RESUMEN
The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell's major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB+) and BCB negative (BCB-) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB- oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB- oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages.
Asunto(s)
Blastómeros/ultraestructura , Mitocondrias/fisiología , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Porcinos/fisiología , Animales , Citoplasma/ultraestructura , ADN Mitocondrial/análisis , Femenino , Fertilización In Vitro , Glucosafosfato Deshidrogenasa/metabolismo , Masculino , Mitocondrias/trasplante , Oocitos/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inyecciones de Esperma Intracitoplasmáticas , Coloración y EtiquetadoRESUMEN
The introduction of nuclear transfer (NT) and other technologies that involve embryo reconstruction require us to reinvestigate patterns of mitochondrial DNA (mtDNA) transmission, transcription and replication. MtDNA is a 16.6 kb genome located within each mitochondrion. The number of mitochondria and mtDNA copies per organelle is specific to each cell type. MtDNA is normally transmitted through the oocyte to the offspring. However, reconstructed oocytes often transmit both recipient oocyte mtDNA and mtDNA associated with the donor nucleus. We argue that the transmission of two populations of mtDNA may have implications for offspring survival as only one allele might be actively transcribed. This could result in the offspring phenotypically exhibiting mtDNA depletion-type syndromes. A similar occurrence could arise when nucleo-cytoplasmic interactions fail to regulate mtDNA transcription and replication, especially as the initiation of mtDNA replication post-implantation is a key developmental event. Furthermore, failure of the donor somatic nucleus to be reprogrammed could result in the early initiation of replication and the loss of cellular mtDNA specificity. We suggest investigations should be conducted to enhance our understanding of nucleo-cytoplasmic interactions in order to improve NT efficiency.
Asunto(s)
Replicación del ADN , ADN Mitocondrial , Desarrollo Embrionario y Fetal/fisiología , Mamíferos/fisiología , Técnicas de Transferencia Nuclear , Adenosina Trifosfato/metabolismo , Alelos , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Diferenciación Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Transcripción GenéticaRESUMEN
The recent introduction of more invasive assisted reproductive techniques offers the possibility to provide a wider treatment profile to patients. However, some of these technologies are of considerable concern as they are fraught with the possible transmission of genetic abnormalities to the offspring they create. To date, much analysis of these technologies has been conducted at the chromosomal DNA level. While some analysis has been conducted on the extranuclear, mitochondrial genome (mtDNA), this has been mainly descriptive. In the vast majority of cases, it appears that mtDNA is maternally inherited. The impact that leakage of sperm mtDNA transmission might have for the offspring is discussed in the light of the recent identification of sperm mtDNA presence in a patient with mtDNA disease. The implications of introducing donor mtDNA into a recipient oocyte through both cytoplasmic and nuclear transfer are also discussed. Again, the implications for offspring survival are discussed and suggestions made as to why the techniques might provide valuable insights into mtDNA transmission, replication and transcription. In order to be confident that patients and their offspring are being offered safe treatment, it is argued that potentially some of these treatments may be of considerable benefit in the future but significant scientific research is required before these treatments can be effectively employed in the clinic.