Effects Of PI3K Specific Inhibitor On Mouse Zygotic Gene Activation And The Interaction Between PI3K/Akt And ERα In Mouse2-Cell Embryos Produced In Vitro

Objective:1. To investigate the effects of PI3K specific inhibitor LY294002on thedevelopmental potential of KM mouse1-cell embryos in vitro, and study theexpression of the genes (e.g. eiF-1A and MuERV-L) related to zygotic geneactivation in2-cell embryos produced in vitro.2. To explore the interaction between PI3K/Akt and ERα in2-cell embryosproduced in vitro.Methods:1. KM mouse1-cell embryos were collected and cultured in M16medium (controlgroup) and M16medium supplemented with different concentrations ofLY294002respectively. The effects of PI3K specific inhibitor LY294002on thedevelopment potential of KM mouse1-cell embryos in vitro were observed andthe percentage of embryos developing from2-to4-cell embryos and blastocystsas evaluation criteria.2. Real-time PCR was used to detect the expression of the genes (e.g. eiF-1A andMuERV-L) related to zygotic gene activation in2-cell embryos cultured in M16medium (control group) and M16medium with50μM LY294002.3. KM1-cell embryos were collected and cultured in M16medium (control group)and M16medium with50μM LY294002.2-cell embryos were obtained for thedetections as follows（1）Laser confocal scanning microscopy was utilized to detect the position ofERα and p-ERα.（2）Western blot was applied to detect the level of p-Akt, ERα and p-ERαprotein. （3）Real-time PCR was used to detect the expression of ERα mRNA.4. KM1-cell embryos were collected and cultured in M16medium (control group)and M16medium with25μM MPP.2-cell embryos were obtained for thedetections as follows:（1）Laser confocal scanning microscopy applied to detect the position ofAkt andp-Akt.（2）Western blot was used to detect the expression changes of Akt and p-Aktprotein.Results1. KM mouse1-cell embryos were cultured in different concentrations of LY294002.The development rate from2-to4-cell embryos had significantly decreasedcompared with the control group. Real-time PCR analysis demonstrated thateiF-1A mRNA level of LY294002group was significantly decreased.2.2-cell embryos were obtained from M16medium (control group) and M16medium with50μM LY294002, and used to further analysis.（1） Laser confocal scanning microscopy displayed that ERα protein wasdistributed uniformly at the nucleus and the cytoplasm of2-cell embryosin M16group, but mainly at the nucleus; p-ERα protein was alsodistributed uniformly at the nucleus and the cytoplasm of2-cell embryosin M16group. However, in LY294002group, relative fluorescenceintensity of p-ERα protein at the nucleus was lower than in M16group,and a few of large fluorescence particles of p-ERα protein were located inthe cytoplasm;（2）Western blot showed that the level of ERα protein increased slightly inLY294002group, but there had no significant difference compared withM16control group(P>0.05), while the level of p-ERα protein wassignificantly decreased compared with M16control group(P<0.05);（3）Real-time PCR analysis demonstrated that the expression level of ERαmRNA was increased slightly in LY294002group, but there had nosignificant difference compared with M16control group (P>0.05). 3.2-cell embryos were obtained from M16medium (control group) and M16medium with25μM MPP, and used to further analysis.（1）Laser confocal scanning microscopy displayed that Akt protein wasdistributed at the plasmalemma, while p-Akt protein was located at theplasmalemma, the cytoplasm and the nucleus of2-cell embryos. There wereno difference of the localization and relative fluorescence intensity of Aktand p-Akt protein between the M16and MPP group.（2）Western blot showed that the level of Akt and p-Akt protein between M16group and MPP group was not significantly different (P>0.05).ConclusionCultured in M16medium with LY294002, The developmental potential of KMmouse1-cell embryos was significantly decreased, and the percentage of embryosdeveloping from2-to4-cell embryos and blastocysts was lower than M16medium.As a marker of ZGA, eIF-1A gene was obviously inhibited by LY294002. Theseindicated that PI3K/Akt signal plays an important role in ZGA.Moreover, the level of p-ERα protein was significantly decreased in mouse2-cellembryos produced in LY294002（50μM） medium in vitro. However, compared withM16control group, the levels of Akt and p-Akt protein were not significantlyobviously changed after MPP（25μM） treatment.The results suggested that ERαactivation was inhibited by blocking PI3K/Akt signaling pathway in mouse2-cellembryos, and PI3K/Akt signaling might not be affected by inhibiting ERα activation.