Effects Of Autophagy On The Clearance Of Donor Mitochondria And Reprogramming In The Early Developmental Stages Of Cloned Embryo In Pigs

Autophagy is one of the basic mechanisms in eukaryotic cells,which degrades cytoplasmic macromolecules and organelles to recycle cellular components for maintenance of cellular homeostasis and embryonic development.Previous studies have shown that autophagy plays an important role in mitochondrial clearance and cell reprogramming.Because of its high incidence of mitochondrial dysfunction and incomplete reprogramming,the cloned embryos have a reduced developmental potential and the cloned offsprings are prone to the postnatal physical abnormalities.This study was designed firstly to explore the possibility of donor mitochondria clearance by autophagy in pig cloned embryos,and secondly to investigate the influence of autophagy on the reprogramming in cloned embryos by evaluating the gene expression.1 Typical LC3-dependent antophagy is not responsible for degradation of dono-r mitochondria in pig cloned embryosIn this section,our results showed that donor mitochondria were gradually scattered to part of the cytoplasm(4 h)from perinuclear area(0 h)after electric activation.No matter at 2-cell(28 h)or 4-cell(52 h)stage,we observed 3 distribution forms of donor mitochondria.And at 2-cell stage of pig cloned embryos,donor mitochondria couldn't be observed in both blastomeres of 21.95%cloned embryos,and 26.83%cloned embryos have donor mitochondria in one blastomere,while 51.22%cloned embryos still have donor mitochondria in both two blastomeres.Even to the blastocyst stage,donor mitochondria still could be observed in few cloned blastocysts.When the fluorescence intensity of LC3,a marker of autophagy,was analyzed from 1-cell(4 h)to 4-cell(52 h)both in cloned and parthenogenetic activation(PA)embryos,results showed that the intensity of LC3 reached the highest level at 2-cell stage in cloned embryos but at 4-cell stage in PA embryos.No matter at 1-cell or at 2-cell stage,the LC3-?/LC3-? of cloned embryos was higher than LC3-II/LC3-I in PA embryos.In addition,LC3-II/LC3-I in cloned embryos was higher at 2-cell stage compared to 1-cell stage.However,no clear co-existing signal of donor mitochondria and LC3 was observed by immunofluorescent staining but the overlapping signal of LC3 with LysoTracker was observed.And despite the presence of more autophagic vacuoles and abnormal mitochondria in cloned embryos compared to PA embyos at 4 h,no visual mitophagy was displayed by transmission electron microscope.Even with 3-MA treatment,the distribution of donor mitochondria in cloned embryos was not affected.2 Cytoplasmic discharge is not spercific for the clearance of donor mitochondriaTo our interest that "redundant" cytoplasm between 2 blastomeres were observed in 87.93%2-cell cloned embryos,in some of which the donor mitochondria could be observed.However,no change of the proportion of donor mitochondria in cloned embryos could be checked with the addition of 5 ?g/mL cytochalasin B(CB).Meanwhile,a similar phenomenon of cytoplasmic discharge with maternal mitochondria were noticed in 2-cell PA embryos.All these results indicated that discharge during the cleavage is not a specific mechanism to remove the donor mitochondria in cloned embryos.3 LC3-dependent autophagy could influence maternal mRNA degradation and the regulation of epigenetic modification in pocine cloned embryoWe examined the possible effect of autophagy on the reprogramming during the maternal to embryonic transition by evaluating genes expression with the regulation of autophagy.By qRT-PCR,our results showed when compared to PA embryos,there is a markedly higher mRNA level of maternal Bmp15,Hlfoo and Dppa3 in cloned embryos at 2-cell stage(P<0.05 or P<0.01),but a significant lower level of LC3,Sox2 and eIF1A at 4-cell stage(P<0.05).Subsequently,the efficient interfering by the LC3 siRNA was performed on the cloned embryos,and results showed that the mRNA level of maternal Cyclin B,Bmp15,Gdf9,c-mos,Hlfoo and Dppa3 was increased significantly(P<0.05)after the knockdown of LC3(P<0.01).And the expression of Dnmtl and Dnmt3b was also upregulated obviously(P<0.05).Although the expression of Sox2 and Oct4 was not changed,the expression of Stat3 decreased significantly(P<0.05).Furthermore,the expression of LC3,eIFIA and Stat3 was significantly increased at 4-cell stage with the treatment of 200 nmol/L rapamycin.In summary,this study found that donor mitochondria distributed unequally in 2-cell and 4-cell cloned embryos,and could be extruded with cytoplasmic discharge in some 2-cell cloned embryos,but the discharge was not specific for the removal of donor mitochondria.And the typical LC3-dependent autophagy in cloned embryos was not directly involved in the clearance of donor mitochondria,but it could affect genome activation with its highest level at 2-cell stage by influencing the degradation of maternal mRNA and regulating the expression of DNA methyltransferase.Our results will offer a reference for improving the understanding of mechanism in early embryonic development and ameliorating the relatively low success rate of cloned embryos.