embryo formation and metabolism

Question 1

at what stage does the activation of the embryonic genome in humans occur?

Answer 1

four cell stage

Question 2

at what stage foes compaction occur?

Answer 2

16-32 cell stage

Question 3

what is compaction dependent on?

Answer 3

calcium

Question 4

what drives compaction?

Answer 4

E-cadherin (a transmembrane cell-cell adhesion molecule)

Question 5

when and where is E-cadherin expressed?

Answer 5

expressed from early cleavage but relocated to regions of cell to cell contact at the time of compaction, probably through the activation of Pk-C

Question 6

what are some cell adhesion molecules/cell junction components in the pre-implantation blastocyst?

Answer 6

E-cadherin
a-catenin
ZO-1
desmoplakin

Question 7

when is JAM-1 staining detected?

Answer 7

in all pre-compact eight cell embryos

at the onset of compaction all eight cell embryos also revealed intense JAM1 staining at the apical poles (outward facing) of the blastomeres as well as contact sited.

Question 8

what is blastocyst formation dependent upon?

Answer 8

–> polarised distribution of Na/K - ATPase confined to the basolateral membrane domain of the trophectoderm.

–>this establishes a trans-trophoectoderm ion gradient that facilitates movement of water across the epithelium facilitated by the presence of both apical and basolateral AQPs.

–> these events combined with the establishment of a trophectoderm tight junctional seal to prevent loss of fluid out of the embryo through parcellular routes results in the expansion of the embryo and the formation of the blastocyst

Question 9

how’s early development regulated?

Answer 9

embryo and uterus produce a large amount of different growth factors or cytokines (e.g. - embryo: IGF-1, IGFII, TGF-alfa, TGF-beta(1-3), FGF-4, IL-1alpha, IL-1beta, IL-6, IL-7)

embryos express growth factor receptors

paracrine and endocrine mechanisms are involved in supporting early stage embryo growth and development

Question 10

what happens to ATP production and the % of ATP from oxidation as the embryo develops?

Answer 10

as number of cells increases ATP production increases

As embryo develops the percentage of ATP from oxidation decreases

Question 11

around the time of compaction what happens to the embryos metabolism?

Answer 11

it switches from dependence on the TCA cycle to a metabolism based on glycolysis.

Question 12

why is glucose needed in the embryo?

Answer 12

provides the pentose moieties for nucleic acid synthesis and is required for phospholipids and non-essential amino acids biosynthesis.

Question 13

what glucose transporter genes are expressed in the oocyte?

Answer 13

glut-1
glut 3
glut-8
SGLT-1

Question 14

what glucose transporter genes are expressed at the 8 cell stage?

Answer 14

glut-1 
glut 3
Glut-5
glut-8
SGLT-1

Question 15

what glucose transporter genes are expressed at the blastocyst stage?

Answer 15

glut-1 
glut 3
Glut-4
Glut-5
glut-8
SGLT-1

Question 16

what stage embryo consumes the most oxygen?

Answer 16

blastocyst stage

Question 17

how does oxygen affect embryo development ?

20% compared with 5%

Answer 17

20% leads to a decrease in glucose uptake

20% leads to a decreased blastocyst formation and decrease in cell number per blastocyst

Question 18

how does glutamine affect human pre-impantation embryo development?

Answer 18

it improves it

increase the morula and blastocyst formation

Question 19

what are the outcomes of fertilisation?

Answer 19

• successful preimplantation embryo development
• two cell arrest - occurs primarily in mouse (cell shrinkage and lysis; possible autophagic death, presence of midline cellular fragmentation, complete embryo fragmentation)
• abnormal embryo development - occurs primarily in human (fragmentation without loss of blastomeres; possible caspase independent, possible oncosis, embryo arrest, fragmentation with loss of blastomeres; caspase dependent process, possible apoptosis)

Question 20

what is a good indicator of good and poor embryo quality?

Answer 20

the ratio of ICM:TE is an important indicator of embryo quality
high number of apoptotic cells is an indicator of poor quality embryos

Question 21

do faster or slower embryos have higher ICM:TE ratio?

Answer 21

faster embryos have significantly higher ICM:TE ration

faster embryos have significantly more inner cell mass cells and subsequently a higher total cell number

Question 22

developmental kinetics of faster embryos
carbohydrate metabolism of faster embryos
amino acid metabolism of faster embryos

Answer 22

developmental kinetics - increased blastocyst number, increased fatal development per implantation
carbohydrate metabolism - increased inner cell mass, increased glucose consumption, decreased lactate production, decreased glycolytic rate.
amino acid metabolism - increase aspirate consumption, glutamate consumption instead of production.

faster embryos consume more aspirate and have a lower glycolytic rate
slower embryos produce glutamate

Question 23

how do fast and slow embryos differ in glucose and lactate production/consumption?

Answer 23

faster embryos consume more glucose and produce more lactate

Question 24

what is time lapse microscopy TLM?

Answer 24

it captures images over time and provides a combination of morphological, dynamic and quantitative information about developmental events.

Question 25

what are the benefits of TLM?

Answer 25

you can see the time taken to for 2 cell embryo (first cleavage
you can see regularity in duration of cell cycles
you can see morphology of embryos and fragmentation
you do not need to open incubators to monitor embryo development, reduces perturbation of embryo enviroment

Question 26

what are the pros of metabolic analysis?

Answer 26

• non invasive
• reproducible
• objective
• rapid
• easy to use routinely
• is it cost effective??
• no ethical questions raised
• independent of other parameters (morphology)

Question 27

what metabolic factors may for the basis of a non invasive test of embryo viability?

Answer 27

uptake:
- glucose, pyruvate, amino acids, other sugars, oxygen
production:
- lactate, ammonium, amino acids, enzymes e.g. LDH, sHLA-G, HOXA2- regulator, PAF, metabolomics/proteomics, other peptides and factors

Question 28

what non-optical spectroscopies can be applied for metabolomics?

Answer 28

NMR spectroscopy and MS which can be coupled with separation methods like HPLC-MS

both NMR and MS can generate info on multiple compounds within an sample and have been applied to the analysis of embryos in culture media

however - expensive equipment, expensive to run, requires trained person, longer analysis time

NMR- nuclear magnetic resonance
MS mass spectroscopy
HPLC high pressure liquid chromatography