Oocyte Quality

Marc-André SirardReproduction and Genomic

Canadian Research Chair

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Plan

• Oogenesis• What makes a good egg ?• The building of the mitochondria population

in oocytes• The “special” mitochondria of the oocyte• Adding or replacing mitochondria

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Oogenesis• 8-10 months growth period• 8-10 days final differentiation and competence

acquisition

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Oogenesis

Full size oocyte

3mm

20mm

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Quality

• 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 place

• cMyc models: all females die < 12 months• The importance of LH receptor

– Path to ovulation– Epithelial to mesenchymal transition in the follicle– Pre-pubertal

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Quality 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 Yes • Hormonal treatment Yes (aneuploidy)

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Oocyte number ?

• All species studied react the same with ovarian stimulation: more aneuploidy

• Logically a mechanism to protect the uterus /mother from too many foetuses

• The uterus can identify the aneuploid embryos and destroy them actively.

• Why does aneuploidy explode with age ?

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Aneuploidy

• Exponential rise with age past 32• (more linear before that period) • Much more pronounced in humans than animals• Like 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.

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Oocyte competence

• Is not often present (average less than 10%) • Goes up and down quickly (24-48 hrs) • Is compromised by ovarian stimulation

– More aneuploidy• Mouse• Sheep- pig bovine• Human

– Less maturity (less days of growth smaller ovulation size)

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Follicle growthOocyte

maturation

Fertilization

Embryodevelopment

Follicle differentiation

Follicle Cumulus Oocyte Embryo

Follicular determinants of oocyte quality

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Oocyte acquires competence in a multi-step fashion

Done in animals….(and for IVM in humans)

Early antral

Full oocyte size0 % competence FSH

Stimulated growth

Low competence

Competencepotential

Low growthLH effectdominant

competent

LH pulsesLH surge

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Follicular quality

• Oocyte quality is influenced by follicular differentiations status

• The oocyte potential is determined before fertilization– Across 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

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Follicular sizehuman(Nivet et al in preparation)

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Oocyte 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, 1484 - 1488 (2008)

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Mitochondrial 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): e1001066. doi:10.1371/journal.pgen.1001066http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001066

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Selection of the good ones or not

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Special Mitochondria

• Very special compared to somatic• Potentially in a sleeping state

– Different morphology – Intense selection pressure – Low membrane potential ? (stain response)– Many ATP enzymes reduced during IVM ?– Alternative energy pathways activated ?

• ATP does go up during maturation

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Impaired function ?

• Morphology indicative of limited capacity (less cristae)

• Decreased expression of Several 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).

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Figure 1 A–E and I are transmission electron micrographs that have been colorized to enhance the visualization of certain subcellular components.

Van Blerkom J Reproduction 2004;128:269-280

© 2004 Society for Reproduction and Fertility

Human oocyte

mitochondria

JC-1 stainGreen lowRed high

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Intracellular reorganization during human oocyte maturation and early embryonic development.

Boumela I et al. Reproduction 2011;141:549-561

© 2011 Society for Reproduction and Fertility

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Manipulating mitochondria

• Replacing them all– Spindle transfer– Pronuclei transfer

• Injecting new ones– From another oocyte– From somatic cells

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Replacing them all

Spindle transfer

Pronuclei transfer

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Replacing some

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Biol Reprod. 2010 Mar;82(3):563-71. Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line. Ferreira CR, et al

Ooplasmic 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 < 0.0001). 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.

Fate of Bos Indicus mitochondria transfer into Bos Taurus oocyte

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Somatic mitochondria in oocytes• Cloning Stem Cells. 2007 Winter;9(4):618-29.• The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.• Ferreira et al • Abstract• The 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.

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mitochondria functionality

• Effect of inhibition or stimulation• Effect of ovarian stimulation• A patient-specific problem ?

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Immediate failure• V.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 development

• Hum. Reprod. (2003) 18 (3): 576-582

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. 576-582.

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Beta-oxidation and competence• Biol Reprod. 2010 Dec;83(6):909-18. doi: 10.1095/biolreprod.110.084145. 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.

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Mitochondria and COS (controlled ovarian stimulation )

• Reprod Fertil Dev. 2012;24(7):945-52. 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.

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Part of the equation

• An oocyte with non functional mitochondria cannot become a viable embryo but conversely a non viable embryo may have functional mitochondria

• What is the ratio of embryos that fail ?More than 90% of the oocytes aspirated

• What is the ratio of embryos that fail due to non functional mitochondria ?

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Selecting embryos afterward

• Morphokinetics – Emerging technology with limited precision

– Insufficient unless obvious phenotype

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Selecting embryos afterward• CGH analysis of trophoblast biopsies

– Would be useful to screen for aneuploidy – But would not detect metabolic mismatch– The biopsies could be used as well for Mito DNA

analysis

1 2 3 4 5 76 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 X Y

Chromosomal position

Log 2 R

atio

Ch1/

Ch2 1.50

1.200.800.50

0.00-0.30

0.30

-0.50-0.80-1.20

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Animal 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.

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Conclusion-competence

• Age and ovarian stimulation impact competence (mainly aneuploidy)

• Competence is labile• Incompetence may be

– Age-induced (ovarian active process)

– Innate or patient specific (genetic)

– Acquired during oogenesis (several adverse conditions)

– Acquired during the last few days (COS)

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Conclusion Mitochondria

• Functional mitochondria are essential• Stimulation or inhibition of mitochondrial

activities impact embryo quality• If transfer is used

– Chimerism possible and not lethal– Distribution difficult to predict as selection occurs– Impact on fertility remains unknown.

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A model depicting the functional interactions between ER and mitochondria in the mouse egg.

Dumollard R et al. Development 2004;131:3057-3067

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The follicular response

Nivet et al 2013. Reproduction

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Blastocyst rate (industry standards)

20 44 68 92Nivet et al 2013. Reproduction