Fertilisation

Question 1

LO

Answer 1

• 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

Question 2

How do cells become haploid and what is meant by this?

Tell me about the major differences between males and females

Answer 2

• 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

Question 3

Tell me the steps to meiosis

Answer 3

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

Question 4

Tell me the steps to mitosis

Answer 4

Question 5

Tell me the stages of the cell cycle

Answer 5

• 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)

Question 6

Tell me about the process of spermatogenesis

Answer 6

• 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

Question 7

Can freshly ejaculated mammalian sperm fertilise eggs?

Answer 7

Freshly ejaculated mammalian sperm cannot fertilize eggs. Need time in female reproductive tract.

Question 8

Whats sperm Capacitation?

Answer 8

• 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

Question 9

Label this sperm

Answer 9

Question 10

What material is IVF carried out on?

Answer 10

IVF – on glass / plastic surfaces in culture raised Ca levels also trigger

Question 11

What does an Acrosome contain?

Answer 11

Acrosome- contains hydrolases, glycodases and proteases

Question 12

Whats an acrosome?

Answer 12

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).

Question 13

Tell me about PGC in female meiosis?

Answer 13

~1000 PGC –> 7million oogonia in gestation but most die, those that survive enter meiosis

Question 14

tell me about female meiosis gestation

Answer 14

~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

Question 15

Female meiosis

Answer 15

Question 16

Whats oogonia?

Answer 16

an immature female reproductive cell that gives rise to primary oocytes by mitosis.

Question 17

About 1,000,000 PGC form what at birth?

Answer 17

oogonia

Question 18

At birth all PGCs have developed into what?

Answer 18

At birth all PGCs have developed to oogonia (or died) and are at the 4N stage – compare with the male

Question 19

Tell me about oogonia meiotic arrest

Answer 19

• 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

Question 20

Are female meiotic divisions symmetric or asymmetric?

Answer 20

Female meiotic divisions are asymmetric

• Two meiotic divisions (unequal sizes) to become haploid. PB1 (first polar body); PB2 (second polar body).

Question 21

Meiosis is evolutionarily conserved, but the timing of fertilisation is not

Tell me about this…

Answer 21

• 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???

Question 22

Tell me about sea urchin eggs

Answer 22

• 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

Question 23

When do sea urchin eggs arrest?

Answer 23

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)

Question 24

What are sea urchin eggs arrested by?

Answer 24

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)

Question 25

What does sea urchin fertilisation trigger?

Answer 25

Fertilization trigger is a calcium wave passing across the egg.

Question 26

Tell me about fast and slow polyspermy block in sea urchin fertilisation

Answer 26

* **Fast polyspermy block**. Fast electrical block (membrane potential): milliseconds * **Slower polyspermy block**. Release of cortical granules and generation of a fertilization envelope: minutes

Question 27

More aspects of sea urchin fertilisation

Answer 27

* 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

Question 28

Calcium is conserved as a fertilisation trigger across the animal kingdom

Answer 28

* 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

Question 29

What do Calcium waves break? What does this alter?

Answer 29

* 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

Question 30

What is the calcium increase caused by? What does calcium do at fertilisation? What does it trigger?

Answer 30

* 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

Question 31

Whats **phospholipase C zeta**?

Answer 31

* Phospholipase C zeta sperm specific protein. * Member of the phospholipase C family (next slide)

Question 32

Give evidence that PLCzeta is responsible for fertilisation

Answer 32

1. Sperm cytosolic extracts (but not those of other cells) trigger events of fertilization when injected into eggs. 2. PLCzeta mRNA/protein can also do this (but not other PLCs). 3. Infertility in some men associated with PLCzeta mutations. 4. Does PLCzeta act alone????? PLC over the acrosome PLC is necessary but??? Sufficient

Question 33

How does PLCzeta release calcium?

Answer 33

1. PIP2 is hydrolyzed by PLC zeta. 2. Generates IP3. and DAG 3. IP3 diffuses into cytoplasm. 4. Binds its receptor on endoplasmic reticulum. 5. Releases calcium ions into cytoplasm.

Question 34

Generation of IP₃ from PIP₂ catalysed by PLC

Answer 34

Question 35

Block from polyspermy

Answer 35

Question 36

How are cortical granules released?

Answer 36

1. Cortical granules (CGs) underneath oocyte plasma membrane released at fertilization. 2. Calcium dependent fusion with plasma membrane 3. CGs block entry of other sperm.

Question 37

Loss of CGs in fertilised egg

Answer 37

Question 38

Tell me about the model to explain polyspermy block

Answer 38

* 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

Question 39

Where is there also a polyspermy block?

Answer 39

There is also a polyspermy block at plasma membrane EGG-SPERM receptor system

Question 40

Tell me about the sperm protein **Izymo1**?

Answer 40

* 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

Question 41

**Checklist**

Answer 41

* **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**