Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice.

Abstract

Does ageing affect the kinetics of the mitochondrial pool during oogenesis and early embryogenesis? While we found no age-related change during oogenesis, the kinetics of mitochondrial DNA content and the expression of the factors involved in mitochondrial biogenesis appeared to be significantly altered during embryogenesis. Oocyte mitochondria are necessary for embryonic development. The morphological and functional alterations of mitochondria, as well as the qualitative and quantitative mtDNA anomalies, observed during ovarian ageing may be responsible for the alteration of oocyte competence and embryonic development. The study, conducted from November 2016 to November 2017, used 40 mice aged 5-8weeks and 45 mice aged 9-11months (C57Bl6/CBA F(1)). A total of 488 immature oocytes, with a diameter ranging from 20μm to more than 80μm, were collected from ovaries, and 1088 mature oocytes or embryos at different developmental stages (two PN, one-cell, i.e. syngamy, two-cell, four-cell, eight-cell, morula and blastocyst) were obtained after ovarian stimulation and, for embryos, mating. Mitochondrial DNA was quantified by quantitative PCR. We used quantitative reverse transcriptase PCR (RT-PCR) (microfluidic method) to study the relative expression of three genes involved in the key steps of embryogenesis, i.e. embryonic genome activation (HSPA1) and differentiation (CDX2 and NANOG), two mtDNA genes (CYB and ND2) and five genes essential for mitochondrial biogenesis (PPARGC1A, NRF1, POLG, TFAM and PRKAA). The statistical analysis was based on mixed linear regression models applying a logistic link function (STATA v13.1 software), with values of P< 0.05 being considered significant. During oogenesis, there was a significant increase in oocyte mtDNA content (P< 0.0001) without any difference between the two groups of mice (P= 0.73). During the first phase of embryogenesis, i.e. up to the two-cell stage, embryonic mtDNA decreased significantly in the aged mice (P< 0.0001), whereas it was stable for young mice (young/old difference P= 0.015). The second phase of embryogenesis, i.e. between the two-cell and eight-cell stages, was characterized by a decrease in embryonic mtDNA for young mice (P= 0.013) only (young/old difference P= 0.038). During the third phase, i.e. between the eight-cell and blastocyst stage, there was a significant increase in embryonic mtDNA content in young mice (P< 0.0001) but not found in aged mice (young/old difference P= 0.002). We also noted a faster expression of CDX2 and NANOG in the aged mice than in the young mice during the second (P= 0.007 and P= 0.02, respectively) and the third phase (P= 0.01 and P= 0.008, respectively) of embryogenesis. The expression of mitochondrial genes CYB and ND2 followed similar kinetics and was equivalent for both groups of mice, with a significant increase during the third phase (P< 0.01). Of the five genes involved in mitochondrial biogenesis, i.e. PPARGC1A, NRF1, POLG, TFAM and PRKAA, the expression of three genes decreased significantly during the first phase only in young mice (NRF1, P= 0.018; POLGA, P= 0.002; PRKAA, P= 0.010), with no subsequent difference compared to old mice. In conclusion, during early embryogenesis in the old mice, we suspect that the lack of a replicatory burst before the two-cell stage, associated with the early arrival at the minimum threshold value of mtDNA, together with the absence of an increase of mtDNA during the last phase, might potentially deregulate the key stages of early embryogenesis. N/A. Because of the ethical impossibility of working on a human, this study was conducted only on a murine model. As superovulation was used, we cannot totally exclude that the differences observed were, at least partially, influenced by differences in ovarian response between young and old mice. Our findings suggest a pathophysiological explanation for the link observed between mitochondria and the deterioration of oocyte quality and early embryonic development with age. This work was supported by the University of Angers, France, by the French national research centres INSERM and the CNRS and, in part, by PHASE Division, INRA. There are no competing interests.