Pre-Implantation Development Flashcards
What are the immediate post-fertilisation events?
- Still in the follicle, the oocyte nucleus breaks down and a microtubule spindle assembles
- Spindle migrates to the oocyte surface and secretes half the homologous chromomoes into the first polar body.
- Remaining chromosomes are captured by a second meiotic spindle, and the egg remains arrested at metaphase II awaiting fertilization
- → the spindle is asymmetric.
- Sperm fuse away from the meiotic spindle and first polar body. Sperm oocyte fusion leads to the Ca2+ transients, cortical granule release and the resumption of meiosis
− CAM kinase II activity stimulated at fertilisaiton
− Phosophorylates Emi2, targeting it for destruction and sets in train the activation of the anaphase promoting complex. - When meiosis is resumed, egg segregate half the remaining sister chromatids into the second polar body. Next, the membrane is reformed around the haploid DNA of the sperm, and the chromosomes form the oocytes second round of meotic division, giving the male and female pronuclei. DNA synthesis occurs.
- Chromsomes become arranged on the mitotic spindle and we get the first cell division – the spindle is derived from the sperm centrioles – provide the asters for condensing chromosomes.
- Cell division gives a 2-cell organism
− Timing of this is crucial to blastocyst formation in IVF and IVF success
− First cleavage furor should be only 14 mins from the beginning to the appearance of 2 cells → may not hold, not used clinically
− The 2 cell stage should last only 11 hours → best indicator
− The cleavage of each of the 2 cells into 4 daughters should occur within 1-2 hours of each other
What are 6 consequences of fertilisation?
- Reconstitution of the normal diploid chromosome complement
- Introduction of the sperm centriole
- Block to polyspermy
• There is no fast electrical block to polysmery in mammals, but there is a slow block
• Increased cytoplasmic Ca2+ induces exocytosis of enzyme-rich cortical granules starting from the point of sperm entry.
− These strip sperm receptors from the plasma membrane
− Harden the zona by a peroxidase mediated oxidation → the zona reaction
− Proteases including Ovastatin cleave ZP-2
− CG enyzymes induce cross-linking of zona proteins, preventing binding and entry of supernumerary sperm. - Reconstruction of the multicellular stage (cleavage asymmetric → symmetric)
- Initiation of the developmental program of the embryo by activating completion of meiosis, paternal genome remodeling and molecular synthesis.
- Determination of the sex of the embryo
How is chromatin remodelled as a consequence of fertilsation?
• Maternal genome is in chromsomes arrested in metaphase II
• Paternal genome is tightly compacted for packaging into the sperm head.
− Achieved by replacing most nucleosomes with protamines rich in positively charged amino acids that form a compled with DNA.
• Increase in Ca2+ after fertilization triggers the sperm genome to undergo decompaction
− Promatines are removed
− DNA is re-wrapped around nucleosomes
• Although now structurally equivalent, the two pronuclei retain some parent-specific histone modifications
• For the zygote to acquire totipotency, the parental genomes must undero extensive epigenetic reprogramming → involves global DNA demethylation (required for EGA)
• However, the entire emryo genome is not de-methylated:
− A small proportion of genes are imprinted (expressed from only one of the parental alleles)
− Imprinted genes have different methylation marks, and these must be maintained
How is chromosome segregation altered as a consequence of fertilisation?
- Transition from meiosis to mitosis involves a dramatic change in the composition of the chromosomal cohesion complex.
- This complex forms a ring that entraps sister chromatids following DNA replication
- Meotic cohesion complexes have the REC8 subunit – essential for reductional chromosome segregation
- Mitotic cohesion complexes contain the SCC1 subunit
How do you switch from the meiotic to the mitotic spindle as a consequence of fertilisation?
Acentrosomal spindle assembly
• Centrosomes are essential for spindle assembly
• Female meiotic cells do not contain canonical centriole-containing centrosomes
• In mice, there are numerios acentriolar MTOCs with similar properties to centrosomes
Transition to centrosomal spindle assembly
• In rodents, the sperm centrioles degenerate during spermatogenesis, and the sperm brings no centrosome material into the egg
− The number of aMTOCs gradually decreases, and some cells begin to show centrin-positive staining consistent with the first appearance of centrioles
• In humans, sperm centrioles are not completely lost, and sperm delivers one intact and one partically degenerate centriole to the egg at fertilization.
− Therefore a switch from meotic to mitotic spindle occurs immediate after fertilization.
How do you switch from asymmetric to symmetric division as a consequence of fertilisation?
Asymmetric spindle positioning
• Meiotic divisions of the oocyte are the most asymmetric cell divisions known in mammals
• Essential to form a large egg containing sufficient storage material for embryonic development
• To divide asymmetrically, mammalian oocytes have to move the spindle from the centre to the cortex using an actin-dependent mechanism
Symmetric spindle positioning
• First mitotic division has to be symmetric to equally distribute storage material between cells
• Key step in centering the spindle is movement of the male and female pronuclei towards the centre
• The male pronucleus is associated with the centrosome → the centrosome nucleates a large microtubule aster, the sperm aster
• The female pronucleus associated with the sperm aster
• Dynein motors pull the aster towards the centre.
What are the metabolic changes occuring during cleavage?
• From the pronucleate eggs to around the 16 cell stage, pyruvate is the main substrate for metabolism
• Later on, as you get compaction and blastocyst formation, glucose is the main substrate and you get an increase in metabolic activity.
• Because in early cells stages, there are lower energy requirements
• However, the blastocyst is made of two cell types
− ICM has low energy requirement and still relies on pyruvate
− Trophectoderm is the more metabolically active
• Implications for IVF – need to use sequential media
How do we get activation of transcription of the embryonic genome?
- Transcription comes to a halt before the end of the growth phase of the oocyte (before maturation) and doesn’t resume until the 4 cell stage
- Therefore, oocyte maturation, fertilization and transition from egg to embryo occur in the absence of transcriptional regulation.
- Post-transcriptional control of gene expression in occytes ensures the storage and timely activaton of maternal factors necessary for egg to embryo transition
- After fertilization, there is a switch from maternal to embryonic control of gene expression
Translational control
• First cell cycles regulated at a post-transcriptional level by maternal mRNA
• During oocyte growth, genome actively transcribed
• Many transcripts are halted and not translated until after fertilization
• These transcripts may be stored in subcortical aggregates functionally related to P bodies
• These dormant mRNAs can be activated for translation by phosphorylation of CPEB (translational repressing complex)
EGA is initiated at the 4-cell stage in humans
• Before EGA, the embryo carries abundant transcripts for proteins that:
− control maternal mRNA translation
− are involve din marking maternal proteins for destruction
Eliminating the maternal stockpile
• Maternal proteins are degraded progressively from the early 4-cell independent of EGA
• Transition from maternal to embryonic control of the genome requires that the amount of maternal transcripts be greatly reduced
• Degredation of excess maternal mRNAs begins at the onset oif oocyte maturation, and continues beyond fertilization, with mRNA levels reaching a minimum at the 2 cell stage in mice
• Transcripts important for oocyte growth and prophase arrest are rapidly degraded, whereas transcripts important for maintaining metaphase II arrest remain intact
• Similarly, transcripts involved in meiotic processes, but not embryonic development, are rapidly degraded upon fertilization
− Degraded by RNA binding protein MYS2
Reactivating the genome
• Beings in the zygote – exhibits low transcriptional activity primarily from the paternal pronucleus
• Followed by a major wave of transcription in the 4 cell embryo
• Transcription is associated with an open chromatin state – chromatin remodeling proposed as key for initiation of EGA
EGA requires:
• Release of chromatin from transcriptionally oppressive environment → demethylation of genome
• Opening up of chromatin
• Protamine → histone exchange
• H3K4dime → indicates transcriptionally active chromatin
• Early transcription of translation initiator proteins, eg) eIF1A
• Synthesis of TF proteins from maternal mRNAs
• Post-translational modification of maternal transcription factors (allowing them to function)
What is the purpose of compaction?
• Purpose is to generate an implantation competent blastocyst containg:
1. Differentiated outer layer of trophectoderm epithelium capable of pumping fluid into the blastocyst cavity and interacting with the uterus for implantation
2. An aggregate of undifferentiated ESCs in the ICM from which the fetus is derived.
• Mouse embryo spends nearly 5 days preparing for implantation
• Early blastomeres can form cells of both ICM and TE:
− Individual 2 or 4 cell blastomeres can give rise to a normal blastocyst and fetus
− Chimeras can be made by combining 2 cleavage stage embryos to form one normal embryo
− Removing one pole of a mouse zygote did not interfere with development
− Microarray analysis shows blastomeres all have similar gene profiles pre-compaction
Describe the general features of polarisation
- 16 cell stage → formation of the morula. Compaction of the 16 cell embryo gives the morula, where individual cells are no longer evident
- At the 16 cell stage, blastomeres have radial symmetry
- Increase in cell-adhesion dependent on Ca2+ and polarization of the outer cells
- Outer cells have apical domain and basolateral domain. Inner surfaces have contact but outer do not → these become trophectoderm
- Inner cells are not polarized. Have contact from all sides → become the ICM.
- Cavitation occurs at the 30 cell stage – morula becomes blastocyst. Presence of the cavity determines the position of the primitive endoderm in the late blastocyst.
- Once the TE and ICM form, they express different TFs:
- Epiblast → Nanog, Sox-2, Oct-4 (The triad TFs of ESCs!)
- Primitive endoderm → Gata4, Gata6, Sox17, Sox7
- Trophoblast → CDX2, Eomes, Gata3
There are two models for how this occurs
• Inside out model → different amounts of cell contact between inside (high) and outside (lower) leads to differences in TF expression
• Polarity model → presence or absence of an outer polar domain leads to differences in TF expression.
How is polarisation set up in blastomeres?
Blastomere flattening
• For compaction, we get an increase in cell-adhesion
• Blastomere flattening is due to formation of adherans junctions between blastomeres
• Only occurs when Ca2+ present in the medium
• Cell adhesion increases via E-cadherin
− E-cadherin KO have defective trophectoderm → cells of the morula compact
Regulation of blastomere flattening
• Components of the adherans junctions localized around the blastomere at the 2 cell stage, but flattening only occurs past the 8 cell stage
− Post-translational modifications maintain the E-cadherin complex inactive
− Cell flattening associated with important phosphorylation events by PKC
− E-cadherin relocated to regions of cell-cell contact at the time of compaction - PKC
− Another candidate for PKC is Ezrin
− Ezrin is a cell membrane protein found in the outer microvillous membrane of the blastocyst
− Relocates to the outer membrane concurrent with polarisaiton
− Rho-family GTPases required for cadherin-mediated cell-cell adhesion in culture cell lines also involved in compaction
− Downregulation of PKC generates the ICM
Blastomere polarization
• Reorganisation of cytoplasmic components is the first sign of blastomere polarization → characterized by accumulation of endosomes under the apical membrane
• Stable microtubules at the base, dynamic microtubules at the apex
• Compaction also associated with the reorganization of the cell surface → removal of microvilli at the basolateral membrane crucial to allow flattening → Ezrin follows the distribution of microvilli.
How is the ICM maintained during implantation development?
Model for ICM-TE Specificaition:
• Hippo pathway not activated in apical cells – trophectoderm forms
• Hippo pathway activated in inner cells – blocks trophectoderm formation
• Inside cells:
− AMOT binds to apical junctions via Nf2, activating the Lats 1/2 kinases
− Active Lats1/2 phosphorylate Yap, resulting in exclusion of Yap from the nucleus.
− Without Yap, TEAD4 cannot induce expression of Cdx2.
− In the absence of Cdx2, Oct-4 is upregulated
• Outside cells:
− Cell polarity sequesters AMOT to the apical protein complex, it cannot bind to the apical junction via Nf2, so does not activate the Lats1/2 kinase
− Yap is not phosphorylated, so Yap is transported into the nucleus where it acts as a co-factor for TEAD4.
− TEAD4 drives the expression of Cdx2
− Cdx2 suppresses the expression of Oct-4
→ Outside cells are thought to additionally be exposed to polarity signals – keeps them polar to maintain apical and basolateral domain!!
• Oct-4 and Cdx-2 are suggested to be selector genes between the ES and TS fate
− Oct-4 is necessary for the maintenance of both the ICM and the ES cells (not possible to derive ES cells from Oct-4 null mutant embryos)
− Not possible to derive TS cells from Cdx2 null embryos, but can derive ES cells
− Forced expression of Cdx2 in ES cells leads to their transformation to TS cells
How is the blastocyst cavity formed?
- Na+/K+ ATPase mediated ion transport driving passive movement of water
- Retention of fluid requires assembly of fully functional tight junctions in a mature TE epithelium
How does the embryo hatch from the zona pellucida?
- Needed in order for implantation to occur
- Stimulated by catechol-estrogen
- Both uterine and embryonic proteases may be involved
- Degradation of the zona occurs through a locally produced hole → the embryo squeezes its way ouf of the hole
How is early development regulated by external factors?
• Low maternal protein diet in the preimplantation period can result in low female birth weight and high blood pressure in adult male rats
External factors influencing early development
- Embryo and uterus produce a large number of GFs eg) IGF, TGF-B, FGF, IL-1
- Embryo expresses GF receptors
• IGF, LIF and FGF functionally important for early development:
− IGF-1 is a survival factor for the human embryo and stimulates protein synthesis
− LIF signaling may help maintain the stem cell population in mouse ICM
− In mice, LIF is necessary for implantation
− HB-EGF stimulated both murine and human TE formation
− FGF-4 maintains the trophoblast stem cell population.
