Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development.
Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.
Spikings
Emma C
EC
The Mitochondrial and Reproductive Genetics Group, Medical School, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
Alderson
Jon
J
St John
Justin C
JC
D000818:Animals; D001755:Blastocyst / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics / Q000502:physiology; D004396:Coloring Agents; D000074002:DNA Polymerase gamma; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D004272:DNA, Mitochondrial; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D046149:Embryo Culture Techniques; D047108:Embryonic Development / Q000502:physiology*; D005306:Fertilization / Q000187:drug effects / Q000502:physiology; D005307:Fertilization in Vitro; D018628:Gene Dosage; D007150:Immunohistochemistry; D009865:Oocytes / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D010078:Oxazines; D012333:RNA, Messenger / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D013552:Swine / Q000196:embryology*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016047:Zalcitabine / Q000494:pharmacology
D004396:Coloring Agents; D004272:DNA, Mitochondrial; D010078:Oxazines; D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; D014157:Transcription Factors; C006797:Brilliant Cresyl Blue; D016047:Zalcitabine; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase