5Quality In mice all fully grown oocytes are competent In human and bovine, only a subset of fully grown oocytes are competent. Why?An oocyte contains all the material to generate an embryo, a mass of totipotent cells that can began to grow at the wrong placecMyc models: all females die < 12 monthsThe importance of LH receptorPath to ovulationEpithelial to mesenchymal transition in the folliclePre-pubertal
6Quality factors Size of the egg ? Yes (80 µm mouse vs 120 µm human) Size of the follicle ? Yes (human- bovine)Age Yes (human)Genetic factors YesHormonal treatment Yes (aneuploidy)
7Oocyte number ?All species studied react the same with ovarian stimulation: more aneuploidyLogically a mechanism to protect the uterus /mother from too many foetusesThe uterus can identify the aneuploid embryos and destroy them actively.Why does aneuploidy explode with age ?
8Aneuploidy Exponential rise with age past 32 (more linear before that period)Much more pronounced in humans than animalsLike menopause, an adaptive mechanism?Data on RNA content of bovine oocyte of different quality points at spindle regulation as a fail-safe mechanism for oocytes coming from compromised follicles.
9Oocyte competence Is not often present (average less than 10%) Goes up and down quickly (24-48 hrs)Is compromised by ovarian stimulationMore aneuploidyMouseSheep- pig bovineHumanLess maturity (less days of growth smaller ovulation size)
10Follicular determinants of oocyte quality Follicle growthOocytematurationFertilizationEmbryodevelopmentFollicle differentiationFollicleCumulusEmbryo
11Oocyte acquires competence in a multi-step fashion Done in animals….(and for IVM in humans)competentLH pulsesLH surgeCompetencepotentialLowgrowthLH effectdominantLowcompetenceFull oocyte size0 % competenceFSHStimulatedgrowthEarly antral
12Follicular qualityOocyte quality is influenced by follicular differentiations statusThe oocyte potential is determined before fertilizationAcross patients (Hamel et al 2008)Within patients (Hamel et al 2010)Some markers can predict implantation failure (Hamel et al 2010)Same phenotype observed in cows
13Follicular size human (Nivet et al in preparation)
14Oocyte mitochondria: building the population The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomesTimothy Wai, Daniella Teoli & Eric A ShoubridgeNature Genetics 40, (2008)
15Mitochondrial DNA (mtDNA) cycle in the mouse germline. Poulton J, Chiaratti MR, Meirelles FV, Kennedy S, et al. (2010) Transmission of Mitochondrial DNA Diseases and Ways to Prevent Them. PLoS Genet 6(8): e doi: /journal.pgen
17Special Mitochondria Very special compared to somatic Potentially in a sleeping stateDifferent morphologyIntense selection pressureLow membrane potential ? (stain response)Many ATP enzymes reduced during IVM ?Alternative energy pathways activated ?ATP does go up during maturation
18Impaired function ? Morphology indicative of limited capacity (less cristae)Decreased expression ofSeveral Complex I-II-III-IV-V (genes) RNA ( n= 47 types) in mature oocytes.(Scantland et al unpublished).(A, B) Oocyte Mitochondria (hooded)(C) regular Mitochondria (Crocco et al. 2011).
24Fate of Bos Indicus mitochondria transfer into Bos Taurus oocyte Biol Reprod Mar;82(3): Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line. Ferreira CR, et alOoplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < ). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.
25Somatic mitochondria in oocytes Cloning Stem Cells Winter;9(4):The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.Ferreira et alAbstractThe mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.
26mitochondria functionality Effect of inhibition or stimulationEffect of ovarian stimulationA patient-specific problem ?
27Immediate failureV.Y. Rawe, S.Brugo Olmedo, F.N. Nodar, R. Ponzio, and P. Sutovsky Abnormal assembly of annulate lamellae and nuclear pore complexes coincides with fertilization arrest at the pronuclear stage of human zygotic developmentHum. Reprod. (2003) 18 (3):Human oocytes which have failed to complete fertilization in vitro (arrest at two pronuclear stage). Arrows denote presence of mitochondria with electron dense material not seen in controls. For further details see pp
28Beta-oxidation and competence Biol Reprod Dec;83(6): doi: /biolreprod Epub 2010 Aug 4.Beta-oxidation is essential for mouse oocyte developmental competence and early embryo development.Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ, Robker RL.Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development.In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage.
29Mitochondria and COS (controlled ovarian stimulation ) Reprod Fertil Dev. 2012;24(7): Impaired mitochondrial function in murine oocytes is associated with controlled ovarian hyperstimulation and in vitro maturation. Ge H, et al.In conclusion, the results of this investigation indicate that non-physiological COH and IVM treatments inhibit mtDNA replication, alter mitochondrial function, and increase the percentage of abnormal cytoskeleton and ROS production. Damage related to the mitochondria may partly explain the low efficiency of IVF and high rate of embryonic loss associated with these clinical procedures.
30Part of the equationAn oocyte with non functional mitochondria cannot become a viable embryo but conversely a non viable embryo may have functional mitochondriaWhat is the ratio of embryos that fail ?More than 90% of the oocytes aspiratedWhat is the ratio of embryos that fail due to non functional mitochondria ?
32Selecting embryos afterward CGH analysis of trophoblast biopsiesWould be useful to screen for aneuploidyBut would not detect metabolic mismatchThe biopsies could be used as well for Mito DNA analysis1.501.200.800.50Log2 Ratio Ch1/Ch20.300.00-0.30-0.50-0.80-1.2012345678910111213141516171819202122XYChromosomal position
33Animal models Total replacement Mouse (shown to work)Monkey (works as well)Infertility treatment (adding mitochondria)Bovine probably the best model for generating oocyte-like stem cells and testing them on experimental oocytes-embryos-foetus-term.
34Conclusion-competence Age and ovarian stimulation impact competence (mainly aneuploidy)Competence is labileIncompetence may beAge-induced (ovarian active process)Innate or patient specific (genetic)Acquired during oogenesis (several adverse conditions)Acquired during the last few days (COS)
35Conclusion Mitochondria Functional mitochondria are essentialStimulation or inhibition of mitochondrial activities impact embryo qualityIf transfer is usedChimerism possible and not lethalDistribution difficult to predict as selection occursImpact on fertility remains unknown.
37A model depicting the functional interactions between ER and mitochondria in the mouse egg. A model depicting the functional interactions between ER and mitochondria in the mouse egg. After Ca2+ is released from the ER into the cytosol via Ins(1,4,5)P3 receptors it can be taken up by the mitochondria. In the mitochondrial matrix, Ca2+ will stimulate the Kreb’s cycle as well as the electron transport chain and the F0/F1 synthase that phosphorylates ADP. ATP is then transported out of the mitochondria via the activity of the adenine nucleotide translocase (ANT) and the cytosolic ATP will be consumed by the SERCA pumps to restore the resting [Ca2+]c and refill the ER.Dumollard R et al. Development 2004;131:
38The follicular response Nivet et al Reproduction
39Blastocyst rate (industry standards) Nivet et al Reproduction