Reproductive aging is associated with changes in oocyte mitochondrial dynamics, function, and mtDNA quantity.
Mitochondria affect numerous aspects of mammalian reproduction. We investigated whether the decrease in oocyte quality associated with aging is related to altered mitochondria. Oocytes from old (12 months) and young (9 weeks) C57BL/6J mice were compared in relation to: mitochondria morphology and dynamics (mitochondria density, coverage, size and shape) throughout folliculogenesis; levels of mitochondrial DNA (mtDNA); mitochondrial stress reflected in the expression of mitochondrial unfolded protein response (mt-UPR) genes; and levels of reactive oxygen species (ROS) under baseline conditions and following H2O2 treatment. In old mice, mitochondria of primary follicle-enclosed oocytes were smaller, with lower mitochondria coverage (total mitochondria μm2/μm2 cytosol area) (p<0.05). Other follicular stages showed a similar trend, but the changes were not significant. Mature oocytes (Metaphase II-MII) from old mice had significantly less mtDNA (p<0.01), and elevated mt-UPR gene Hspd1 expression (p<0.05), compared with those from young mice. ROS levels in aged MII oocytes were also higher following pretreatment with H2O2 (p<0.05). Aging is associated with altered mitochondrial morphological parameters and decreased mtDNA levels in oocytes, as well as an increase in ROS under stressful conditions and elevated expression of mitochondrial stress response gene Hspd1. Delineation of the mechanisms underlying mitochondrial changes associated with ageing may help in the development of diagnostic and therapeutic tools in reproductive medicine.
Babayev E
，Wang T
，Szigeti-Buck K
，Lowther K
，Taylor HS
，Horvath T
，Seli E
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Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice.
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 5-8 weeks and 45 mice aged 9-11 months (C57Bl6/CBA F(1)). A total of 488 immature oocytes, with a diameter ranging from 20 μm to more than 80 μm, 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.
May-Panloup P
，Brochard V
，Hamel JF
，Desquiret-Dumas V
，Chupin S
，Reynier P
，Duranthon V
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Maintaining mitochondrial DNA copy number mitigates ROS-induced oocyte decline and female reproductive aging.
Oocytes play a crucial role in transmitting maternal mitochondrial DNA (mtDNA), essential for the continuation of species. However, the effects of mitochondrial reactive oxygen species (ROS) on mammalian oocyte maturation and mtDNA maintenance remain unclear. We investigated this by conditionally knocking out the Sod2 gene in primordial follicles, elevating mitochondrial matrix ROS levels from early oocyte stages. Our data indicates that reproductive aging in Sod2 conditional knockout females begins at 6 months, with oxidative stress impairing oocyte quality, particularly affecting OXPHOS complex II and mtDNA-encoded mRNA levels. Despite unchanged mtDNA mutation load, mtDNA copy numbers exhibited significant variations. Strikingly, reducing mtDNA copy numbers by reducing mtSSB protein, crucial for mtDNA replication, accelerated reproductive aging onset to three months, underscoring the critical role of mtDNA copy number maintenance under oxidative stress conditions. This research provides new insights into the relationship among mitochondrial ROS, mtDNA, and reproductive aging, offering potential strategies for delaying aging-related fertility decline.
Long S
，Zheng Y
，Deng X
，Guo J
，Xu Z
，Scharffetter-Kochanek K
，Dou Y
，Jiang M
... -
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Mitochondrial unfolded protein response: a stress response with implications for fertility and reproductive aging.
Mitochondria play a central role in the regulation of energy metabolism in oocytes and preimplantation embryos, where the number and morphology of mitochondria and mitochondrial DNA (mtDNA) content are tightly regulated. A number of mouse models with mitochondrial dysfunction result in infertility, further confirming the key role of mitochondria in female reproductive function. When cells and organisms detect mitochondrial dysfunction they use response mechanisms directed at recovering salvageable mitochondria and eliminating mitochondria that can no longer be rescued. Among these mechanisms, mitochondrial unfolded protein response (UPRmt) has recently been linked with prevention of aging, as compromised mitochondrial stress response contributes to age-related accumulation of damaged proteins, reduced oxidative phosphorylation, and increased reactive oxygen species (ROS) production. These mechanisms seem to be especially relevant for reproduction, as targeted deletion of the UPRmt-regulatory gene Clpp results in female infertility, with impaired oocyte maturation and two-cell embryo development, and failure to form blastocysts. In addition, absence of CLPP results in accelerated depletion of follicles, and a phenotype similar to premature reproductive aging. Further studies will provide novel mechanistic insights for physiologic and pathologic control of oocyte and early embryonic mitochondrial function, which can be exploited for the development of novel therapeutic approaches for the promotion of fertility during the aging process.
Seli E
，Wang T
，Horvath TL
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