W4L2 Tue Early Embryo development

Question 1

Oocyte nutrient consumption

Answer 1

Oocytes stay quiescent for ~40 years (metabolically adapted so don’t require substantial energy)
Ø Low oxygen consumption, can’t use glucose (creates ROS)
Ø Take nutrients from cumulus cells in form of pyruvic acid + amino acid

Question 2

Cleavage (mammals)

Answer 2

Cleavage: initial divisions form multicellular embryo (slow, 12-24 hours/division)
Ø Holoblastic: cells equal + completely separate
Ø Rotational: cleavage changes axis each division
Ø Produces developmentally equivalent blastomeres (clones)
Ø Mitotic cell cycle without G1 + G2 phases
Ø Cells become smaller with each cell division = restoration of nuclear:cytoplasm ratio
§ Little or no yolk: embryo surrounded by oviduct + uterine fluids
§ Initial steps depend on mRNA + proteins stored in egg

Question 3

Embryonic Genome Activation

Answer 3

-embryonic genome activates at different stages in different species with activation of embryo genome
-destruction of pre-stored mRNA
-but pre-stored proteins may continue to function and regulate development for some time

Question 4

Compaction, morula creation

Answer 4

8-cell embryo – cadherin mediates adhesion between blastomeres
-cadherin is a Ca++ dependent cell adhesion molecule
-cell outlines coalesce to form a “morula”
defines polarity of blastomeres à inside and outside

Question 5

Blastulation

Answer 5

-cleavage of blastomeres results in some cells enclosed within the inner compartment of the morula
-fluid begins to accumulate between blastomeres > cavitation
-results in formation of a blastocoel
-outer layer of cells à trophoblast
-Inner cell> inner cell mass

Question 6

Zona Hatching

Answer 6

-zona prevents cell-cell contact of embryo and oviduct wall
-premature hatching cause tubal/ectopic pregnancy
-hatching occurs in uterus just before implantation
-blastocyst secretes proteases that weaken the zona failure to hatch can cause infertility
- Uterus produces proteases to expose zona from outside
- Glycoprotein zona pellucida is digested + hatched blastocyst can implant into endometrium of uterus
§ Fluid cavity creates enough turgor pressure to allow embryo to emerge from zona pellucida

Question 7

Zygote energy consumption

Answer 7

low QO 2 (relatively quiescent).
limited capacity to utilize glucose.
generates energy from low levels of oxidation of pyruvate
and/or lactate with aspartate (and other amino acids).

Question 8

Blastocyte energy consumption

Answer 8

-high QO 2 (metabolically very active).
-high capacity to utilize glucose.
generates energy from both aerobic glycolysis and the oxidation of glucose (this pattern of metabolism is typically associated with invasive tumours).

Question 9

Pronuclei

Answer 9

-Pronuclei are haploid + seen as 2 spherical objects
§ Female pronucleus slightly larger + closer to 2nd polar body

Question 10

Syngamy

Answer 10

§ 21 hours post fertilisation: pronuclei come together on 1st mitotic plate
§ Embryo is in ‘syngamy’ = union of male + female germlines
§ Embryo undergoes restrictive mitosis (no cell growth) + 1st paternal transcripts detected

Question 11

cleavage stages

Answer 11

-After around 44h the cells of the embryo (known as blastomeres) divide to the 4-cell stage through further restrictive mitosis. This period is referred to as the “cleavage stages”
- Between the 4- and 8-cell stage a large number of embryonic genes begin to be transcribed
-Between the 8- and 16-cell stage the blastomeres become polarised and form an epithelium. This is referred to as compaction. The time is now around 70h post fertilisation

Question 12

Blastocoel Ionic Gradient

Answer 12

Na/K ATPase located in basolateral membrane of trophectoderm, forms an ionic gradient + water flows into embryo through aquaporins
1. 3Na+ and 1ATP from inside cell bind to Na/K ATPase
2. Hydrolysis of ATP phosphorylates Na/K ATPase = shape change
3. Releases Na+ outside cell + enables 2K+ to bind to pump
4. Release of Pi returns pump to original shape + releases K+ inside cell
§ Osmosis: water follows Na+ out of cell to from blastocoel cavity, down the concentration gradient

Question 13

Experiment module: Transgenic
Animals

Answer 13

§ Transgene: gene that has been transferred naturally from one organism to another
§ Transgenesis: DNA injected into male pronucleus – some DNA enters chromosomes = transgene
§ Male mice X chromosome tagged with GFP
Ø Female embryos exhibit green fluorescence from 2-cell stage, can be used for analysis (cell count/embryo transfer

Question 14

Dolly the Sheep

Answer 14

§ Cells removed from Dorset ewe (female sheep) + egg removed from Scottish blackface ewe
§ Nucleus removed from egg
→ mature udder (somatic) starved of nutrients so that it is in G1 phase
→ udder cell + enucleated egg are fused
→ stimulating mitotic inducers cause cell to divide
§ Early embryo develops + is transplanted into an ewe’s uterus = Dolly (genetically identical to Dorset ewe)

Question 15

Therapeutic Cloning

Answer 15

§ Nucleus removed from patient egg
→ patient nucleus implanted into an enucleated egg
→ egg is stimulated to divide + form a blastocyst
→ embryonic stem cells removed
→ ESCs induced to form differentiated cells (bone, muscle, nerve tissue)
→ transplanted into patient (no risk of rejection)