (ROS) block on the meiotic resumption of goat oocytes.
Goat oocytes from 2 to 4 and 0.8 to 1.2-mm follicles were freed (DOs) or not (COCs) of cumulus cells and cultured for different times in an inhibition medium supplemented with different concentrations of roscovitine (ROS). At the end of culture, oocytes were either cultured in a maturation medium for 24 hr and activated chemically for embryo development, or examined for GV chromatin configurations. Nuclear status was checked at different time points during maturation culture. Although both 200 and 250 microM ROS maintained 78-85% of oocytes at the GV stage for 24 hr, only oocytes blocked with 200 microM ROS developed to MII stage at a high rate after maturation culture. While few oocytes blocked with 200 microM ROS for 24 hr developed into morulae and none into blastocysts after activation, percentages of oocytes developing into morulae and blastocysts increased to the level of the control oocytes when the block time was reduced to 8 hr. While the GV and pMI stages were shortened with MI, and A/TI unaffected after oocytes were blocked for 8 hr, all the stages but A/TI were shortened after 24 hr of block. The sizes of nucleoli diminished with time and the GV chromatin configuration changed during ROS block. Significantly more DOs than COCs were blocked with 200 microM ROS, but none of the blocked DOs matured after drug withdrawal. However, maturation of the DOs improved significantly when ROS concentration was reduced to 150 microM or DOs were co-inhibited with COCs. The GV intact percentages of DOs did not differ after ROS inhibition with or without eCG, but those of COCs decreased significantly after ROS inhibited in the presence of eCG. When MII-incompetent oocytes from 0.8 to 1.2-mm follicles were inhibited with ROS for 8 and 24 hr prior to maturation culture, nuclear maturation improved significantly, activation rates were as high as that of the control oocytes, and some of the activated developed to 4- or 8-cell stages. It is concluded that (i) the efficiency and reversibility of ROS block was both drug concentration and exposure-time dependent; (ii) cumulus cells alleviated the toxicity of ROS on goat oocytes; (iii) eCG released goat oocytes from ROS block through the mediation of cumulus cells; (iv) ROS block quickened the nuclear maturation of goat oocytes and improved the developmental competence of meiosis-incompetent oocytes, possibly due to a sustained nuclear activity during inhibition culture; (v) oocyte nuclear maturation and activation did not depend upon cumulus expansion, but the embryo development occurred in association with cumulus expansion.
Han
Dong
D
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian City, P.R. China.
Lan
Guo-Cheng
GC
Wu
Yan-Guang
YG
Han
Zheng-Bin
ZB
Wang
Hui-Li
HL
Tan
Jing-He
JH
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005260:Female; D006041:Goats; D008540:Meiosis / Q000187:drug effects / Q000502:physiology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D006080:Ovarian Follicle / Q000378:metabolism; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D013997:Time Factors
D002843:Chromatin; D003470:Culture Media; D011687:Purines; D000077546:Roscovitine