Background During oocyte growth, extensive mitochondrial biogenesis leads to a substantial increase in mitochondrial DNA (mtDNA) copy number, from approximately 5000 in a primordial oocyte to 200,000 in a fully grown mouse oocyte. In mammalian cells, individual mtDNA molecules are packaged with mitochondrial transcription factor A (TFAM) into nucleoprotein structures termed nucleoids, which serve as the fundamental units of mtDNA organisation and replication. However, it remains unclear whether all nucleoids contribute equally to mtDNA replication. We hypothesised that mitochondrial nucleoids exhibit heterogeneity, with mtDNA replication restricted to a distinct subset during oocyte growth.
Methods Mouse oocytes at different growth stages were analysed for mtDNA copy number. Temporal dynamics of mtDNA replication were assessed by 5-ethynyl-2′-deoxyuridine (EdU) labelling, followed by anti-DNA and TFAM immunostaining. Confocal microscopy and quantitative image analysis were used to assess the proportion, spatial distribution and TFAM content of nucleoids. Data are presented as mean ± SEM, and statistical analyses were performed using Student's t-test or one-way ANOVA with Tukey's multiple comparisons test, as appropriate.
Results mtDNA copy number increased markedly during oocyte growth, from approximately 4,750 ± 363 in primordial oocytes (15 μm) to 38,416 ± 2595 in growing oocytes (50 μm).The proportion of EdU-positive nucleoids increased progressively over time, reaching approximately 60% after 24 h. The proportion of replicating nucleoids was highest in the perinuclear region compared with cytoplasmic (P=0.01) and cortical regions (P=0.004). Furthermore, EdU-positive nucleoids displayed greater TFAM abundance and higher TFAM/mtDNA ratios than EdU-negative nucleoids (0.97 v 0.51; P=0.0007).
Conclusion In growing oocytes mtDNA replication is spatially organised and restricted to a subset of nucleoids. TFAM enrichment in replicating nucleoids implicates nucleoid composition in regulating replication competence. These findings reveal spatial and functional heterogeneity among mitochondrial nucleoids and provide new insights into mitochondrial genome maintenance in the female germline.