Involvement of mitochondrial transcription factor (TFAM) in porcine gametogenesis and preimplantation embryo development

Mitochondrial Transcription Factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA) within the mitochondrial nucleoid. Heterozygous TFAM knockout mice exhibit depletion of mtDNA and a homozygous knock-out is embryo-lethal, implying that TFAM has an essential role in mitochondrial biogenesis and sustenance during preimplantation embryonic development. We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig ( Sus scrofa), a livestock species of major agricultural and economic significance. We hypothesized that TFAM will be expressed at high levels in oocytes and embryos to support their development. We also anticipated that sperm mitochondria should be devoid of TFAM since they are destined for degradation after fertilization. The content of TFAM mRNA increased considerably during porcine oocyte maturation and preimplantation development of porcine embryos. TFAM protein accumulated in the cytoplasm of porcine oocytes during in vitro maturation and was reduced by proteolysis after fertilization. This pattern of TFAM accumulation was not mirrored by parthenogenetically activated oocytes and zygotes reconstructed by somatic cell nuclear transfer (SCNT), suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. The appropriate band of 25 kDa was detected by western blotting of both meiotically mature ova and zygotes, and was also present in ejaculated boar spermatozoa. Boar sperm extracts also displayed several bands of lesser mass, indicative of proteolytic degradation, and several bands >25 kDa suggestive of posttranslational modification by ubiquitination. Ubiquitination of sperm TFAM was confirmed by affinity purification of ubiquitinated proteins using matrix immobilized ubiquitin-binding protein p62, followed by TFAM western blotting. The accumulation of TFAM protein was observed in the cytoplasmic lobes of late stage elongating spermatids on boar testicular tissue sections. TFAM immunoreactivity was absent from the mitochondrial sheath, and relegated to the sperm tail principal piece and cytoplasmic droplet in fully differentiated spermatozoa. Isolation of sperm heads and sperm tails confirmed that TFAM protein is present in the principal piece of the sperm tail. These data suggest that mitochondrial transcription factor TFAM is developmentally regulated in porcine gametes and embryos, and may exert a critical role in gametogenesis and preimplantation embryo development. Sperm TFAM protein could be marked for proteolysis by the ubiquitin-proteasome pathway during spermatid elongation to facilitate the degradation of paternal mitochondria after fertilization.