Abnormal Mitochondriogenesis During Early Inter-specific Cloned Embryonic Development

The inter-specific somatic cell nuclear transfer(iSCNT)embryo developmental block is a common phenomenon in mammals.The failure of iSCNT embryo zygotic genome activation(ZGA)may be caused by abnormal mitochondriogenesis and mitochondrial dysfunction.In the present study,the bovine-bovine intra-specfic and sheep-bovine inter-specfic cloned embyros at 8-cell stages were collected and conducted the transSOFptomic sequencing(RNA-Seq)analyses.The results showed that the genes associated with mitochondrial function were abnormally expressed in the inter-specific embryos compared to the intra-specific cloned embyros.The differentially expressed genes included the mitochondrial fusion genes(Mfn1 and Mfn2),mitochondrial ribosomal large subunit genes(Mrpl),mitochondrial outer membrane genes(Tomm)and mitochondrial inner membrane genes(Timm).RT-qPCR analysis showed that,in comparson with the intra-specfic embryos,Mfn1,Mfn2,Timm22,Timm23,Tomm40 and Mrpl48 in the inter-specfic cloned embryos were expressed at lower levels,wheras the expressions of caspase-2 genes were at higher levels.The expression levels of the Tomm40,Timm22,Timm23 and Mrpl48 in the intra-specfic cloned embryos were 15.38,11.01,9.38 and 2.61 times of that in the inter-specific cloned embryos,respectively.The Na~+/K~+transporter(ATP1B3)and mitochondrial fusion protein(MFN1 and MFN2)were with lower expressions in the inter-species embryos,and no specific differences were observed between mitochondrial ribosome large subunit protein(MRPL48)and mitochondrial outer membrane protein(TOMM40).These results indicated that the abnormal mitochondrial function during mitochondriogenesis played key roles in the initiation of ZGA,especially the abnormal expression of Mfn1 and Mfn2,which could lead to embryo development block.