Fertilisation Flashcards
LO
- Understand the concept of two divisions during meiosis
- Explain how PLC zeta and Ca2+ cause egg activation.
- Explain how polyspermy is prevented.
- Understand what is meant by capacitation
How do cells become haploid and what is meant by this?
Tell me about the major differences between males and females
- Meiosis - the most revolutionary invention of the eukaryote- the halving of genetic material to allow sexual reproduction. Meiosis is an initial duplication of the whole genome followed by 2 cell divisions with no intervening DNA replication
- Commitment to differentiation - Primary spermatocytes/ oogonia enter a pre-meiotic S-phase (all chromosomes replicated, = 4n) MALES after puberty, FEMALES in utero(arrest)
- Primary spermatocytes/ oocyte (4n) undergoes a first meiotic division to form…
- Secondary spermatocytes/ oocyte (2n) undergoes a second meiotic division to form, FEMALES ovulate (arrest)
- Spermatid or ovum (=n, haploid- in female this completes at fertilization)
- Major differences in male and female - female needs to produce an egg with sufficient nutrients and organelles for later development – hence 4 sperm: 1 egg
Tell me the steps to meiosis
1. Prophase I
- prior to prophase, chromosomes replicate to form sister chromatids
- NE disintegrates and the chromosomes condense
- spindle fibres appear
- homologous chromosomes exchange parts of themselves such that one chromosome contains both maternal and paternal DNA (crossing-over)
2. Prometaphase I
- spindle fibres attach to the centromeres
- chromosomes continue to condense
3. Metaphase I
- chromosomes align along the equator of the cell
- independent assortment occurs
4. Anaphase I
- homologous chromosomes pulles in opposite directions towards poles as the spindle fibres retract to divide the DNA between the two cells which will be formed
5. Telophase/ cytokinesis I
- Telophase: NE reforms, spindle fibres disappear
- cytokinesis: two, haploid cells
6. Prophase II
- same as prophase I
7. Prometaphase II
- same as prometaphase I
8. Metaphase II
- chromosome line up in single file along the equator of the cell
9. Anaphase II
- sister chromatids pulled to opposite poles of the equator
10. Telophase/ cytokinesis II
- Telophase: same as telophase I
- cytokinesis: 2 non-identical haploid daughter cells
net= 4 non-identical haploid daighter cells
Tell me the steps to mitosis
Tell me the stages of the cell cycle
- Meiosis – allows recombinatory rearrangement at prophase 1
- S phase – DNA replicated
- Males – symmetry, Females – asymmetrical– (fish/ chicken egg has to supply all nutrients till free living, mammal till implantation)
Tell me about the process of spermatogenesis
- A1-4 as they divide here
- Recombination occurs as part of the first meiotic division – first meiotic division after S phase duplication of genome
- Increased testosterone production (intertiail cells) at puberty- via FSH leads to retinoic acid formation by sertoli cells
- Spermatids mature physically – nucleus condenses, mitochondria coalesce in mid piece and tail forms – Spermiogenesis- but not functional
Can freshly ejaculated mammalian sperm fertilise eggs?
Freshly ejaculated mammalian sperm cannot fertilize eggs. Need time in female reproductive tract.
Whats sperm Capacitation?
- Freshly ejaculated mammalian sperm cannot fertilize eggs. Need time in female reproductive tract.
- Capacitation name given to this process. Can be triggered in vitro by raising sperm calcium levels.
- Hyperactivation capacitated sperm swim faster. May help sperm swim to egg.
- Capacitation – also destabilises the acrosomal membrane breaks down the Zona Pellucida
Label this sperm
What material is IVF carried out on?
IVF – on glass / plastic surfaces in culture raised Ca levels also trigger
What does an Acrosome contain?
Acrosome- contains hydrolases, glycodases and proteases
Whats an acrosome?
The acrosome is an organelle that develops over the anterior half of the head in the spermatozoa (sperm cells) of many animals including humans. It is a cap-like structure derived from the Golgi apparatus. In Eutherian mammals the acrosome contains degradative enzymes (including hyaluronidase and acrosin).
Tell me about PGC in female meiosis?
~1000 PGC –> 7million oogonia in gestation but most die, those that survive enter meiosis
tell me about female meiosis gestation
~3-7 months gestation- remain paused at 4n (diplotene) stage as primary oocytes –for up to 50 years
- at onset of puberty waves of primary oocytes resume meiosis due to the action of LH
Female meiosis
Whats oogonia?
an immature female reproductive cell that gives rise to primary oocytes by mitosis.
About 1,000,000 PGC form what at birth?
oogonia
At birth all PGCs have developed into what?
At birth all PGCs have developed to oogonia (or died) and are at the 4N stage – compare with the male
Tell me about oogonia meiotic arrest
- Arrest at 3 month stage for 12-50 yrs!
- Activate due to variable numbers of granulasa cells – reduce cAMP (have gap junctions to oogonia) if falls enough LH receptors form and oogonia leave arrest – usually 4-5/cycle – competition for FSH most mature follice produces estrogen in induces down regulation of FSH
- At ovulation –Arrests again in the second meiotic division at ovulation until fertilized- sperm breaks that arrest
Are female meiotic divisions symmetric or asymmetric?
Female meiotic divisions are asymmetric
- Two meiotic divisions (unequal sizes) to become haploid. PB1 (first polar body); PB2 (second polar body).
Meiosis is evolutionarily conserved, but the timing of fertilisation is not
Tell me about this…
- I and II are positions of meiotic arrest –All species show S phase arrest in the first meiosis but there is great variation after this and Fertilisation* can occur are different points during meiosis
- Clam Starfish – has no second arrest
- Note Clam – 5 copies of genome!! Before mitosis restarts
- Mouse -2 (?3)
- In all cases except star fish – breaking of arrest caused by fertilisation – how???
Tell me about sea urchin eggs
- Large eggs 150-200um diameter
- Quantity
- Simple to use (keep in seawater)
- Very simple system – inj KCl – releases millions of eggs into sea water – very much used experimental system
- Sold as fish roe!!!- sushi
When do sea urchin eggs arrest?
Eggs arrest having completed meiosis. Eggs are shed and fertilized at interphase (G1) of the first mitotic cell cycle (mammals arrest but in second meiotic cycle)
What are sea urchin eggs arrested by?
Arrest by cytoplasmic acidification. Unfertilized egg cytoplasm is acidic. Fertilization increases egg pH 0.3 units (1-4 min). Sperm activates a Na+/H+ exchanger in the plasma membrane (mammalian arrest does not cause by this mechanism)
What does sea urchin fertilisation trigger?
Fertilization trigger is a calcium wave passing across the egg.
Tell me about fast and slow polyspermy block in sea urchin fertilisation
- Fast polyspermy block. Fast electrical block (membrane potential): milliseconds
- Slower polyspermy block. Release of cortical granules and generation of a fertilization envelope: minutes
More aspects of sea urchin fertilisation
- Fertilisation trigger, Ca ion waves – fully conserved – even in plants
- Block is important to prevent trisomy which is lethal – needed in sea urchins as spawning marine organisms have huge numbers of sperm –
- In mammals (all internal fertilisation) very few sperm reach the egg 5-10 so not so important to have a fast block??? IVF
Calcium is conserved as a fertilisation trigger across the animal kingdom
- All show Ca signal can be single and simple – seconds in sea urchin
- Multiple and hours in mammals++ 4 hrs in mammal- drives cells the leave arrest and enter mitosis
What do Calcium waves break?
What does this alter?
- Ca- Waves – breaks arrest – and alters fertilisation envelope lowers colloidal OP – pulls the fertilisation envelope away from the plasma membrane so penetrating sperm head don’t contact the ova membrane
- Also alters plasma membrane to prevent fusion
What is the calcium increase caused by?
What does calcium do at fertilisation?
What does it trigger?
- The Ca increase is caused by delivery of a sperm protein (PLC zeta) at fertilization
- Necessary and sufficient trigger for egg activation (wakes it up, arrested in meiosis, allows haploid embryo to go further into mitosis and trigger development – without trigger dies in ~48hrs)
- Activates calmodulin dependent protein kinase II (CaMII kinase)
- Triggers degradation of proteins keeping egg arrested.
- Triggers release of cortical granules to block entry of other sperm
- Ubiquitous enzyme – Ca depended, Ca binds Calmodulin which then binds and actives the protein kinase
- ME2(inhibitory cyclin proteins) – is phosphorylated leading to its degradation
- cortical granules – release proteases, glycosidases and hydrolases
Whats phospholipase C zeta?
- Phospholipase C zeta sperm specific protein.
- Member of the phospholipase C family (next slide)
Give evidence that PLCzeta is responsible for fertilisation
- Sperm cytosolic extracts (but not those of other cells) trigger events of fertilization when injected into eggs.
- PLCzeta mRNA/protein can also do this (but not other PLCs).
- Infertility in some men associated with PLCzeta mutations.
- Does PLCzeta act alone?????
PLC over the acrosome
PLC is necessary but??? Sufficient
How does PLCzeta release calcium?
- PIP2 is hydrolyzed by PLC zeta.
- Generates IP3. and DAG
- IP3 diffuses into cytoplasm.
- Binds its receptor on endoplasmic reticulum.
- Releases calcium ions into cytoplasm.
Generation of IP3 from PIP2 catalysed by PLC
Block from polyspermy
How are cortical granules released?
- Cortical granules (CGs) underneath oocyte plasma membrane released at fertilization.
- Calcium dependent fusion with plasma membrane
- CGs block entry of other sperm.
Loss of CGs in fertilised egg
Tell me about the model to explain polyspermy block
- Glycosidase and protease activities in the content of the CGs change ZP structure.
- Exact details still unclear!
- ZP1-3, 3 glycoproteins
- Sperm can’t attach – and acrosomal enzymes don’t cleave
Where is there also a polyspermy block?
There is also a polyspermy block at plasma membrane EGG-SPERM receptor system
Tell me about the sperm protein Izymo1?
- Sperm protein Izumo1, tethered to sperm membrane.
- Forms adhesion complex with its receptor protein, Juno.
- Fertilization does not take place in the absence of this complex.
- After fertilization, Juno is lost from the egg’s membrane, due to fusion of cortical granules exiting in extracellular vesicles.
- Juno – protein of love
Checklist
- Can summarize the process of the meiotic divisions leading to a mature sperm and egg.
- Can summarize main events of sea urchin fertilization and can contrast with mammal. Conservation of a calcium signal to break egg arrest and to block polyspermy.
- Can give a molecular explanation of how PLC zeta functions at fertilization.
- Can explain how a calcium signal causes the block to polyspermy at the.
- Can explain role of zona pellucida changes and loss of Juno in plasma membrane block to polyspermy.
- Can define sperm capacitation
