PPARGC1A regulates transcriptional control of mitochondrial biogenesis in early bovine embryos

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.

Keywords: NRF; PGC-1α; TFAM; bovine blastocyst; mitochondrial DNA.

FIGURE 1

PGC-1α mRNA and protein expression levels from signal cell zygote to day-8 bovine blastocyst. (A) The mRNA levels of PPARGC-1A in bovine embryos at PN, 2-cells, 4-Cells, 8-cell, 16-cells, and blastocysts stages were examined via qRT-PCR. (B) Expression level and localization of TFAM (Green FITC) and PGC-1α (Red TRITC) in bovine embryos at PN, 2-cell, 4-cell, 8-cell, 16-cell, and blastocysts stages were examined via immunofluorescence. Scale bar = 20 μm. (C) The relative mRNA expressions of DUXA, GSC, SP-1, and PGC-1α in the 8-cell stage and 16-cell stages of bovine embryos. (D) Immunofluorescence images of NRF-1 (Green FITC) and SIRT-1 (Red TRITC) in the day-8 bovine blastocysts. The data are indicated as the mean ± SEM for the indicated proteins (n = 10/group). Scale bar = 20 μm. The data are indicated as the mean ± SEM and the n = 3 per each group. Data are represented as mean ± standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Effect of PGC-1α knockdown on bovine embryo developmental competence. (A) PGC-1α was silenced via siRNA microinjections to the signal cell zygotes and developed to the blastocysts stage. Representative Western blot image of PGC-1α in control and PGC-1α knockdown day-8 bovine blastocysts. β-actin was used as a loading control. The bands were quantified using ImageJ software, and histograms represented the differences. The data are the mean ± SEM for the indicated proteins (20 blastocysts per group). (B) Relative mRNA expression of PGC-1α in the control and PGC-1α KD samples. The mRNA was isolated from the PN stage to the day-8 blastocyst stage. PN (20), 4-cell (10) and 16-cell stage embryos (10), and blastocysts (5) were used for each sample. (C) Representative images of pronuclear zygotes (PN), 4-cell stage, 16-cell stage, and day-8 blastocysts of control and PGC-1α KD samples. The scale bar represents 20 μm. (D) Day-8 bovine blastocysts of control, PGC-1α KD, and PGC-1α KD + Hesperetin samples were immunolabeled with anti-mtTFA (green) and anti-NRF1 (red) antibodies. DAPI (blue) was applied to visualize DNA. Images were obtained using confocal microscopy. Scale bar = 20 μm. The data are the mean ± SEM (n = 10/group). The data are indicated as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS indicate non-significant.

FIGURE 3

PGC-1α Knockdown effects on mitochondrial dynamics. (A) Relative mRNA expression of mtDNA from single cell zygote to day-8 blastocyst in the control and PGC-1α Knockdown samples. (B) Histograms represent the relative mRNA expressions of mtTFA, ARE, CREB, and NRF2. Five blastocysts per sample were taken to isolate mRNA, and the experiment was repeated a minimum of three times. (C) Images of day eight bovine blastocysts from PGC-1α KD and control samples stained with JC-1. 10 blastocysts were used per group. Scale bar = 20 μm *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

PGC-1α knockdown reduced the expression of mitochondrial genes and activated apoptosis. (A) The histogram represents the relative mRNA expressions of ATPase 8, ND1, ND3, and ND5 in the control and PGC-1α KD bovine day-8 blastocysts. (B) Relative mRNA expressions of CYTB, COX1, and COX2 in the control and PGC-1α KD samples. (C) Representative immunofluorescence images showed significantly downregulated BCL2(green, FITC) and upregulated Cyto-C (red, TRITC) expression in day-8 bovine blastocysts in the PGC-1α KD group than in the control group. Scale bar = 20 μm. (D) Representative immunofluorescence images showed significantly downregulated Caspase 3 (green, FITC) and upregulated p-NF-kB (red, TRITC) expression in day-8 bovine blastocysts in the PGC-1α KD group than in the control group. Scale bar = 20 μm *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

PGC-1α KD disrupts lipid metabolism in bovine blastocysts. (A) Immunofluorescence images of Mitotracker green and Nile red stainings of control and PGC-1α KD blastocysts. Low mitochondrial activity (green) and high lipid content (red) showed the low quality of PGC-1α KD blastocysts. Scale bar = 20 μm. (B) Immunofluorescence images of PPAR-δ (green, FITC) and CPT-1 (red, TRITC) in control and PGC-1α KD blastocysts. The nuclei of blastocysts were counterstained with DAPI (blue) to visualize DNA. Scale bar = 20 μm. (C) The histograms represent the relative mRNA expressions of AGTL, PLIN-2, LMF2, and LPL. The data are the mean ± SEM for n = 5 blastocysts per group. N.S, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.