Fertilization & Early Embryo Development Flashcards
How long does it take for sperm to reach the fallopian tube?
30 minutes to 6 days
How long does it take for the fertilized embryo to reach the uterine lumen?
3-4 days
Where does fertilization occur?
The ampulla - isthmus junction of the fallopian tube (where it narrows)
How many sperm reach the site of fertilization?
Only 3-500 sperm reach the site of fertilization (out of 2-300 million)
What are the stages of fertilization?
- Capacitation > penetration of the corona radiata (expanded cumulus)
- Acrosome reaction > penetration of the zona pellucida
- Fusion of oocyte (oolemma) and sperm cell membranes > zona / cortical reaction
Where does capacitation occur?
In the fallopian tube
What does the PH-20 enzyme do?
Digest hyaluronic acid in the corona radiata during capacitation, allowing sperm penetration
What is capacitation and what processes occur during it? How long does it take?
The process sperm must undergo to become capable of fertilization (and the initial step of corona radiata penetration)
Takes 7 hours of capacitation > then sperm may penetrate corona radiata.
What occurs during capacitation:
- Removal of glycoprotein coat and seminal plasma proteins from the sperm membrane overlying the acrosome
- Altered metabolism
- Changes in protein phosphorylation
- Elevation in intracellular pH
- Elevation in intracellular Ca2+
- Hyperpolarization of membrane potential > hyperactivation of sperm: hypermotility of flagella (wave to whip-like motion) to allow penetration
- Digestion of hyaluronic acid in cumulus cells of corona radiata via PH-20 enzyme
Is the corona radiata high or low in hyaluronic acid?
High
What is ZP3?
A glycoprotein on the surface of the zona pellucida that attaches to sperm head receptors > induces acrosome reaction
Describe the acrosome reaction
- Sperm head ZP3 receptors bind to ZP3 glycoprotein on the zona pellucida
- Sustained influx of Ca2+ > stimulates release of lysosomal enzymes from sperm head (primary enzyme: acrosin from the acrosome) which remove plasma membrane from sperm head
- > sperm can penetrate the zona pellucida (stage 2 of fertilization)
What does acrosin do?
Released from the acrosome of the sperm head during the acrosome reaction phase of fertilization, acrosin is the primary lysosomal enzyme that removes the plasma membrane from the head of the sperm, enabling the sperm to penetrate the zona pellucida
Describe oocyte-sperm membrane fusion
Oocyte integrins :: sperm disintegrins interact
Sperm head and tail enter (after acrosome reaction, the sperm plasma membrane on the sperm head has been removed)
Plasma membrane fusion actually occurs at the posterior sperm head
The flagella and mitochondria in the sperm tail remain outside the oocyte and disintegrate (this is why only maternal mitochondrial DNA is passed down to offspring)
Describe the zona reaction
When does it occur?
What is its function?
AKA cortical reaction
Once oocyte/sperm plasma membranes fuse
Zona reaction occurs to prevent polyspermy
Metabolic activation of the egg: sperm PLCz (zeta) > IP3 > open Ca2+ channels
Cortical granules are released from the oocyte cortex just within the oocyte plasma membrane via exocytosis. Enzymes within cortical granules:
- depolarize membrane
- inactivate species-specific sperm receptors on the ZP
- harden ZP
When does the resumption of meiosis II occur in the oocyte?
Describe the process of oocyte activation and meiosis II resumption
After oocyte-sperm plasma membrane fusion during fertilization
Metabolic activation of the egg: sperm PLCz (zeta) > IP3 > open Ca2+ channels > influx of Ca2+ > releases cortical granules via exocytosis (zona/cortical reaction). Enzymes within cortical granules:
- depolarize oocyte membrane
- harden ZP
- digest ZP3 receptors for sperm
Resume meiosis II from metaphase II > extrusion of 2nd polar body
Establish female pronucleus
What happens to the sperm after oocyte-sperm fusion?
Cytosolic oocyte environment uncoils sperm DNA (de-condensation)
New membrane forms around uncoiled sperm DNA (male pronucleus)
Describe syngamy
Sperm centriole organizes microtubules to direct pronucleus migration and rotation within cytoplasm to achieve proper orientation to oocyte 2nd polar body
Pronuclei (male and female) each undergo haploid DNA replication
Mitotic potential is inherited paternally (sperm centrosome)
Once 2 PN are close to each other, nuclear membranes break down > chromosomes align on common metaphase plate (restoration of diploid, sex determination) > 1st embryonic cleavage to 2-cell zygote / mitosis (24 hours post fertilization)
When does the embryonic genome become activated?
What else occurs in preparation for genomic activation of the embryo?
Between D2-D3 (between 4 to 8 cell stage)
Nucleoli also form at this time (sites of synthesis of pre-rRNA which are essential to translation after activation of the embryonic genome)
How long are male and female pronuclei apparent?
Appear 6-8 hours after sperm penetration of the egg
Persist 10-12 hours until 1st embryo cleavage at 24 hours post fertilization
When does mitosis of the embryo occur?
24 hours after fertilization (1st cleavage) = 2-cell zygote
When does the 2nd cleavage of the embryo occur?
Where?
2 cell > 4 cell (equally sized cells known as blastomeres) occurs at 40 hours post-fertilization (by D2) (of note, size of embryo remains the same)
Still in the fallopian tube
When does 4 > 8-cell cleavage occur?
~ 72 hours post-fertilization (by D3)
When are embryo cells totipotent?
Until 8-cell stage
When does embryo compaction begin?
8-cell stage > 16-cell morula (“mulberry-like”)
Cell-to-cell tight junctions between blastomeres form
What processes occur between 8 > 16 cell stage?
When does this occur?
96 hours s/p fertilization (D4)
Embryo compaction to morula
Cell-to-cell tight junctions between blastomeres form
Cell differentiation into ICM vs trophectoderm
What occurs during early blastocyst formation?
When does this occur?
D4-6
D4-5: Morula > early blastocyst: formation of fluid between blastomeres (blastocyst) using Na+/K+/ATP-ase channels which INCREASES salt concentration > increasing fluid
D5-6: blastocyst: cell replication and blastocyst formation expand the embryo and the trophectoderm flattens in preparation for hatching > implantation
When does implantation occur?
D6 after conception
When is IVF or ICSI completed after egg retrieval?
IVF/ICSI completed 4-7 hours after egg retrieval
When can fertilization be observed after IVF? After ICSI?
After IVF: 16-20 hours post-IVF
*Will occur generally 1 hour longer than after ICSI fertilization
So generally, fertilization assessment is performed 18 +/- 1 hour after IVF/ICSI
What must be performed after fertilization to observe pro-nuclei? (hint: does not have to be done after ICISI)
Cumulus stripping (via micropipetting or needle dissection)
*Already done during ICSI
What is seen microscopically when normal fertilization occurs?
- 2 pronuclei (2PN)
- 2 polar bodies
- Regular round shape
- Intact zona pellucida
- Healthy appearing cytoplasm
- Clear halo (5-10 um thick) in peripheral cytoplasm is an indication of oocyte activation and re-initiation of meiosis
Polyspermy
More than one spermatozoon penetrates the zona pellucida > multiple pronuclear formation when DNA decondensation occurs
What are various ways 3PN can occur?
- Polyspermy
- Diploid sperm/ovum
- If only 1 PB: failure to exude 2nd polar body (usually the cause of 3PN after ICSI, as polyspermy is excluded with single sperm injection)
What is an example of normal fertilization that does not appear with 2PN + 2PBs?
- 1 PN + 2 PBs
- Seen with IVF as exact fertilization timing is unclear
- Possibly normal, s/p syngamy
- Preferentially not transferred if other 2 PN embryos are available, but can be cultured/watched/vitrified
Is 1 PN / 1 PB normal fertilization?
No
Not typically considered for transfer
Which embryos with abnormal fertilization can continue to be watched in culture?
0 PN
1 PN + 2 PB
What is zygote scoring?
What is considered the best score? What characterizes this score?
Zygote scoring evaluates the pronuclei
Scores patterns 0-5
Patterns 0-1 are best
An even number of nucleolar polar bodies (NPB) should be observed (split evenly between male/female)
What should be done in the lab in event of failed fertilization?
- Evaluate sperm motility in dish
- Are sperm bound to ZP?
- Compare to other eggs for the patient; any other fertilized?
- Are there any abnormalities in the egg?
- Cortical granule clearing?
For ICSI fertilized oocytes:
1. Normal PB number/atretic?
2. Cytoplasmic fragmentation?
3. Oocyte damage?
4. Presence of sperm? Inside oocyte?
