lecture 12: early embryo development

Question 1

What is the process of mammalian embryo development?

Answer 1

• very complicated and not entirely understood

Question 2

What was William Harvey’s suggestion?

Answer 2

• 1651
• De Generatione Anamalium
• Ex ovo omnia - everything comes from the egg
• in a way he was right - e.g. all mitochondria come from the egg

Question 3

What is the first week of ‘life’?

Answer 3

• preimplantation embryo development
• from fertilisation up to implantation
• amazing journey
• embryo undergoes massive changes
• egg is the largest cell in female body
• capable of sitting in ovary for up to 40 years and generating a viable embryo - very quiescent cell
• doesn’t use glucose
• when the sperm activates development (via fertilisation) the egg is already passing through the oviduct
• cleavage of the cells - they start to get smaller - restrictive mitoses
• so while the embryo is technically ‘growing’ there is no increase in diameter of the zygote
• no actual net growth of mass until around implantation
• at about the 8 cell stage you are essential looking at the cleaving egg because none of the paternal genes are active
• undergoing process based on what is stored in the egg
• around day 4 one of the first major morpho-events takes place: formation of an epithelium
• generation of an epithelium is important because you can create an inside and an out
• up until this point the zona is holding the embryo together
• around day 5 we get differentiation into 2 cell types
  
  • outer layer called trophectoderm
    
    • maintain fluid filled cavity called the blastocoel
    • get rid of zona
    • turn into placental tissue
    • this layer of cells is not embryonic
  • inner cell mass
    
    • from where foetal tissue derives
• zona pellucida serves two purposes:
  
  • binding to the sperm
  • holds embryo together
• but trophectoderm and endometrium have to come together in order for blastocyst to invade the tissue so need get rid of physical block - the glycoprotein coat of zona - in order for this to occur
• endometrium helps the blastocyst do this
• both secrete proteases so zona is literally digested from the inside and the outside
• and at the same time the embryo gets bigger
• this process is very similar in the mouse
• there are two uterine horns
• occurs under a similar time frame

Question 4

What is cleavage in the mammal?

Answer 4

• cleavage is slow: 12 to 24 hour per division
• little or no yolk (why? - mum is providing the nutrients, obesity can negatively affect through epigenetic modifications gametes)
• cleavage holoblastic, rotational
• first cleavage produces blastomeres that are developmentally equivalent
• intial steps depend on mRNA and proteins stored in egg
• rapid cell division (??)
• cytoplasm of zygote is repackaged into smaller and smaller cells
• no increase in size of embryo
• initial divisions to form the multicellular embryo
• cleavage stage cells = blastomeres
• mitotic cell cycle without G1 and G2 phases
• cells become smaller with each division → restoration of nuclear:cytoplasm ratio
• holoblastic cleavage → cells equal and completely seperate

Question 5

What is embryo splitting?

Answer 5

• embryo splitting produces 2 (or more) embryos that are genetic clones
• this is used in agriculture to maximise yield of offspring from high genetic value eggs
• cattle embryos can be worth up to $10,000 each - very fiscal componet
• take elite embryos and bisect them

Question 6

What is embryonic genome activation?

Answer 6

• 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
• in human this is about 8 cell stage
• gradual

Question 7

What is compaction?

Answer 7

• 8-cell embryo – cadherin → membrane
  
  • cadherin is a Ca²⁺ dependent cell adhesion molecule
• cadherin mediates adhesion between blastomeres
• cell outlines coalesce to form a “morula”
• defines polarity of blastomeres → inside and outside

Question 8

What is blastulation?

Answer 8

• 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 → trophoblast / trophectoderm
  
  • trophectoderm → placenta
• inner cells → inner cell mass
  
  • ICM → embryo
• polarity

Question 9

What is zona hatching?

Answer 9

• zona prevents cell-cell contact of embryo and oviduct wall
• premature hatching → tubal pregnancy
• hatching occurs in uterus just before implantation
• blastocyst secretes proteases that weaken the zona
• failure to hatch can cause infertility

Question 10

What is the changing physiology of the embryo during the preimplantation period?

Answer 10

• the preimplantation period is highly dynamic during which there are many significant changes in embryo physiology
• around the time of compaction glucose becomes a primary nutrient
• embryos have similarities to cancer - invasive etc
• if you want to kill a cancer glucose starvation is one effective method - works on embryo
• glucose is a key molecule in biosynthesis
• if you are a rapidly dividing tissue you need to synthesise lots of DNA - requires glucose
• need glucose for lipid biosynthesis

Question 11

What are features of the zygote in regards to energy sources?

Answer 11

• low QO₂ (relatively quiescent)
• limited capacity to utilise glucose
• generates energy from low levels of oxidation of pyruvate and/or lactate with aspartate (and other amino acids)

Question 12

What are features of the blastocyst in regards to energy sources?

Answer 12

• high QO₂(metabolically very active) → as active as skeletal muscle
• QO2 is amount of oxygen consumed/tissue - so a good way to compare
• high capacity to utilise glucose
• generates energy from both aerobic glycolysis and the oxidation of glucose
  
  • this pattern of metabolism is typically associated with invasive tumours

Question 13

What is the pronucleate oocyte? What happens to it? What occurs at the different stages of cell division in an embryo?

Answer 13

• the pronuclei are haploid and can be seen as two spherical objects
• the female pronucleus is typically slightly larger than the male and is closer to the 2nd polar body
• after around 21h post fertilisation, the pronuclei come together on the first mitotic plate
• at this stage the embryo is said to be in “syngamy”
• this represents the union of the male and female germlines
• in many countries, in the eyes of the law syngamy represents the beginning of life
• the embryo then undergoes a restrictive mitosis (no cell growth)
• first paternal transcripts can be detected
• 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
• the time is now around 70h post fertilisation
• once an embryo has compacted, and formed tight junctions between blastomeres, the embryo is known as a morula
• the morula can control inside and out, a significant step in development
• through Na/K ATPase activity on the basolateral membrane, an ionic gradient is formed and water flows into the embryo through aquaporins
  
  • 3 Na into blastocoel, 2K out
  • water follows the sodium in
• the embryo is now known as a blastocyst and contains two cell types:
  
  • the inner cell mass (ICM), which goes on to form predominantly foetal tissue
  • the trophectoderm, which form extra embryonic tissue
• through the action of apical proteases (and also through uterine proteases) the glycoprotein zona pellucida is digested and the hatched blastocyst is free to implant into the endometrium of the uterus

Question 14

How do you get monozygotic twins vs fraternal twins?

Answer 14

• if the embryo or ICM splits you get monozygotic
• fraternal twins are formed from two eggs

Question 15

How can we “humanise” other mammalian species?

Answer 15

• through molecular biology and embryology we can “humanise” other mammalian species
• can get integration of exogenous DNA at pronucleate oocyte stage → transgenic animal
• transgenic animals are an extremely common technology

Question 16

How can you sex embryos? Why do it?

Answer 16

• GFP
• female embryos will exhibit green fluorescence aat ~480nm from 2 cell stage
• embryos can be used for further analysis such as cell count, embryo transfer etc
• much faster than PCRing the blastocyst
• important due to sex-related physiology of the preimplantation embryo
• 2 active copies of the X-chromosome in female mouse embryos resulting in an altered proteome and metabolome
• following implantation you get formation of barr-bodies and so on, but both are active beforehand

Question 17

What is cloning?

Answer 17

• therapeutic and reproductive cloning
• Dolly is the most famous sheep in the world - a reproductive clone
  
  • mature udder cell (starved so that it is in G1 phase of cell cycle) is put into unfertilised egg with nucleus removed (enucleated cell)
  • new “zygote” placed in sheep’s uterus
  • Embryo develops into Dolly → the 1 out of 277 that worked
• have now cloned many types of animals
• therapeutic cloning
  
  • take your somatic cell etc generate
  • don’t insert blastocyst into a womb
  • put it on a plate and let it propagate to form stem cells
  • use this to generate any tissue and then use that tissue to treat disease
  • we are not there yet but that is the goal

Question 18

What do I expect you to learn from this lecture?

Answer 18

• how the embryo forms its first epithelium
• identify the two cell types of the blastocyst and what is their fate
• how the embryo derives its energy prior to implantation
• the temporal and spatial events during the first week of life