Acrosome Reaction, Sperm Penetration, Polyspermy

Question 1

What is the first step in fertilization after 100-200 sperm reach the fallopian tubes and “find” the egg?

Answer 1

species-specific binding to oocyte via a receptor linked to ZP3 oligosaccharides which is present on sperm surface and exposed during capacitation. It binds to the C-terminus of the ZP3 which contains glycosylated Ser residues (sperm recognition site) and stimulate acrosome reaction

Question 2

What is the second interaction between the sperm and oocyte?

Answer 2

Acrosome reaction - sperm passes through loose cumulus layers around oocyte and stimulate acrosome reaction

Question 3

Compare mammalian oocyte “coatings” to the jelly coat of sea urchins and frogs:

Answer 3

1. Thick jelly in sea urchins is made of glycoprotein network that stimulates acrosome reaction and fusion between sperm and egg.
2. In mammals the cumulus oophorus is HETEROGENOUS and has mucusy matrix with hyaluronic acid stimulated by gonadotropes, which appears to also stimulate acrosome reactions in sperm.

Question 4

Label a four-step sperm pathway in mammals vs. sea urchins

Answer 4

Mammals:
1. Female tract capacitate, activate, and attract sperm into gaps in cumulus cells
2. Zona pelucida (thicker than in sea urchins) causes acrosome rxn to occur
3. Sperm digests a hole in ZP
4. Perivitaline space is invaded by sperm, which adheres to oocyte and membrane fuses with its membrane
Sea Urchins:
1. Activated sperm = chemotaxically attracted to cell
2. Jelly stimulates acrosome rxn, which releases proteolases
3. Sperm adheres to vitaline layer and lyses hole in it
4. Sperm adheres to oocyte plasma membrane and fuses with it, ready to inject pronucleus

Question 5

What are six important things to remember about the acrosome rxn in mammals?

Answer 5

1. ZP3 carbohydrates are cross-linked to sperm with GalTase (lacking this gene = no acrosome rxn in mice)
2. ZP3 and GalTase binding causes sperm G protein activation (opening Ca2+ influx) which cause exocytosis of acrosomal vesicle.
3. Sperm has to stay motile at this phase to push forward during its digestion of ZP
4. sperm usually enters at oblique angle
5. After binding, protons are released (increased pH)
6. Ca2+ is SUPER important and CAUSES the acrosomal rxn to take place!

Question 6

What three parts of the oocyte-cumulus-complex cause the sperm, which is fully in-tact upon arrival, to undergo the acrosomal reaction?

Answer 6

1. Cumulus oophorus
2. Corona radiata
3. ZP

Question 7

How long does sperm penetration through the ZP take?

Answer 7

5-20min

Question 8

After ZP3 binding, what secondary binding occurs?

Answer 8

ZP2 (binds sperm more tightly because outer acrosome is lost and narrow hole through ZP is made for sperm head to penetrate)

Question 9

The acrosome can be compared to what organelle in a regular cell and why?

Answer 9

A lysosome because it contains lysing (degrading) enzymes like acrosin (maybe sperm-ZP binding) and hyaluronidase (dissolves hyaluronic acid matrix linking cumulus cells) - doesn’t explain how frogs, without hyaluronidase, can penetrate hamster cumulus oophoros

Question 10

Describe the sperm nucleus.

Answer 10

1. Condensed DNA with disulphide cross-links
2. Protamine proteins hold nucleus together with S=S as well
3. Has equatorial segment which binds to the ZP and plasma membrane of oocyte
4. Studies show that after equatorial segment is lost hours after acrosomal rxn finishes the spermatozoa no longer has the ability to bind to the ZP

Question 11

The sperm plasma membrane covers what three structures inside?

Answer 11

1. Outer acrosomal membrane
2. Inner acrosomal membrane
3. Nucleus (with centrioles)

Question 12

What is the difference between the terms “vesiculation” and “fenestration” and which term more accurately depicts the processes occurring during the acrosomal reaction?

Answer 12

1. Vesiculation describes the appearance of membrane vesicles between the fusion points where the outer acrosomal membrane and the overlying sperm membrane (it makes “holes” where the acrosome’s contents can escape from), but this is an optical illusion from 2-D electron microscopes
2. Fenestration means “windows” and better depicts the fusion points from which the acrosomal contents can escape as the spermatozoa pushes through the ZP (the penetrated acrosomal remnants are left behind or disentegrate and laves the sperm’s nucleus only protected by its nuclear membrane, which is now in the front)

Question 13

What process signals development of embryo to begin?

Answer 13

sperm-egg fusion

Question 14

What evidence suggests that sperm-egg fusion is similar to viral invasion?

Answer 14

1. PH-30/fertilin is a sperm surface protein that shares biochemical characteristics with viral fusion proteins
2. PH-30/fertilin has an alpha and beta subunit and the alpha one has a fusion peptide like viral fusion proteins; its beta one has a soluble intern ligand domain that’s also present in snake venom
3. Therefore, both alpha and beta PH-30/fertilin subunits are postulated to have similar fusion and binding functions based on membrane topology (fancy word; don’t like it!)

Question 15

What evidence suggests that PH-30/fertilin is important for binding rather than fusion of oocyte?

Answer 15

1. It’s a transmembran protein found in the posterior plasma membrane of sperm head and this area is involved in binding to the egg plasma membrane.
2. A monoclonal antibody binding to fertilin was found to inhibit binding and fusion of sperm and egg.
3. It sticks to the alpha6beta1 integrin protein on the egg’s plasma membrane; studies show that oocyte interns are necessary for sperm-oocyte interaction (and they think this is the receptor for PH-30/fertilin)
4. ADAM protein family found to have finding qualities.

Question 16

What is CD-9?

Answer 16

A protein somehow related to oocyte interns which is distributed ALL over the oocyte surface EXCEPT for meiotic spindle region (sperm can’t bind here and there is no surface microvilli here) and believed to be involved in sperm fusion with oocytes.

Question 17

Studies show that epididymal protein DE on sperm interact with what on the oocyte during fusion?

Answer 17

1. CD9

2. glycosyphosphatidylinositol (GPI)-anchored proteins

Question 18

What is Izumo and why is it important?

Answer 18

A gene that is responsible for sperm fusion-related antigens in mice and it is important because null mice are sterile despite producing normal-looking sperm (also using an anti-izumi antibody made the sperm fail at fusion with zona-free hamster eggs).

Question 19

What parts of a sea urchin’s sperm enter the egg?

Answer 19

1. Sperm nucleus
2. Mitochondria (will be removed later on)
3. Centriole
4. Flagellum

Question 20

What are two fusenogenic proteins in sea urchin sperm-egg binding?

Answer 20

1. Bindin

2. Fertilin

Question 21

In animals that have centrioles in their sperm, what is its function?

Answer 21

1. Enters egg with sperm nucleus and tail and a centrosome forms around it.
2. In humans it replicates and organizes assembly of 1st mitotic spindle in zygote
3. Multipolar or extra mitotic spindles form in cases of polyspermy because more than one sperm has contributed a centriole to the eg.

Question 22

What are two defenses against polyspermy?

Answer 22

1. Physical barrier between extra sperm and egg

2. Limiting sperm-egg fusion

Question 23

What are two levels of blocks to polyspermy?

Answer 23

1. Fast electrical block

2. Cortical block (slow)

Question 24

Describe the mechanism of the fast electrical block to polyspermy.

Answer 24

An ionic change occurs during the fusion of sperm to egg, which reverses in 0.1s and prevents any more sperm from fusing (not in mammals–only in sea urchins and frogs).

Question 25

Describe the mechanism of the slow cortical block to polyspermy.

Answer 25

Cortical rxn starts with Ca2+ release within egg and causes egg membrane to depolarize. Then cortical granules discharge and their contents swell which makes a fertilization membrane (in mammals these enzymes released from the cortical granules actually block sperm receptors in the ZP and is the only block in humans)

Question 26

How efficient is the polyspermy block in humans and is the incidence of polyspermy likely to be the same in vivo or in vitro?

Answer 26

This is probably a test question b/c she put it on the slide with no answer.

Question 27

Sea urchin fast-block potential uses what ions and involves what voltage?

Answer 27

1. Resting potential is -70mV
2. Sperm contact makes voltage +20mV
3. Caused by influx of Na+ ions–nothing else can fuse to oocyte

Question 28

Fast-block to polyspermy doesn’t work when?

Answer 28

1. Too little sodium in water (not enough available for influx, resulting in polyspermy)
2. Forcing membrane potential to remain at -70mV
3. Forcing membrane potential to remain at +20mV

(somehow the change in membrane potential must cause conformational change in a fusogenic protein on egg surface)

Question 29

Is the slow block to polyspermy physical or chemical?

Answer 29

Both

Question 30

In sea urchins, what mediates the slow block to polyspermy?

Answer 30

cortical reaction (cortical granules found beneath plasma membrane of underutilized eggs and fuse with plasma membrane when there is an increase of free calcium, which releases their contents into perivitelline space)

Question 31

What four things are in cortical granules?

Answer 31

1. Proteases (digest connections between vitelline membrane and plasma membrane, cut bindin receptors)
2. Mucopolysaccharides (cause accumulation of water in perivitelline space)
3. Peroxidase enzyme (crosslinking tyrosine proteins on fertilization envelop to harden it)
4. Hyalin proteins (coasts the plasma membrane which forms an extracellular layer called the hyaline layer which supports the embryonic cells during cleavage)

Question 32

Do mammals have both slow and fast block mechanisms to polyspermy?

Answer 32

No, only slow (cortical).

Question 33

Do mammals have a fertilization membrane?

Answer 33

No, but the rest of the cortical rxn is the same (close enough).

Question 34

Describe cortical granules in mammals.

Answer 34

CONTENTS of cortical granules diffuse quickly across thin perivitelline space and react with ZP

1. Cortical granules are lysosome-like and have trypsin-like enzymes
2. Enzymes (N-acetylglucosamineadases) modify ZP3 structure (cleaving terminal sugars) so spermatozoa can’t bind/enter through the zone
3. ZP2 cleavage by proteases may also render it inactive

Question 35

When are mammalian cortical granules released?

Answer 35

After calcium release travels around egg cortex.

Question 36

Why is the mouse a bad model for human zona-drilling?

Answer 36

Because it has fast egg plasma blocks followed by slow ZP block of polyspermy and accessory spermatozoa winds up swimming in the perivitelline space, unable to fuse with egg plasma membrane.

Question 37

Where does the Ca2+ used in the cortical reaction come from?

Answer 37

It was stored in the endoplasmic reticulum.

Question 38

What three experiments were performed on calcium to test its involvement in the cortical reaction?

Answer 38

1. Raise intracellular levels (no effect on rxn)
2. Lower sea water levels (no effect on rxn)
3. Lower intracellular levels (blocks cortical rxn)

Question 39

Describe the calcium wave necessary for fertilization.

Answer 39

1. Starts at point of sperm entry
2. Creates “spikes” or oscillations (with special amplitude and frequency) that, if altered or missing, prevent normal fertilization.
3. Rise in Ca2+ is necessary and enough for cortical granule release.