The role of mitophagy during oocyte aging in human, mouse and Drosophila; implications for oocyte quality and mitochondrial disease

There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria, may be involved, but studies are scarce. And although assisted reproductive technologies (ART) can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems.

Aged mouse ovarian immune milieu shows a shift towards adaptive immunity and attenuated cell function

The immune system plays a major role in maintaining many physiological processes in the reproductive system. However, a complete characterization of the immune milieu in the ovary, and particularly how it is affected by maternal aging, is still lacking. In this work, we utilize single-cell RNA sequencing and flow cytometry to construct a complete description of the murine ovarian immune system and its changes along with pre-estropause aging. We show that the ovarian immune cells composition undergoes an extensive shift with age towards adaptive immunity. We analyze the effect of aging on gene expression and chemokine and cytokine networks and show an overall decreased expression of inflammatory mediators together with an increased senescent cells recognition. Our results reveal the changes in the aging ovarian immune system of the fertile female as it copes with the inflammatory stimulations during repeated cycles and the increasing need for clearance of accumulating atretic follicles.

P–204 Dynamics of cavitation as a potential non-invasive predictor of mammalian blastocyst quality

Study question We wished to investigate whether dynamics of cavity formation can be used in embryo quality assessment. Summary answer Dynamics of mouse embryo cavitation reflects to certain extent blastocysts’ developmental capabilities. It can be potentially used as a biomarker of mammalian embryo quality. What is known already During cavity expansion blastocyst pulsates, i.e. changes its volume in an oscillatory way. Recent studies performed on a mouse model have shown, that dynamics of cavitation, biomechanical properties of the trophectoderm (TE) and embryo size are intertwined. Presence or absence of blastocyst contractions has been linked to particular parameters related to positive outcome of the in vitro fertilization procedures, but the data on influence of contractions on human embryos’ developmental capabilities is often contradictory. Moreover, mostly in those studies only strong contractions (leading to a high volume loss) have been taken into consideration. Study design, size, duration We tested how postovulatory (in vitro or in vivo) or maternal aging of mouse oocytes affects dynamics of cavity formation and expansion in the resulting embryos (n = 27, n = 26 and n = 30, respectively). Furthermore, we also analyzed almost 100 mouse blastocysts in order to correlate dynamics of their cavitation with their ability to form correct outgrowths (in vitro model of implantation). Participants/materials, setting, methods Mouse oocytes subjected to postovulatory (either in vivo or in vitro) or maternal aging were fertilized in vitro. Dynamics of cavity formation and expansion was assessed by time-lapse imaging; equatorial images were taken every 10 minutes. Blastocyst area was measured over time and compared to the outcome from control embryos. In another set of experiments, after the filming mouse blastocysts were cultured for additional 4 days to test their ability to form outgrowths. Main results and the role of chance We noticed, that mouse embryos which represent limited developmental potential (obtained from either postovulatory or maternally aged oocytes) and blastocysts developed from freshly fertilized young females’ oocytes differ in terms of some parameters related to dynamics of cavitation, e.g. time of the initiation of cavity formation, frequency of contractions or mean loss of blastocyst’s area during contraction. We observed that embryos obtained from oocytes subjected to maternal or postovulatory aging have distinct dynamics of cavitation. Moreover, we noticed slightly different effect on particular parameters related to cavitation between in vivo and in vitro version of postovulatory ageing. We also showed that blastocysts, which are unable to create proper outgrowths (i.e. too small or without epiblast cells), differ from embryos that differentiate into correct outgrowths in terms of certain parameters of cavitation dynamics. Our data indicates, that dynamics of cavity formation and expansion might be related to developmental potential of mouse embryo. Limitations, reasons for caution Further studies with extended group size and testing embryos’ ability to implant in vivo are required to confirm our results. Moreover, we examined dynamics of cavitation only in a mouse model, so additional studies performed on other mammalian species are needed. Wider implications of the findings: Our data proves, that dynamics of embryo cavitation reflects, to certain extent, developmental capabilities of mouse blastocysts. Therefore, it is possible that it can be a biomarker of embryo quality (in combination with parameters provided by other methods or solely) of other mammalian species, including humans. Trial registration number Not applicable

Maternal age affects equine Day 8 embryo gene expression both in trophoblast and inner cell mass

Background: Increased embryo loss as mares become older is a major consideration for breeders as older animals are currently used for reproduction in the equine industry. Lower embryo quality has been pointed out as partly responsible for this reduced fertility. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post ovulation embryos were collected by uterine flushing from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner cell mass enriched (ICMandTE) trophoblast were obtained by embryo bisection and paired end, non-oriented RNA sequencing (Illumina, NextSeq500) was performed on each hemi-embryo. To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the ICMandTE dataset. Differential expression was analyzed (DESeq2) with embryo sex and diameter as cofactors using a false discovery rate < 0.05 cutoff. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. Results: Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity but modified gene expression in equine D8 embryos. The expression of genes in the ICM seemed to be more altered by maternal aging than in the TE. In both compartments, the expression of genes involved in mitochondrial function, translation and transcription due to chromatin modification were disturbed by maternal age. Mitosis, signaling and adhesion pathways and embryo development were particularly decreased in the ICM hemi-embryos from old mares. Finally, in TE, ion movement related genes were affected. Conclusions: This is the first study showing an effect of maternal age on gene expression in the equine blastocyst at Day 8 post ovulation. Maternal age, even for mares as young as 10 years old, disturbs mitosis, translation and transcription, cell signaling and adhesion as well as mitochondrial function and cell commitment of horse embryos. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

Equine maternal aging affects oocyte lipid content, metabolic function and developmental potential

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.

FKBP25 Regulates Meiotic Apparatus During Mouse Oocyte Maturation

FK506 binding proteins 25 (FKBP25) has been shown to function in ribosome biogenesis, chromatin organization, and microtubule stability in mitosis. However, the role of FKBP25 in oocyte maturation has not been investigated. Here, we report that oocytes with FKBP25 depletion display abnormal spindle assembly and chromosomes alignment, with defective kinetochore-microtubule attachment. Consistent with this finding, aneuploidy incidence is also elevated in oocytes depleted of FKBP25. Importantly, FKBP25 protein level in old oocytes is significantly reduced, and ectopic expression of FKBP25 could partly rescue the aging-associated meiotic defects. In addition, by employing site-specific mutagenesis, we identify that serine 163 is a major, if not unique, phosphorylation site modulating the action of FKBP25 on meiotic maturation. In summary, our data indicate that FKBP25 is a pivotal factor for determining oocyte quality, and may mediate the effects of maternal aging on female reproduction.

The Impact of Unbalanced Maternal Nutritional Intakes on Oocyte Mitochondrial Activity: Implications for Reproductive Function

Accumulating evidence on the effect of nutrition on reproduction is emerging from both animal and human studies. A healthy dietary pattern and nutrient supplementation, especially during the peri-conceptional period, might be helpful to achieve a live birth, although the mechanisms implicated are not fully understood. The endocrine system and the ooplasmic organelles apparatus, in particular the mitochondria, are clearly key elements during oogenesis and subsequent embryo development, and their proper functioning is associated with nutrition, even beyond maternal aging. Several studies in animal models have reported various adverse effects on mitochondria caused by unbalanced dietary intakes such as high fat diet, high fat high sugar diet, and low protein diet. The alterations produced might include mitochondrial intracellular distribution, content, structure, biogenesis, and functioning. This review summarizes the key role of mitochondria in female reproduction and the effects of different dietary macronutrient compositions on oocyte mitochondrial activity with their possible short-, medium-, and long-term effects.

Mitochondria-targeted therapeutics, MitoQ and BGP-15, reverse aging-associated meiotic spindle defects in mouse and human oocytes

STUDY QUESTION Do mitochondria-targeted therapies reverse ageing- and oxidative stress-induced spindle defects in oocytes from mice and humans? SUMMARY ANSWER Exposure to MitoQ or BGP-15 during IVM protected against spindle and chromosomal defects in mouse oocytes exposed to oxidative stress or derived from reproductively aged mice whilst MitoQ promoted nuclear maturation and protected against chromosomal misalignments in human oocytes. WHAT IS KNOWN ALREADY Spindle and chromosomal abnormalities in oocytes are more prevalent with maternal aging, increasing the risk of aneuploidy, miscarriage and genetic disorders such as Down’s syndrome. The origin of compromised oocyte function may be founded in mitochondrial dysfunction and increased reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION Oocytes from young and old mice were treated with MitoQ and/or BGP-15 during IVM. To directly induce mitochondrial dysfunction, oocytes were treated with H2O2, and then treated the MitoQ and/or BGP-15. Immature human oocytes were cultured with or without MitoQ. Each experiment was repeated at least three times, and data were analyzed by unpaired-sample t-test or chi-square test. PARTICIPANTS/MATERIALS, SETTING, METHODS Immature germinal vesicle (GV) stage oocytes from 1-, 12- and 18-month-old mice were obtained from preovulatory ovarian follicles. Oocytes were treated with MitoQ and/or BGP-15 during IVM. GV-stage human oocytes were cultured with or without MitoQ. Mitochondrial membrane potential and mitochondrial ROS were measured by live-cell imaging. Meiotic spindle and chromosome alignments were visualized by immunofluorescent labeling of fixed oocytes and the 3-dimensional images were analyzed by Imaris. MAIN RESULTS AND THE ROLE OF CHANCE MitoQ or BGP-15 during IVM protects against spindle and chromosomal defects in oocytes exposed to oxidative stress and in oocytes from aged mice (P &lt; 0.001). In human oocytes, the presence of MitoQ during IVM promoted nuclear maturation and had a similar positive effect in protecting against chromosomal misalignments (P &lt; 0.001). LIMITATIONS, REASONS FOR CAUTION Our study identifies two excellent candidates that may help to improve fertility in older women. However, these potential therapies must be tested for efficacy in clinical IVM systems, and undergo thorough examination of resultant offspring in preclinical models before utilization. WIDER IMPLICATIONS OF THE FINDINGS Our results using in-vitro systems for oocyte maturation in both mouse and human provide proof of principle that mitochondrially targeted molecules such as MitoQ and BGP-15 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities. STUDY FUNDING/COMPETING INTEREST(S) The project was financially supported by the National Health and Medical Research Council and Australian Research Council, Australia. U.A.-Z. was supported by the Iraqi Higher Education and Scientific Research Ministry PhD scholarship and O.C. was supported by TUBITAK-1059B191601275. M.P.M. consults for MitoQ Inc. and holds patents in mitochondria-targeted therapies. R.L.R. is an inventor on patents relating to the use of BGP-15 to improve gamete quality. TRIAL REGISTRATION NUMBER N/A