Do small differences in oxygen concentration influence oocyte and embryo developmental competence?

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1 Jonathan Van Blerkom Department of Molecular, Cellular and Developmental Biology University of Colorado, Boulder Do small differences in oxygen concentration influence oocyte and embryo developmental competence? Disclosure information: nothing to declare

2 EARLIER OVARIAN CLUB THEME: IT S ALL ABOUT ENERGY STILL MOSTLY IS

3 Bioenergetics and Competence in Clinical IVF Re-energized Focus on [oxygen] and developmental competence: primarily, relationship to mitochondrial ATP generation as a central developmental determinant Henry Leese s Quiet Hypothesis too much detrimental mtdna content related to developmental competence at blastocyst----mtdna quantitation and Mitoscore for selection lower content better? controversial, seems not to be supported by recent studies Oxygen consumption/depletion as proxy for competence (oxygen electrodes Embryoscope) for selection however, direct relationship with outcome questionable om human IVF, difficult to perform in routine IVF programs.

4 What is Cellular Energy? Energy=Work: can it be stored in cells and why ATP? energy for work occurs or is stored in certain high energy chemical bonds for ATP (GTP), the orthophosphate bond (energy released by hydrolysis of ATP to ADP + Pi) is highest state but half-life of ATP in cell is measured in seconds and typically it is used where generated. Glucose is storable but as a substrate, i.e., potential bioenergy source if converted to ATP.

5 POTENTIAL DEVELOPMENTAL CONSEQUENCES OF AN ENERGY DEFICIT: Too few mitochondria, low mtdna content, mtdna mutations, disproportionate segregation Mechanical Work- moving things in cells chromosomes, mitochondria, cytoplasmic streaming aneuploidy, nonuniform cell divisions, abnormal organelle clustering, breakdown of cytostructure, mitochondrial segregation, etc. Chemical Work- energy released form hydrolysis of ATP (GTP) is coupled with thermodynamically unfavorable reactions and moving molecules against concentration gradients: membrane pumps, transporters, receptors, enzymes such as [Pi]-dependent kinases often have ATPase/GTPase domains and function fails when energy deficiency ---arrested cell division, fragmentation, dysfunctional signaling, apoptosis, pathological cell death, failure to cavitate or form expanding blastocyst cavity

6 ASSOCIATED WITH OVERPRODUCTION: ROS toxicity However, ROS needed for oxygen sensing by HIF pathway and other ROS-driven signaling pathways, and levels produced by leakage during ETP while detectable with certain fluorescent probes may be normal and nontoxic questions need to suppress by exogenous anti-oxidant supplementation, unless ROS scavenger functions are known or suspected of being compromised- maternal age?

7 Regulators of Mitochondrial ATP Production Substrate availability oxygen, glucose, fatty acids Free calcium levels- (SER, CiCr and mcicr signaling) Local cytoplasmic REDOX potential Intracellular [oxygen]: function of energy-independent diffusion rate across plasma membrane: passive diffusion of gas molecular across membrane determined by surface area, distance from source, presence of a concentration gradient, and in biological (live) membranes, its structure and composition. Allosteric regulation AMPK pathway, ATP/AMP mitochondrial transmembrane potential (ΔΨm), which is influenced by ambient ph, proximity to other mitochondria, redox state, free calcium CiCr and mcicr

8 Potential Confounding Factors with [O2] and Energetics as Related to Competence Mitochondrial structure in oocyte and early embryo Species differences Genetic background Kinetics of gas diffusion

9 (ΔΨm)

10 Mitochondrial mass vs. mtdna copy number 18-20K organelles* 40K- 1000K mtdna copies Organelles replicate after implantation in contrast to mtdna *Van Blerkom, 2008, RBMO Van Blerkom, 2011, Mitochondrion Makabe and Van Blerkom, 2006

11 Mitochondrial association with Smooth ER in human MII oocyte-- and example of functional compartmentalization of ATP production? SER Makabe and Van Blerkom 2006.

12 Species and Genotype 4-5%. vs. 21% (ambient) Rabbit development to expanded blastocyst, 8-10 mm, 1x10 6 cells on day 6-7, is significantly improved at higher O2 with respect to LB trophoblast cells have potent anti-oxidation mechanisms: SOD, catalase (paracrystals), glutathione. Van Blerkom et al, 1973 and unpublished

13 Mouse: Genetic Background Swiss-Webster, BALB/c: development to blastocyst and LB after ET better at 12% than 5%, but not at ambient O2. C3H; C57BL/6J; Short Sleep (SS)and Long Sleep (LS) better at 4-5% than higher. ob/ob and db/db embryos do better to blastocyst in 2-3%O2 compared to 5-6%O2 or higher. Mouse may not be best model to test O2 and early development with respect to human IVF

14 Perifollicular Blood Flow and Oocyte Ploidy Geeta Nargund et al: Linda Gregory et al,, Van Blerkom et al (1997: HR)

Aneuploidy by conventional cytogenetics at MII Color Pulsed Doppler- /Spectral Imaging of PFBF A: high flow, 3-4% O2 lowest frequency of spindle disorders and aneuploidy by

15 Aneuploidy by conventional cytogenetics at MII Color Pulsed Doppler- /Spectral Imaging of PFBF A: high flow, 3-4% O2 lowest frequency of spindle disorders and aneuploidy by conventional cytogenetics BB: medium flow, 1-2% O2 >C<A C: Low flow, <1% O2 highest frequency of spindle disorders and aneuploidy Van Blerkom et al, 1997, Hum Van Blerkom et al, HR, 1997 Reprod

Kinetics of oxygen diffusion in serum/ high protein concentration as in FF The maintenance of a given rate of oxygen utilization may be critically depended on fluid depth and on the

16 Kinetics of oxygen diffusion in serum/ high protein concentration as in FF The maintenance of a given rate of oxygen utilization may be critically depended on fluid depth and on the solubility and rate of diffusion of oxygen in (culture) fluid. * *McLimans et al (2004) Kinetics of gas diffusion in mammalian cell culture systems. II. Theory. Biotechnology and Bioengineering

17 Partial list of genes whose expression is up-or down regulated in cumulus oophorus by 2% change in oxygen concentration: (Van Blerkom 2012) In cumulus oophorus, the level of expression of over 300 genes at the mrna level is up or down regulated by 2% incremental changes in O2 in vivo Coronal cells may not have have a similar response. which ones are important for oocyte developmental competence a few, some, all?

18 Early Mouse Oocyte Microarray-Based Gene Expression Studies* Expression level of nearly 350 genes apparently up- (301) or down- (49) regulated in day 4.5 mouse blastocysts between culture at 5% and 18% O2. Extent of differences may be strainassociated. reviewer comments: elevator science, developmental relevance? most assigned genes had unknown functions in cells and clearly unknown in early development difficult to know which are developmentally relevant---presumably all were---interconnected pathways, functions and regulation. (return when you know their specific functions in follicle, oocyte or embryo) *

19 Regulators of Mitochondrial ATP Production Substrate availability oxygen, glucose, fatty acids Free calcium levels- (SER, CiCr and mcicr signaling) Local cytoplasmic REDOX potential Intracellular [oxygen]: function of energy-independent diffusion rate across plasma membrane: passive diffusion of gas molecular across membrane determined by surface area, distance from source, presence of a concentration gradient, and in biological (live) membranes, its structure and composition. Allosteric regulation AMPK pathway, ATP/AMP mitochondrial transmembrane potential (ΔΨm), which is influenced by ambient ph, proximity to other mitochondria, redox state, free calcium CiCr and mcicr

20 Is energy production and utilization uniform or spatially localized in the ooplasm?

21 ATP production bursts observed during oocyte maturation are stage specific and spatially localized within ooplasm: Yu et al (2010) in Redistribution of Mitochondria Leads to Bursts of ATP Production During Spontaneous Mouse Oocyte Maturation. J Cell Physiol. 224: :--- suggests that that mitochondrial ATP production is stimulated by microfilament-driven, subcellular targeting of mitochondria. Other studies show that ATP production in oocyte regulated by focal changes in free calcium levels: SER/mitochondrial functional relationships. (see reviews by Van Blerkom 2008, RBMO; 2009 Sem. Cell and Dev. Biol. 2011: Mitochondrion

22 Spatial Functional Compartmentalization [O2], ΔΨm and average net ATP content JC1 and J- aggregates red to orange fluorescence=high ΔΨm green= lower ΔΨm proxy for relative ATP generation activity

23 What level of dissolved oxygen does the oocyte see in the follicle, is it uniform within the follicular fluid or occur as a gradient, and how does this level relate to outcome in clinical IVF?

24 Hypothesis: hypoxic to near anoxic conditions exist in oocyte and inner cumulus oophorus and coronal cells (that communicate with the oocyte) are normoxic such that small differences in oxygen concentration may have functional consequences for the the oocyte at the molecular and oolemmal levels. How test this hypothesis?

Reduction, Oxidation Loss of Quenching Hypoxyprobe-1 or pimonidazole HCl, produces pimonidazole adducts in hypoxic tissues designed to be introduced in vivo, and is highly water soluble and can be

25 Reduction, Oxidation Loss of Quenching Hypoxyprobe-1 or pimonidazole HCl, produces pimonidazole adducts in hypoxic tissues designed to be introduced in vivo, and is highly water soluble and can be used in vitro. Adducts form with thiol groups in proteins such that side-chain of the 2-nitroimidazole is Retained and visualized by anti-adduct immunofluorescence. Hypoxia Probe LOX-1 e Sciences), rapid response to changing po2 in vitro -added to culture medium 4-amino-8-N-(2-(2-hydroxyethoxy)ethyl)-naphthalimide- N,N-dimethylaniline in vitro use, fluorescent signal quenched at normoxic level Image-iTTM Hypoxia Probe (Thermo- Fisher) iridium-based for in vitro use

26 gas out gas mix in sampling port oxygen electrode FF or culture medium

27 In vitro modulation of O2 detected with Hypoxia Probe LOX-1 in mouse 2-cell embryo

28 Detection of relative hypoxia level at follicular aspiration with Image-iTTM Hypoxia Probe* *Thermo-Fisher At follicular aspiration, human oocyte and cumulus oophorus are normally severely hypoxic

29 For Cumulus Oophorus Among others, expression at protein level for HIF, VEGF, leptin, heat shock proteins 70,86,90,mitochondrial CoA components, NADH-dehydrogenase, beta-oxidation components, and others involved in bioenergetics including AMPK and GTPase(s) was function of small differences in [O2].

30 For Oocyte: Single cell genomics/proteomics is the next step to test the hypothesis that ambient oxygen concentration may be a critical regulatory driver during oogenesis and preimplantation embryogenesis

31 Are these results meaningful in relating developmental competence and outcome [O2]?

32 Cumulus oophorus outgrowth phenotypes in 6-12 h: potential bio-indicator of hypoxia level and ganglioside GM1 organization consistent with fertilization 3-4% 2% <1% Van Blerkom and Caltrider,2013, RBMO human: Van Blerkom and Zimmermann, 2016, RBMO, mouse <1%

33 Affect of [O2] on GTPase Content GTPase signaling pathways -protein signaling pathways, tubulin polymerization, myosin molecular motors Golgi-derived secretory vesicle formation, membrane anchoring (tethering) and exocytosis Endosome trafficking

34 [oxygen] and GTPase expression at protein level

35 Take Home Message For the oocyte, hypoxia bordering on anoxia is normoxia and as such may be a critical driver of fertilization and developmental competence. For oocyte and embryo, 2-3% may be optimal concentration from MII-D6 blastocyst.

36 Acknowledgement Kyle Caltrider Sarah Zimmermann

CELLS. Cells. Basic unit of life (except virus)

CELLS. Cells. Basic unit of life (except virus) Basic unit of life (except virus) CELLS Prokaryotic, w/o nucleus, bacteria Eukaryotic, w/ nucleus Various cell types specialized for particular function. Differentiation. Over 200 human cell types 56%