Improving Nutritional Conditions Enhance Porcine Oocyte Cytoplasmic Developmental Competence By Regulation Of Gene Transcription During Inhibition Of Meiosis Recovery

The developmental capacity of in vitro matured oocytes is inferior to that of the in vivo matured.In vivo,there are germinal vesicle breakdown inhibitors in the follicular fluid that inhibits oocytes in the diplonema of the meiosis and does not cause premature recovery of meiosis,during which oocytes experienced a fairly long transcription and post-transcriptional translation process to gradually acquire cytoplasmic maturation,until the LH peak induces oocyte recovery after meiosis.In vitro,however,a premature meiotic resumption without adequate cytoplasmic maturation is induced by transfer of oocytes from follicles into culture medium.Only oocytes complete nuclear maturation and cytoplasmic maturation at the same time to support the development of embryos,so that in vitro matured oocyte's poor cytoplasmic maturity is the caused of poor developmental.In order to improve the cytoplasmic maturation of in vitro matured oocytes and thus improve it development,Cultivating porcine oocytes using "Two Steps" : First during the meiotic arrest maintenance(MAM)adding the inhibitor in the culture medium to inhibit GVBD of the oocyte,in vitro extending the time of the oocyte in the germinal vesicle period,giving the oocyte sufficient time to synthesize some of the necessary substances to support the development;and then in the normal mature culture medium to mature,so as to improve the development potential of oocytes.In order to study the effect of the composition of the medium during the inhibition on the subsequent developmental capacity of the oocytes,Set up four culture systems: the Ctrl group,199 + PFF,199 and MEM groups.The Ctrl group was not inhibited and directly maturing 44 h,199 + PFF,199 and MEM group was first inhibited24 h then maturation normaly.199 + PFF group was inhibited during the addition of pig follicular fluid,199 group was inhibited during the absence of follicular fluid,MEM group is simplified MEM without follicular fluid.The results are as follows:(1)The effect of three different inhibitors of db-cAMP,IBMX and Roscovitine on porcine oocytes was studied with MEM with a simple composition.Compared with other inhibitors,25 ?M of Ros can more effectively inhibit the recovery of oocyte meiosis;(2)Changes in the composition of the culture medium during the inhibition.The addition of follicular fluid during the inhibition period has a crucial effect on improving the development of the oocytes after inhibition.Compared with the Ctrl group,the blastocyst rate and blastocyst number of the 199 + PFF group were significantly increased compared with the Ctrl group,while the developmental ability of the 199 group and the MEM group was significantly lower than that of the Ctrl group;(3)Each period of the oocyte nucleus process,which was inhibited compared to the Ctrl group of oocytes,was shortened and the most pronounced was the 199 + PFF group;(4)Compared with the Ctrl group oocytes,the migration rate of the cortical granules of the treated oocytes was significantly accelerated regardless of the culture components during the inhibition,and the most accelerated was the 199 + PFF group;(5)After the maturation culture,the oocytes of the Ctrl group and the 199 + PFF group showed 3 or 4 expansion.However,only a small part of the oocytes in the 199 group showed a 3-stage expansion,and all of the MEM group oocytes have a grade 1 or 2 expansion.In the Ctrl group and 199 + PFF group,the proportion of cumulus apoptosis was relatively low,but the proportion of cumulus cells in 199 group and MEM group was significantly increased.The ratio of Bcl2/Bax in the cumulus cells of the Ctrl group and the 199 + PFF group was higher,while the ratio of Bcl2/Bax in 199 group and MEM group was lower;(6)Detecte of Calcium storage,active oxygen and glutathione content of oocytes after maturation culture.The calcium storage and the ratio of GSH / GSSG in the 199 + PFF group was higher than that in the untreated Ctrl group and the 199 and MEM groups.The level of reactive oxygen species in 199 + PFF group was lower than that of the untreated Ctrl group also the 199 and MEM groups,indicating that the addition of follicular fluid during inhibition can increase oocyte maturation by increasing calcium storage and antioxidant capacity;(7)The oocytes were observed GV chromatin configuration and global RNA transcription immediately after the end of culture in different inhibitory media.In the Ctrl group,most of the cells were in the IN and SN types,but many of the SN-configured oocyteswere transformed into the RDC configuration after inhibition culture in the 199 + PFF group.Transcription is detected both in RDC and IN types oocyte,indicating that oocytes were still able to continue transcription during inhibition;(8)After the maturation culture,the expression level of the genes was detected by real-time fluorescence quantitative PCR.The expression of Bcl2/Bax,Nfe2l2,Mater,Zar1 and PCNA were the highest in the 199 + PFF group,the lowest in the MEM group,the middle of the Ctrl group and the 199 group.Indicating that the PFF in the inhibition medium can increasing the expression of anti-apoptotic gene and oocyte development ability correlated gene to improve the ability of oocyte development.Together,the results showed that prolonging the time of oocyte in GV phase in the culture medium containing follicular fluid could further improve the cytoplasmic maturity of oocytes and improve the developmental ability of oocytes.