Study On Synchronized Maturation Of Nuclear And Cytoplasm Of Goat Oocytes In Vitro

The maturation of nuclear and cytoplasm of oocytes was obvious not synchronous because the nuclear maturation inhibition mechanism in vivo was ineffective when oocytes were cultured in vitro. The nuclear maturation was earlier than the cytoplasm maturation. The process of nuclear maturation affects the cytoplasm maturation as well as the maturity of cytoplasm promotes the nuclear maturity. Thus, both nuclear and cytoplasm maturation are required in order to ensure fertilization and subsequent embryo development capacity. Roscovitine (ROS) is a purine known as a specific inhibitor of cyclin-dependent protein kinases that prevents P34cdc2 dephosphorylation through reversible competitive inhibition ATP bind to P34cdc2, further inhibits MPF activity, delays the process of nuclear maturation, therefore the nuclear and cytoplasm maturation in vitro near in time eventually.In the present study, the goat oocytes were in vitro maturated by using of different protocols, including nuclear maturation inhibition for different time in the whole cultural process (8 h, 16 h, 24 h), and inhibition in stages (8 h, 16 h) in normal maturation medium containing different concentration of ROS following maturation culture for 16 h, 8 h. Based on this, assessed the selected inhibition protocol to examine the incubated oocytes maturation in vitro and parthenogenetic activation thus parthenogenetic embryo developmental competence of early embryo, further confirmed the protocol utilizing ROS to induce the synchronized maturation of nuclear and cytoplasm of goat oocytes. The results in this study are listed as follows:1. The oocytes in vitro maturation in the normal maturation culture conditionThe percentage of extrusion of the polar body 1 were gradually increased with prolonged culture time when the goat oocytes cultured in normal maturation medium within 24 h, and reached the highest point at 24 h (67.13±3.10%), whereas the percentage were decreased with prolonged culture time when oocytes were cultured from 26 h to 28 h.2. Whole process of nuclear maturation inhibiting culture of the oocytesThe goat oocytes were cultured in the normal maturation medium containing different concentration of ROS for 8 h, 16 h and 24 h, respectively. The rates of nuclear maturity of all nuclear maturation inhibition groups and the control group were low when goat oocytes were incubated for 8 h, it indicated that the oocytes didn't access the nuclear maturation process at this stage. The rates of nuclear maturity of the experimental groups were considered to be not statistically significant comparing with the control group (P>0.05) when oocytes were incubated in the maturation medium containing 10μmol/L, 20μmol/L and 40μmol/L of ROS for 16 h. However, the rate were significant lower than the control group when oocytes were incubated in the maturation medium containing 80, 120, 160μmol/L and 200μmol/L ROS (P<0.05). Furthermore, when oocytes were incubated in the medium adding 10μmol/L and 20μmol/L of ROS for 24 h, the rates of nuclear maturation had no statistic significant difference comparing with the control group (P>0.05), interestingly, the rates were significant lower than control group when adding 40μmol/L or higher concentration of ROS (P<0.05) and it demonstrated the correlation between the dose and effect.3. Phased nuclear maturation inhibiting culture following the normal maturation culture of the oocytesGoat oocytes were incubated in medium containing different concentration of ROS for 16 h then transferred to normal maturation medium for 8 h (16 h+ 8 h), and inhibited by ROS for 8 h then transferred to normal medium for 16 h (16 h+ 8 h) compared with the normal maturation cultured group, the results showed that the inhibition of ROS on nuclear maturation of goat oocytes were dose and time-dependent.4. Process of the oocyte nuclear maturation cultured the optimal inhibition maturation condition in vitroThe results showed it was the optimal protocol of nuclear and cytoplasm synchronized maturation of oocytes in vitro when oocytes were cultured in the medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture. Further, using this protocol to examine the results of nuclear maturation inhibition culture of oocytes and analyzed the duration of nuclear maturity. Oocytes were incubated in normal medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture, the percentage of extrusion of the polar body 1 was no statistic significant difference comparing with the control group within 16 h. And the percentage of experimental groups when transferred to normal culture to 18 h, 20 h and 22 h (32.80±1.79%, 35.93±2.95%, 43.35±2.57%) were significant lower than the control groups (45.60±2.19%, 51.62±2.19, 62.16±1.78%). Further, the percentage of the experimental group (61.01±5.98%) was very close to the control group(67.13±3.10) (P>0.05). It showed it effectively arrested meiotic resumption about those oocytes meiosis occurred at 18 h~22 h, approximately postponed the nuclear maturation of those oocytes to 24 h.5. The results of parthenogenetic activation and embryos development from mature goat oocytes in maturation inhibiting medium with different concentration roscovitineWhen oocytes were cultured in the maturation inhibiting medium with different concentration ROS for 8 h then transferred to maturation culture to 24 h, the cleavage rate and 4-8 cells embryo rate were no significant difference between 40μmol/L ROS group and the control group, the marulae and blastocyst rate of 40μmol/L ROS group were 69.33±1.83% and 41.33±4.43%, respectively, higher than the control group (65.19±2.70% and 33.09±3.54%) of the parthenogenetic embryo from the activated mature goat oocytes. The developmental competence of the parthenogenetic embryos of all the other experimental groups except the 40μmol/L ROS group were lower than the control group, and no blastocyst was found. The results showed that the developmental competence of parthenogenetic embryos were improved when oocytes were cultured in the medium with 40μmol/L ROS for 8 h then transferred to normal maturation culture to 24 h.In conclusion, it was the optimal protocol of nuclear and cytoplasm synchronized maturation of goat oocytes that oocytes were cultured in the normal maturation medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture to 24 h; It effectively arrested meiotic resumption, achieved nuclear and cytoplasm synchronized maturation at 24 h thus improved the quality of oocytes cultured in vitro.