Abstract
Extensive mitochondrial replication during oogenesis and its role in oocyte maturation and fertilization indicate that the amount of mitochondrial DNA (mtDNA) may play a significant role in early embryonic development. Early embryos express peroxisome proliferator-activated receptor gamma co-activator alpha (PPARGC1A/PGC-1a), a protein essential for mitochondrial biogenesis. This study investigated the role of PGC-1α from a single-cell zygotic stage to day-8 bovine blastocyst and the effect of PGC-1a knockdown (KD) on embryo mitochondria and development. PGC-1α KD via siRNA injection into single-cell zygotes does not substantially affect embryonic cleavage up to the morula stage but considerably reduces blastocyst development (18.42%) and hatching than the control (32.81%). PGC-1α regulates transcription of the gene encoding mitochondrial transcription factor A (TFAM), and immunofluorescence analysis indicated significantly lower TFAM expression in the 16-cell KD embryos and day-8 KD blastocysts. Reduction in NRF1 protein's nuclear localization in bovine blastomeres was also observed in PGC-1α-KD embryos. Furthermore, to understand the effect of PGC-1α-KD on the mitochondrial genome, we found a low mtDNA copy number in PGC-1α-KD day-8 bovine blastocysts. Several genes related to mitochondrial functioning, like ND1, ND3, ND5, ATPase8, COI, COII, and CYTB, were significantly downregulated in PGC-1α-KD embryos. Moreover, high mitochondrial depolarization (ΔΨm) and abnormal lipid depositions were observed in the PGC-1α KD blastocysts. SIRT1 is the upstream regulator of PGC-1α, but SIRT1 activation via Hesperetin does not affect PGC-1α-KD embryonic development considerably. In conclusion, PGC-1α plays a critical role in early embryo mitochondrial functioning, and any perturbation in its expression significantly disrupts early embryonic development.