Abstract
Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.
Highlights
The developmental capacity of in vitro matured oocytes is markedly lower than that of their in vivo matured counterparts [1,2]
A significant species difference in the carrying-over effect of germinal vesicle breakdown (GVBD) blocking with roscovitine on postblocking meiotic progression (PBMP) was observed between mouse and pig oocytes
To study the mechanisms for this species difference, we observed the dynamics of cyclin B, the pre-M-phase promoting factor (MPF) to MPF conversion and the activity of the epidermal growth factor (EGF) signaling cascade during the blocking culture
Summary
The developmental capacity of in vitro matured oocytes is markedly lower than that of their in vivo matured counterparts [1,2]. In vivo, oocytes acquire cytoplasmic maturity after a long series of preparatory processes involving transcription and translation of transcripts during the meiotic prophase [4,5], whereas in vitro, a premature GVBD without adequate cytoplasmic maturation is induced by transfer of oocytes from follicles into culture medium. To improve the quality of in vitro matured oocytes, attempts have been made to increase the ooplasmic maturation by temporary inhibition of meiotic resumption [6,7,8]. Many procedures aimed at regulating gene expression of maturing GV oocytes such as gene knockdown or over expression and RNA interference often involve oocyte culture with meiosis arrested, and such procedures will definitely benefit from knowledge on the molecular events during GVBD blocking
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