Preliminary Studies Of The Effects Of MG132 On Porcine Oocytes In Vitro Maturation And Development Of Debao Black Porcine Somatic Cell Nuclear Transfer Embryos

The low efficiency of somatic cell nuclear transfer(SCNT)is the most important factor that limits the application of this technology.It had been reported that there were lots of problems in SCNT embryos,including epigenetic modification,maternal-to-zygote transition(MTZ),maintenance of pluripotency,which were different with normal in vivo and in vitro fertilization(IVF)embryos.Ubiquitin-proteasome pathway(UPP)is a major pathway for degradation of intracellular protein that exists in eukaryotic cells.UPP involves in progress of cell cycle,regulation of transcription,epigenetic modification,spermatogenesis,oogenesis,and embryogenesis.Some reports had demonstrated that proteasome inhibitor MG 132 could improve the development potential of clone embryos,and the pregnancy rate of clone animals.It was suggested that UPP might play an important role in clone embryonic development.In order to further understand the mechanism of UPP to regulate the porcine oocytes in vitro maturation and Guangxi Debao black porcine SCNT embryonic development.In present study,an optimized MG132 treatment condition was explored,the effects of proteasome inhibitor MG132 on the in vitro maturation of porcine oocytes,developmental potential,ZGA and histone epigenetic modification of Debao black porcine SCNT embryos were systematically investigated.Moreover,the full term development potential of porcine clone embryos after MG132 treatment in vivo was examined.The results would help us to improve the efficiency of Debao black porcine SCNT.1.Effects of MG132 treatment on in vitro maturation of porcine oocytes and development of Debao black porcine SCNT embryos.First,the effects of MG 132 treatment on the nuclear and cytoplasm maturation of porcine oocytes were evaluated by adding different concentrations of MG 132 into maturation medium during IVM 30-42 h.The acetic orcein staining results showed that the rate of M? oocytes of different treated groups had no difference(P>0.05).Then,the levels of reactive oxygen species(ROS)in matured oocytes were detected.It was found that,with the increase of MG132 concentration,ROS levels first decreased,then increased both in the samples of treated and non-treated groups.The ROS levels in matured oocytes of 1,2.5,5 ?mol/L groups were significantly decreased in comparision with that of in non-treated group(P<0.05),but 10 ?mol/L group had no difference with other groups(P>0.05).The distribution of cortical granules(CGs)was detected by PNA-FITC staining.The results showed that 1 ?mol/L MG132 treatment improved the rate of the type I oocytes(CGs spreaded completely in the cortex),but no difference with that of non-treated oocytes(P>0.05).It was also found that a higher concentration(2.5,5,10 ?mol/L)of MG132 would significantly decrease the rate of type I oocytes(P<0.05).Subsequently,the optimal MG132 treatment condition was explored:First,the immature oocytes were treated with different MG 132 concentrations,the matured oocytes were used for SCNT.It was found that 1?mol/L group had a higher cleavage rate than that of non-treated,2.5,and 5 ?mol/L groups(69.61%vs 58.91%,60.32%,60.87%,P<0.05).More embryos developed to blastocysts than that of 2.5,5and 10 ?mol/L groups(14.71%vs 9.52%,8.70%,6.57%,P<0.05),but no difference with non-treated group(14.71%vs 11.02%,P>0.05).Second,non-treated oocytes were used for SCNT,post-fusion and activation reconstructed embryos were treated with different MG132 concentrations.The results indicated that 5 ?mol/L MG 132 group had a higher blastocyst rate than that of non-treated group,land 10 ?mol/L groups(26.16%vs 15.50%,18.01%,16.67%,P<0.05),but no difference with that of 2.5 ?mol/L group(21.39%)(P>0.05).Finally,immature oocytes which treated with 1?mol/L MG132 were used for SCNT,and the reconstructed embryos were treated with different MG 132 concentrations for 2 h after fusion and activation.The reconstructed embryos with no MG132 treated were set as control group.The results showed that 5 ?mol/L group had a higher blastocyst rate than control,1,2.5,10 ?mol/L groups(36.51%vs 14.40%,23.33%,21.54%,7.02%,P<0.05)and more total cell numbers in blastocytsts than control,1 and 10 ?mol/L groups(52.5±4.01 vs 37.33±0.51,41.11±1.72,31±1.15,P<0.05).1 ?mol/L +5 ?mol/L MG132 combination treatment was used in following experiments.2.Effects of ubiquitin-proteasome pathway on the expression of important maternal factors in porcine oocytes and Debao black porcine SCNT embryos.To evaluated if MG132 treatment would regulate the expression of maternal factors(MFs)in porcine oocytes and SCNT embryos,important maternal proteins,Tet3 and NLRP5 were first examined by immunohistochemistry technique.The results showed that,MG 132 treatment could significantly improve the expression of Tet3 and NLRP5 proteins levels in M II oocytes and SCNT embryos at pronucleus stage(P<0.05),when compared with non-treated group.There were no 5-mC signals in non-treated and MG132-treated embryos at pronucleus stage,but it appeared at 2-cell stage,and then increased from 4-cell to blastocyst stage.Moreover,to found out if MG132 treatment had effects on the dynamic changes of maternal proteins at early pronucleus stage,SCNT pronucleus embryos at 6 h and 12 h were selected to analyze by immunohistochemistry.The results showed that,MG132 treatment improved the Tet3:5-mC ratio when compared with non-treated embryos at 6 h and 12 h pronucleus stages(P<0.01),which also significantly increased the NLRP5 protein levels in SCNT embryos at 6 h and 12 h pronucleus stages(P<0.05).Subsequently,the expression levels of NPM2,Zar1,NLRP5 and Tet3 genes were analyzed by qRT-PCR.It was found that NPM2,Zar1,NLRP5 and Tet3 genes had a higher expression levels in GV to 2-cell stage samples of non-treated and MG132-treated groups(P<0.05),but they decreased from 2-cell to blastocyst stage,declined to nadir at blastocyst stage.Meanwhile,it was also found MG132 could significantly improve the expression of NPM2,Zar1,NLRP5 and Tet3 genes in M? oocytes and pronucleus SCNT embryos,which were consistent with the results of immunohistochemistry analysis.3.Effects of MG132 treatment on histone modification and the expression of ZGA-related genes in porcine oocytes and SCNT embryos.First,the protein levels of H3K4me3 and H3K9me3 in porcine oocytes were evaluated by immunohistochemistry.The results showed that,in non-treated group samples,the H3K4me3 levels decreased from GV to M? stage,declined to nadir at GVBD stage;The H3K9me3 levels increase from GV to GVBD stage,reached to a peak at GVBD stage,but disappeared from Pre-MI to M ?stage.It was found that MG132 could significantly increase the H3K4me3 levels of oocytes at AT-? and M ? stage,there was no H3K9me3 signals in oocytes at AT-? and M? stages.Subsequently,to better understand the mechanism of MG 132 treatment on the development of Debao black porcine SCNT embryos,SCNT embryos were treated with TSA and MG 132 respectively.It was found that both MG 132 and TSA could significantly improve the 4-cell rate(64.94%,60.66%vs 49.61%,P<0.05),blastocyst rate(29.91%,23.77%vs 13.95%,P<0.05)and total cell numbers in blastocyst(49.56 ± 4.11,47.32 ± 3.72 vs 32.57 ± 1.55,P<0.05)when compared with non-treated group,but no difference between MG132-treated and TSA-treated groups(P>0.05).The RNA pol II levels of SCNT 2-and 4-cell embryos which treated with MG132 or TSA were higher than that of non-treated embryos(P<0.05).In comprasion with non-treated group,MG132 could significantly increase the expression of eIF3A and TFIIA in SCNT 2-and 4-cell embryos(P<0.05),the expression levels of eIF3A and TFIIA in embryos of TSA-treated group were lower than that of MG132-treated group at 4-cell stage(P<0.05).Finally,the H3K4me3,H3K9me3 and H3K9ac levels in Debao black porcine SCNT embyos were analyzed by immunohistochemistry.The results showed that,in comparison with non-treated group,both MG 132 and TSA could significantly improve H3K4me3 levels,decreased H3K9me3 levels in SCNT 1-cell and 4-cell embryos(P<0.05).MG132 and TSA treatments could increase the H3K9ac levels in SCNT 1-cell embryos(P<0.05),TSA also induced a higher H3K9ac levels in SCNT morula and blastocysts than that of MG132-treated and non-treated groups(P<0.05).The qRT-PCR results showed that,the expression profiles of HAT1,HDAC1,HDAC2,ASHL2,SMYD3,KDM5A and KDM5B genes in MG132-treated SCNT embryos at 1-to 4-cell stage were similar with that of TSA-treated embryos.The expression of HAT1,HDAC1,HDAC2,KDM5A and KDM5B genes were decreased in TSA-treated SCNT embryos at blastocyst stage when compared with that of non-treated group.At morula and blastocyst stage,the expression levels of those genes in MG132-treated embryos were significant higher than that of TSA-treated and non-treated embryos(P<0.05).4.Effects of MG132 treatment on the quality of Debao black porcine SCNT embryos.The apoptotic cell numbers of Debao black porcine SCNT blastocysts from different treatments were detected by TUNEL assay.It was found that MG 132 treatment could significantly decrease the apoptotic cell numbers in SCNT blastocysts in comparision with non-treated group(P<0.05),but no difference between TSA-treated and non-treated blastocysts(P>0.05).Furthermore,the status of pluripotency and formation of ICM and TE in Debao black porcine blastocysts were examined by immunostaining.The result showed that,in comparison with non-treated group,MG132 and TSA could remarkably improve the Oct4 levels in porcine SCNT blastocysts(P<0.05),MG132 could induce a higer Oct4 levels in SCNT blastocysts than that of TSA-treated blastocysts(P<0.05).The immunostaining results of CDX2 indicated that both MG132 and TSA could induce more total cell numbers in SCNT blastocysts than that of in non-treated blastocysts(51.2±5.8,51.0±0.6 vs 35.7±4.2,P<0.05),ICM cell numbers had no difference between MG132 and TSA treatments(11.43±4.3 vs 9.71 ±2.8,P>0.05).The results of ICM:total cell number ratio presented that TSA-treated blastocysts had a higher ratio than that of MG132-treated and non-treated blastocysts(29.03%vs 23.27%,18.01%,P<0.05).To evaluate the molecular mechanism of MG132 treatment on development of porcine SCNT embryos,the apoptotic-related and development-related genes were analyzed by qRT-PCR.The results showed that Bcl-2,Nanog,Oct4 and CDX2 increased their expression Bax decreased in MG132-treated blastocysts in comparison with that of non-treated blastocysts(P<0.05).TSA treatment could significantly increase the expression levels of Nanog,Oct4,significantly decrease the expression of Bax and CDX2 in SCNT blastocyst in comparision with non-treated group(P<0.05).Finally,the full-term development of MG132-treated Debao black porcine SCNT embryos was examined.The results showed that,abortion fetus was obtained from transferring MG132-treated embryos.The genetic relationship of abortion fetus,donor cells,recipient sow and randomized control was analyzed by DNA microsatellite assay.It was found that the genetic relationship of abortion fetus was consistent with donor cell.It was preliminarily confirmed that abortion fetus were cloned Debao black procine.In conclusion:(1)MG132 treatment would not influence the nuclear maturation process ofporcine oocytes,but it decreased the ROS levels and promoted CGs spread completely in the cortex of matured oocytes.(2)MG 132 treatment could regulate the expression of maternal proteins and maternal effect genes in M? oocytes and Debao black porcine SCNT pronucleus embryos,and promote the DNA demethylation of pronucleus embryos;(3)MG132 treatment promoted the initiation of ZGA and regulated the histone epigenetic modification in Debao black porcine SCNT embryos;(4)MG132 treatment decreased the apoptotic cell numbers,promoted the differentiation of TE and ICM and maintained the pluripotency of Debao black porcine SCNT blastocysts;(5)The optimized MG 132 treatment could produce clone Debao black porcine.