L5: Embryo Metabolism and Blastocyst Formation

Question 1

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

Question 2

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

Question 3

How often does cleavage occur?

Answer 3

• Asynchronous
• Every 10-12 hrs

Question 4

Define MZT and ZGA:

Answer 4

• 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)

Question 5

List the 4 types of epigenetic modifications in eukaryotes:

Answer 5

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

Question 6

Outline the process of compaction:

Answer 6

• 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

Question 7

What molecules are involved in adherens junctions (AJ)?

Answer 7

• 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)

Question 8

Consequence of e-cadherin knockout?

Answer 8

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

Question 9

How is e-cadherin expressed during embryogenesis?

Answer 9

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

Question 10

Zonula adherens vs zonula occludens:

Answer 10

• 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)

Question 11

Junction permeability during tight junction biogenesis:

Answer 11

• 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)

Question 12

What changes occur in osmolarity during blastulation?

Answer 12

• 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

Question 13

Adverse fertilisation outcomes: 2-cell arrest

Answer 13

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

Question 14

Adverse fertilisation outcomes: Abnormal embryo development

Answer 14

• 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)

Question 15

Arrested embryo material in ART:

Answer 15

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

Question 16

Key metabolic switch during compaction:

Answer 16

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

Question 17

Consequences of cumulus cell expansion:

Answer 17

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

Question 18

Detail on embryo metabolism before compaction:

Answer 18

• 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

Question 19

Detail on embryo metabolism after compaction:

Answer 19

• 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)

Question 20

Pathway for glucose uptake in COCs:

Answer 20

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

Question 21

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

Answer 21

• 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)

Question 22

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

Answer 22

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

Question 23

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

Answer 23

• 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)

Question 24

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

Answer 24

• 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.

Question 25

How does exposure to atmospheric oxygen impact embryos in vitro:

Answer 25

* 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%)

Question 26

Relationship between oxygen exposure and glucose uptake in embryos:

Answer 26

* 5% O2 -> greater glucose uptake by embryos than those in 20%

Question 27

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

Answer 27

* 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)

Question 28

How does glutamine affect embryo and foetal growth?

Answer 28

* 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

Question 29

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

Answer 29

* 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

Question 30

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

Answer 30

* Biopsy of... * Polar bodies (pronuclei stage) * Blastomeres (cleavage stage) * Trophoectoderm (blastocyst stage) * Blastocoel fluid (blastocentesis)

Question 31

Give 4 non-invasive methods of assessing embryo quality:

Answer 31

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

Question 32

Benefits of metabolomic analysis in clinics:

Answer 32

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

Question 33

What non-optical spectroscopies can be applied for metabolomics?

Answer 33

* NMR * MS * (Often coupled with HPLC-MS for separation)

Question 34

Cons of non-optical spectroscopies for metabolomics:

Answer 34

* Expensive to install and run * Require trained personnel * Longer analysis time

Question 34

What information can TLM technology capture?

Answer 34

* Time to formation of 2-cell embryos (first cleavage) * Regularity in duration of cell cycles * Morphology of embryos * Fragmentation

Question 34

What secreted factors have been investigated for embryo metabolic asssessment?

Answer 34

* sHLA-G (technical controversy over measurement) * Follicular IL-15, GM-CSF

Question 35

Further indicators for embryo quality:

Answer 35

* High ICM:TE ratio * Lower number of apoptotic cells