Approximately two billion years ago, the ancestor of eukaryotic cells established an endosymbiotic relationship with aerobic bacteria, which were subsequently incorporated as the organelle mitochondria, a theory widely known as the endosymbiotic hypothesis [1,2]. A key piece of evidence emerged in the early 1960s, when Margit Nass-Edelson and Sylvan Nass at Stockholm University discovered mitochondrial DNA (mtDNA) in chicken embryos [3,4], providing strong support for its bacterial origin. In mice, mtDNA exists as a circular, multicopy genome of approximately 16.3 kilobases (kb), with copy numbers ranging from tens to thousands per cell. Typically, mtDNA is packaged into nucleoprotein complexes called nucleoids, whose structure is maintained by mitochondrial topoisomerases and associated proteins. These factors regulate mtDNA topology, replication, and transcription, thereby ensuring genomic stability [5].

Epidemiological studies have shown that the mtDNA copy number (mtDNAcn) derived from peripheral blood declines with age [6,7] and is inversely associated with all-cause mortality [8]. In fact, blood-derived mtDNAcn has been shown to correlate with gene expression across multiple tissues and to reflect tissue-specific energy metabolism [9]. In addition, cord blood mtDNAcn has been linked to perinatal outcomes such as birth weight and gestational age. A recent three-generation cohort study demonstrated that cord blood mtDNAcn was negatively correlated with gestational age, birth weight, and umbilical cord length, and that maternal factors such as folic acid supplementation could modify neonatal mtDNAcn [10]. From the perspective of the developmental origins of health and disease (DOHaD) [11,12], mtDNAcn is therefore considered a potential mediator linking early-life biological stressors to disease risks in later life [13].

Ionizing radiation represents a well-characterized source of changes in cellular redox balance that can perturb mitochondrial homeostasis [14]. The mtDNA encodes key subunits of the electron transport chain (ETC), which is essential for oxidative phosphorylation and cellular energy production. Unlike nuclear DNA, mtDNA genes lack introns and are supported by more limited DNA repair mechanisms, rendering them particularly vulnerable to ionizing radiation. Damage to mitochondrial ETC components has been associated with altered electron transport and increased production of reactive oxygen species (ROS) [15]. Rather than being repaired efficiently, mtDNA frequently responds to such stress through compensatory replication, resulting in altered copy number and potential shifts in heteroplasmy. Similar mtDNA alterations have been observed after various environmental exposures, including chemical pollutants and oxidative agents, suggesting that mitochondrial genomic responses may represent a common adaptive response to environmental stress.

In experimental studies, dose-dependent increases in mtDNAcn have been observed in irradiated murine gut and bone marrow tissues in mice [16]. Clinically, elevated mtDNAcn levels in peripheral lymphocytes have also been reported in patients with acute lymphoblastic leukemia following total-body irradiation [17]. In a recent work, X-ray exposure was shown to elevate mtDNAcn while reducing intact mtDNA ratios in both human cells and mice. Notably, offspring born to female mice following preconception exposure displayed reduced mtDNAcn at two weeks of age [18]. To our knowledge, this was the first experimental evidence of radiation-induced intergenerational effects on the mammalian mitochondrial genome.

Despite these findings, the specific consequences of irradiation during pregnancy remain insufficiently characterized. Early gestation represents a particularly sensitive developmental stage, as mitochondrial dynamics are closely intertwined with organogenesis and fetal growth. Whereas the earlier work examined preconceptional exposure, it has remained unclear whether in utero exposure induces distinct, dose-dependent effects on mtDNA regulation in mothers and their offspring. To capture dose-dependent mitochondrial responses across environmentally and medically relevant exposure levels, we selected a dose range of 0.05–2 Gy. In particular, the lowest dose (0.05 Gy) was chosen to represent a low-dose exposure range relevant to environmental, occupational, and diagnostic medical contexts, where subtle biological effects may occur in the absence of overt tissue damage. To address the research question, the present study investigated the impact of X-ray exposure at gestational day 8 (GD8), a critical window of organogenesis [19,20], in C57BL/6N mice, evaluating pregnancy outcomes, maternal mtDNA indices, and offspring endpoints including mtDNAcn, intact mtDNA ratio, sex ratio, and body weight. Through these analyses, we aim to clarify dose-dependent mitochondrial genome responses to prenatal X-ray exposure and provide new insights into mitochondrial genomic responses during early development, with implications for mitochondrial biology and the developmental origins of health and disease (DOHaD).