Type 2 diabetes increases oocyte mtDNA mutations which are eliminated in the offspring by bottleneck effect

Diabetes mellitus (DM) has become a worldwide problem. It brings reproductive problems for both men and women. Women with diabetes mellitus often show low quality oocytes, increased chances of miscarriage, and congenital malformations of offspring [1, 2]. Diabetes creates a complex environment, which could cause permanent damage to the oocytes and embryos [3].

Mitochondria are exclusively maternally inherited. All mitochondria in mature oocytes come from primordial germ cells (PGCs), which have only a few hundred mitochondria [4]. During the one-cell zygote development to preimplantation blastocysts, cells divide persistently with no mitochondrial replication. Mitochondria transmission to offspring from only a small proportion of mitochondria in maternal PGCs is referred to as the mitochondrial genetic ‘bottleneck’. During embryo development, continuous cleavage with limited maternal mitochondria produces cells with different mitochondria resulting in organ and tissue heterogeneity. Therefore, there are bottlenecks during both oogenesis and cleavage stages. Studies proposed two mechanisms of mtDNA bottleneck transmissions. One is unequal partitioning of mtDNA during cell division and the other is selection of a small subpopulation of mitochondria. This could produce rapid genotypic shifts within a single generation [5].

Mitochondrial DNA (mtDNA) has a circular double stranded configuration comprising approximately 16,569 base pairs in rodent mature oocytes; mtDNA copy number must be 40,000–50,000 in order to sustain embryo development [6]. Since it has no repair system, mtDNA is easily mutated and its mutation rate might be 5–10 fold of that of nuclear DNA (nDNA) [7]. Generally, mtDNA in cells is homogeneous, but when mtDNA mutation occurs it becomes heterogeneous. mtDNA has thousands of copies in a cell, and coexistence of a few mtDNA mutations with wild type mtDNA does not cause abnormality, unless the mutation ratio reaches a threshold usually as high as 60–90% [8, 9], with some exceptions [10].

Many studies have proved that in both normal human cells and tumor cells, mtDNA mutations exist [11, 12]. The mtDNA bottleneck exerts a strong purifying selection in the maternal germ line to avoid mtDNA mutation transmissions to offspring [13, 14]. Diabetes affects oocyte quality and fertility by affecting mitochondrial distribution and activity, but whether it causes mtDNA mutations, and if it does, whether the mtDNA mutations are transmitted to offspring, are not known. In this study, we generated a diabetes mouse model to explore mtDNA mutations in oocytes in diabetic mice and their offspring. We showed that mtDNA mutations increased in the T2D mouse model, but the mitochondrial bottleneck could bring clearance of mtDNA mutations in the offspring.

All C57BL/6 mice were bought from SPF (Beijing) Biotechnology Co., Ltd. and maintained at 12D:12 L in a temperature-controlled room. All procedures described were reviewed and approved by the ethical committee of the Institute of Zoology, Chinese Academy of Sciences. All animal care and use procedures were in accordance with guidelines of the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences.

Diabetes mellitus is a chronic endocrine metabolic diseases, which can cause many complications, such as diabetic eye disease, diabetic nephropathy and diabetic foot disease [21, 22]. Many studies suggested that diabetes caused reproduction ability decline, both for males and females [23‐27], and it also induced diabetic embryopathy [1, 28]. Diabetes can foster a eutrophy environment, high glucose, hyperlipidemia, which impair mitochondrial function. Whether diabetes causes mitochondrial mtDNA mutations is not known. We generated a T2D mouse model, and showed that, in addition to glucose intolerance, insulin resistance, and low fecundity, T2D also increased mtDNA mutation sites and mutation numbers. The mitochondrial bottleneck, existing in oogenesis and embryo cleavage periods for clearance of mtDNA mutations [13, 14], eliminated mtDNA mutations in T2D-F1 mice.

Offspring of T2D diabetic individuals have a high rate of glucose intolerance, insulin resistance, and obesity. For T2D-F1 mice, a lower birth weight was seen compared to the T2 DC-F1 group. At 3 weeks female T2D-F1 mice also showed a lower body weight and blood glucose level. When the two groups were further fed at the same nutrition level, the body weight of T2D-F1 showed a rapid growth phenotype. This might be related to catch-up growth and protective effects of estrogen [29, 30]. Diabetes often occurs at an older age. In our study, offspring of T2D did not develop obesity or high blood glucose, and this may be because they were in a post-adolescence stage of life, not in an advanced stage.

In 1988, it was found for the first time that mtDNA mutations could cause human diseases [31, 32]. In humans, 1 in 400 individuals within populations could inherit maternal mtDNA mutations [33], and 1 in 4300 individuals within populations might have mtDNA diseases [34, 35]. Studies in humans suggested that not only in patients and aging individuals mtDNA mutations exist, but normal healthy individuals also harbor mtDNA mutations [12]. However, individuals with mtDNA mutations usually display normal phenotypes. When a threshold exists mtDNA mutations may induce diseases. Studies showed that an mtDNA deleterious threshold was 60%, and that of point mutations was 90%. But a clinical case suggested that mtDNA mutations <25% had affected tissues [10]. In our study, we detected increased mtDNA mutations in oocytes of T2D mice, suggesting that diabetes might induce mtDNA mutations in reproductive cells.

Mitochondria are maternally inherited. In theory, maternal mtDNA mutations may also be transmitted to the offspring. But mtDNA diseases occur in only 1 in 4300 individuals within human populations. This is owing to maternal mtDNA mutation clearance and in vivo mtDNA mutation threshold effects. mtDNA mutation threshold was usually 60–90%. In view of this fact, it is hard to reach mtDNA mutation threshold in vivo. There are several mechanisms to eliminate mtDNA mutations [36‐38]. Mitochondria may be in an active state of fusion, biogenesis and mitophagy, which could eliminate mtDNA mutations. In vivo, mtDNA mutations could be recovered via recombination-mediated repair which may only exist in plants, and in many cases mtDNA mutation elimination is achieved through autophagy and bottleneck-mediated selection [39]. Our study showed that T2D-F1 oocytes displayed fewer mtDNA mutation sites and mtDNA mutation numbers compared to the maternal oocytes. T2D-F1 mice showed almost the same mtDNA mutation level as that seen in the control, which means that bottleneck mediation might eliminate mtDNA mutations in T2D offspring.

In conclusion, oocytes from T2D mice have increased mtDNA mutation sites and mtDNA mutation numbers compared to the counterparts, while these adverse effects can be eliminated by the bottleneck effect in their offspring. This is the first study using a small number of oocytes to examine mtDNA mutations in diabetic mice.