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M6112 > L5: Embryo Metabolism and Blastocyst Formation > Flashcards

L5: Embryo Metabolism and Blastocyst Formation Flashcards

1
Q

What changes take place in embryo metabolism during the preimplantation period?

A
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2
Q

How can metabolomics be used to assess embryo quality? (e.g.)

A
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3
Q

How often does cleavage occur?

A
  • Asynchronous
  • Every 10-12 hrs
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4
Q

Define MZT and ZGA:

A
  • Maternal to zygotic transition: Period during which zygotic genes are activated and maternal transcripts are cleared
  • Occurs between 2-cell stage and morula
  • 2 pathways: mediated by maternal factors (M-decay) or zygotic factors (Z-decay)
  • This decay is followed by two eaves of zygotic genome activation (minor and major ZGA)
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5
Q

List the 4 types of epigenetic modifications in eukaryotes:

A
  • DNA methylation (Methylation of CpG islands)
  • Histone modifications (acetylation, ub, methyl’n etc)
  • RNA modification (generally m6A)
  • Chromatin 3D remodelling
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6
Q

Outline the process of compaction:

A
  • Morula becomes compacted as it enters uterus
  • Cell-cell adhesion increases (driven by e-cadherin expression)
  • Outer cells become polarised (progenitors of trophoectoderm)
  • This process is calcium dependent
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7
Q

What molecules are involved in adherens junctions (AJ)?

A
  • alpha and beta catenins attach to actin cytoskeleton
  • catenins acts as tether to cadherin which mutually bind in adjacent cells
  • Binding between cadherins: calcium dependent homophilic association
  • Association is dynamic (mechanism of regulation unclear)
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8
Q

Consequence of e-cadherin knockout?

A
  • Cells of morula start to compact then partially dissociate at morula stage
  • Trophoectoderm unable to form
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9
Q

How is e-cadherin expressed during embryogenesis?

A
  • Expressed from early cleavage (largely unlocalised)
  • At time of compaction, gets relocated to regions of cell-cell contact
  • Process likely mediated by PkC activation
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10
Q

Zonula adherens vs zonula occludens:

A
  • Adherens: E-cadherin binds apposite cells in ca-dependent manner, attaching to actin cytoskeleton via a, b, y-catenin complex
  • Occludens: Occludin binds adjacent cells, binding via ZO-1, ZO-2 complex to cingulin which attaches to actin cytoskeleton (N.B. a.k.a Tight junction)
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11
Q

Junction permeability during tight junction biogenesis:

A
  • Permeable at 16-cell stage
  • Becomes impermeable at blastocyst due to tight junction formation (ZO)
  • It is at this point that occludin begins to be expressed (key ZO component)
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12
Q

What changes occur in osmolarity during blastulation?

A
  • Sodium influx increases [Na+] concentration gradient (this requires polarised distribution of Na/K-ATPase on the basolateral membrane of the trophoectoderm)
  • Water enters trophoblast via aquaporins on apical surface, then passes through aquaporins on basolateral surface to enter blastocoel-> blastocoel cavity becomes fluid filled
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13
Q

Adverse fertilisation outcomes: 2-cell arrest

A
  • Primarily observed in mice
  • (a) Cell shrinkage and lysis -> possible autophagic cell death
  • (b) Presence of midline cellular fragmentation
  • (c) Complete embryo fragmentation
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14
Q

Adverse fertilisation outcomes: Abnormal embryo development

A
  • Occurs primarily in humans (at morula)
  • (a) Fragmentation without loss of blastomeres (caspase independent -> oncosis)
  • (b) Embryo arrest
  • (c) Fragmentation with loss of blastomeres (caspase-dependent -> apoptosis)
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15
Q

Arrested embryo material in ART:

A
  • Fragmented arrested embryos considered toxic
  • Will be remove during ART procedures
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16
Q

Key metabolic switch during compaction:

A
  • Embryo switches from dependence on TCA cycle to glycolysis
  • Requirement for pyruvate replaced with glucose
17
Q

Consequences of cumulus cell expansion:

A
  • Prompted by LH surge
  • Allows detachment from follicular wall
  • Oocyte can be released from ruptured follicle
18
Q

Detail on embryo metabolism before compaction:

A
  • Before compaction, the embryo has a metabolism based on low levels of oxidation of pyruvate, lactate and specific AAs
  • CCs actively produce pyruvate and lactate from glucose
  • Embryo characterised by high ATP:ADP level (low glucose supply, high pyruvate -> predominantly TCA cycle is producing ATP)
  • This in turn inhibits PFK, limiting flux of glucose through glycolytic pathway
19
Q

Detail on embryo metabolism after compaction:

A
  • Rapidly increasing energy demand
  • After compaction, embryo shows greater oxygen consumption and ability to metabolise glucose
  • ATP:ADP ratio reduces and AMP levels increase
  • Positive effect on PFK -> greater rate of aerobic glycolysis of glucose
  • While this switch is less energetically efficient, it does ensure the PPP has maximum substrate availability at all times -> production of GSH, key antioxidant, and biosynthetic precursors
  • Potential key role for malate-aspartate shuttle activity
  • There is diversity in metabolic activity of trophoblasts vs inner cell mass (purely glycolytic in mice)
20
Q

Pathway for glucose uptake in COCs:

A
  • Circulating glucose enters cumulus cell through GLUT tranporters
  • Conversion to pyruvate in CC
  • Pyruvate enters oocyte via gap junctions (passing through ZP)
21
Q

+ Localisation and expression pattern of different GLUT transporters on trophoectoderm:

A
  • GLUT3, 8: Apical surface
  • GLUT1: Cell-cell contacts (basolateral surface)
  • Some GLUT are ubiquitous through embryonic development whereas some are coordinated at key stages e.g. GLUT4 is expressed during initiation of blastulation (which cannot proceed without it)
22
Q

Why is glucose a good substrate for biosynthesis of non essential AAs?

A
  • Provides pentose moieties for nucleic acid synthesis
  • Occurs via pentose phosphate pathway (PPP)
23
Q

Why is a comprehensive understanding of embryonic metabolomics invaluable in clinics?

A
  • Avenue for development of non-invasive quality assessment of blastocysts
  • e.g. quantifying lactate production, utilisation of AAs etc
  • e.g. viable embryos found to have higher glucose uptake
  • e.g. respiratory activity of the blastocyst affects the outcome of embryo transfer (cattle)
24
Q

Outline the hypothetical model of oxygen gradient in early embryo development:

A
  • Early cleavage stage embryos encounter a physiological O2 concentration during transport through the oviduct, where a minimal gradient is encountered by individual blastomeres.
  • However, postcompaction stage embryos encounter a significantly lower (hypoxic) O2 environment in utero, potentially creating a more marked gradient within the embryo and resulting in the establishment of spatial relationships between blastomeres.
25
Q

How does exposure to atmospheric oxygen impact embryos in vitro:

A
  • Altered gene expression
  • e.g. Culture in 5% O2 associated with fewer perturbations in global pattern of gene expression -> more closely resembles that of in vivo embryo
  • -> Affects embryonic proteome (separate study)
  • 5% culture -> signficantly greater % progress to blastocyst stage and higher cell no. (compared with 20%)
26
Q

Relationship between oxygen exposure and glucose uptake in embryos:

A
  • 5% O2 -> greater glucose uptake by embryos than those in 20%
27
Q

Roles of dietary AAs in embryo and foetal growth (with examples):

A
  • Increasing fetal growth and viability (e.g. glutamine)
  • Improving antioxidant function, embryo survival (e.g. methionine)
  • Anti-inflammatory properties (e.g. taurine)
  • -> Both essential and non-essential AAs have key role in development
  • Buffer (e.g. glycine)
28
Q

How does glutamine affect embryo and foetal growth?

A
  • Increases foetal growth and viability (alongside alanine)
  • Also shown to improve human pre-implantation embryo development (greater % progression to morula and blastocyst stage)
  • Used as an energy source by the early embryo
29
Q

How can amino acid profiling be useful in quality selection? (Give 3 pieces of evidence)

A
  • Lower turnover of certain AAs (Glu, Gly, Ala) in viable vs non-viable embryos (measured in capacity to reach blastocyst stage)
  • Similar effect seen in 5% vs 20% oxygen (at blastocyst stage but not cleavage stage)
  • Correlation between AA turnover on day 2 and mean DNA damage in human blastocysts
30
Q

Give 4 invasive methods for testing embryo metabolism: (including stage in embryogenesis)

A
  • Biopsy of…
  • Polar bodies (pronuclei stage)
  • Blastomeres (cleavage stage)
  • Trophoectoderm (blastocyst stage)
  • Blastocoel fluid (blastocentesis)
31
Q

Give 4 non-invasive methods of assessing embryo quality:

A
  • Time-lapse system (light issues)
  • Spent culture media
  • On-chip analysis (genome/transcriptome/proteome/metabolome)
  • Live cell-imaging system
32
Q

Benefits of metabolomic analysis in clinics:

A
  • Non-invasive!
  • Reproducible
  • Objective
  • Rapid
  • Independent of other parameters (i.e. morphology)
  • However: Not necessarily cost effective
33
Q

What non-optical spectroscopies can be applied for metabolomics?

A
  • NMR
  • MS
  • (Often coupled with HPLC-MS for separation)
34
Q

Cons of non-optical spectroscopies for metabolomics:

A
  • Expensive to install and run
  • Require trained personnel
  • Longer analysis time
34
Q

What information can TLM technology capture?

A
  • Time to formation of 2-cell embryos (first cleavage)
  • Regularity in duration of cell cycles
  • Morphology of embryos
  • Fragmentation
34
Q

What secreted factors have been investigated for embryo metabolic asssessment?

A
  • sHLA-G (technical controversy over measurement)
  • Follicular IL-15, GM-CSF
35
Q

Further indicators for embryo quality:

A
  • High ICM:TE ratio
  • Lower number of apoptotic cells

M6112 (15 decks)

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