The Study Of Mouse Denuded Oocytes Developmental Potential During In Vitro Culture

Denuded oocytes have not been widely used for its poor ooplasmic quality and low development competence. The inhibition effect of high level cyclic adenosine monophosphate (cAMP) in oocytes arrests meiosis resumption, and the maturation delay is able to elevate ooplasmic quality. The cAMP level decreases at the point when ooplasm matures, at the same time, maturation promotion factor (MPF) is activated. Thus, inhibition of meiosis synchronizes the maturation of ooplasm and nuclear, which does good help to the early embryonic development. The study aimed at establishing an in vitro maturation system of mouse denuded oocytes (DOs), using the agent addition mode which mimicked the maturation mechanism in vivo. We observed the ooplasmic quality and early embryonic development competence of DOs through statistic analysis, including maturation rate, the activation rate and blastocyst rate after parthenogenetic activation (PA), the cleavage rate and blastocyst rate after in vitro fertilization (IVF). We also analyzed the feasibility of our system by immunofluorescence staining and quantification-PCR technologies. The experiment was divided into two parts:Experiment 1:Natural mouse DOs at GV stage were cultured at different concentrations of forskolin, a cAMP activator, for different time-addition (3 h,12 h,24 h), then were transferred into ordinary culture media for different time culture (21 h,12 h,0 h). The results of orthogonal experiment showed the maturation rate of DOs were 54%,51%,54%, whose culture condition was in 10,50,100μM forskolin culture media for 3 h firstly, then in the ordinary media for 21 h; the maturation rate of DOs were 49%,74%,50%, whose culture condition was in 10,50,100 μM forskolin culture media for 12 h firstly, then in the ordinary media for 12 h; the maturation rate of DOs were 62%,49%,56%, whose culture condition was in 10,50,100μM forskolin culture media for 24 h. The maturation was highest when culture condition was forskolin 3 h-50μM (P<0.05). Immunofluorescence staining showed fine morphologies of spindle microtubules in MII DOs. However, the MII DOs were not able to be activated after PA or to be fertilized after IVF.Experiment 2:Natural mouse DOs at GV stage were cultured under 50μM forskolin concentration condition for different time (3 h,12 h), then were incubated in PD166285 (a MPF activator) for different time (21 h,12 h). Results showed that there were no significant difference of maturation rate between different culture conditions:the maturation rates of DOs cultured in forskolin for 3 h firstly, then in PD 166285 for 21 h at different concentrations (2.5μM,5μM,10 pM) were 76%,63%,85%; the maturation rates of DOs cultured in forskolin for 12 h firstly, then in PD 166285 for 12 h at different concentrations (2.5μM,5μM,10μM) were 73%,74%,74%. Immunofluorescence staining showed fine morphologies of spindle microtubules in MⅡ DOs. In the following PA experiment, the blastocyst rate of DOs harvested from the 3 h forskolin-culture, then the 2.5μM,5μM,10μM 21 h PD166285-culture condition were 18%、9%、3%; blastocysts could only be found from the MⅡ DOs which were cultured under 2.5μM PD166285 concentration condition, when forskolin addition time was 12 h. The 1CM rate of blastocyst from PA was 32.39%, which was close to the control group (P>0.05). In addition, in the following IVF experiment, blastocysts could only be found from the MⅡ DOs which were cultured under 2.5μM PD 166285 concentration condition, when forskolin addition time was 3 h, the blastocyst rate was 5.65%, the ICM rate was 25.06%. The expressions of cyclin B1 (Ccnbl) and cyclin O (Ccno), which were the related genes of MPF, were lowest in the MⅡ DOs harvested from 3 h forskolin-2.5μM PD166285 culture condition. And the expression of pro-apoptotic gene Bax in MⅡ DOs was lowest in the same condition.In conclusion, our study had established a new method to harvest embryo from mouse DOs with the combination of forskolin and PD 166285 during IVM. Addition of forskolin only enhanced the maturation rate of DOs. PD 166285 addition following 3 h forskolin effect could enhance the future embryonic development, leading blastocysts generation.