Fertilization Part 1

Question 1

What is fertilization?

Answer 1

Fertilization is the process whereby two sex cells (gametes) fuse together to create a new individual whose genome is derived from both parents.

Question 2

What are the twofunctions fertilization ?

Answer 2

• Thus, the first function of fertilization is to transmit gens from parents to the offspring; and
• The second function is to initiate in the egg cytoplasm those reactions that permit development to proceed.

Question 3

What are the four main parts of fertilization?

Answer 3

1. Contact and recognition between sperm and egg (in most cases, this ensures that the sperm and egg are of the same species)
2. Regulation of sperm entry into egg: prevent polyspermy
   - This is usually accomplished by allowing only one sperm to enter the egg and inhibiting any others from entering
3. Fusion of sperm and egg genetic material
4. Activation of egg metabolism to start developmental process

Question 4

What is the complex dialogue between a sperm and egg?

Answer 4

The egg activates the sperm metabolism that is essential for fertilization, and the sperm reciprocates by activating the egg metabolism needed for the onset of development.

Question 5

Explain the significance of Anton Van Leeuwenhoek’s studies/findings.

Answer 5

Anton van Leeuwenhoek, the Dutch microscopist who co-discovered sperm cells in 1678, first believed them to be small parasitic animals living within the seminal fluid

He originally assumed that they had nothing at all to do with reproducing the organism in which they were found, but he later came to believe that each sperm contained a preformed embryo

Question 6

Explain the significance of Nicolaas Hartsoeker’s studies/findings.

Answer 6

Leeuwenhoek was continually disappointed in his attempts to find the preformed embryo within the sperm cells

Nicolaas Hartsoeker, the other co-discoverer of sperm, drew a picture of what he hoped to find: a preformed human (“homunculus”) within the human sperm (in 1694)

• Like many other biologist in his time, Hartsoeker believed in “spermism”, the theory that information to create a fetus was transmitted by the miniature human form lodged inside sperm, which then grew into a child within the womb

Question 7

Explain the significance of Oscar Hertwig and Herman Fol’s studies/findings.

Answer 7

It was only in 1876 that Oscar Hertwig (German zoologist) and Herman Fol (Swiss zoologist) independently demonstrated sperm entry into the egg and the union of the two cells’ nuclei

• Hertwig had sought an organism suitable for detailed microscopic observations, which turned out to be the Mediterranean purple sea urchin, Paracentrotus lividus
• Not only was it common throughout the region and sexually mature throughout most of the year, but its eggs were available in large numbers and were transparent even at high magnifications
• After mixing sperm and egg suspensions together, Hertwig repeatedly observed a sperm entering an egg and saw the two nuclei unite
• He also noted that only one sperm was seen to enter each egg, and that all the nuclei of the embryo were derived from the fused nucleus created at fertilization
  Fol made similar observations and detailed the mechanism of sperm entry

Significance: Fertilization was at last recognized as the union of sperm and egg, and the union of sea urchin gametes remains one of the best-studied examples of fertilization

Question 8

Explain the methodology and significance of Victor Vacquier and colleagues’ studies/findings (1977).

Answer 8

Victor Vacquier and colleagues isolated bindin protein from the acrosome of the purple sea urchin and red sea urchin and found each to be capable of binding to dejellied eggs of the same species.
This species-specific binding was looked at through agglutination assays.
Bindin isolated from acrosomes of purple sea urchin binds to its own dejellied eggs but not to those of red sea urchin.
Bindin isolated from acrosomes of red sea urchin binds to its own dejellied eggs but not to those of purple sea urchin.

Question 9

Explain the methodology and significance of Gary Moy and Victor Vacquier’s studies/findings (1979).

Answer 9

Gary W. Moy and Vacquier demonstrated that bindin is located specifically on the acrosomal process exactly where it should be for sperm-egg recognition (using immunological techniques).

Rabbit antibody was made to the bindin protein, and this antibody (also known as rabbit anti-bindin) was incubated with sperm that had undergone the acrosomal reaction.

If bindin was present, the rabbit antibody would remain bound to the sperm.

After any unbound antibody was washed off, the sperm were treated with swine (pig) antibody (also known as swine anti-rabbit) that had been covalently linked to peroxidase enzymes.

The swine antibody binds to the rabbit antibody, placing peroxidase enzymes wherever bindin was present.

Peroxidase catalyzes the formation of a dark brown precipitate from
diaminobenzidine (DAB) and hydrogen peroxide (H2O2)
- Thus, this precipitate formed only where bindin was present.

Question 10

What is polyspermy?

Answer 10

Polyspermy is the fertilization of an egg by more than one sperm.

Question 11

What is a proucleus?

Answer 11

Pronucleus is the haploid nucleus of a male or female gamete (such as an egg or sperm) up to the time of fusion with that of another gamete in fertilization.

Question 12

What is spermatozoon?

Answer 12

Spermatozoon is the mature motile sex cell of an animal, by which an ovum is fertilized. It has a head, midpiece and a tail region

Question 13

What is an acrosome?

Answer 13

An acrosome is a secretory vesicle that is located over the anterior part of the pronucleus (derived from the Golgi, it is an organelle containing digestive enzymes that enable the pronucleus to enter the egg).

Question 14

What is an axoneme?

Answer 14

An axoneme is a microtubular-based cytoskeletal structure that consists of nine peripheral microtubule doublets surrounding a central pair of microtubules – 9 + 2 microtubule structure.

Question 15

What are cortical granules?

Answer 15

Cortical granules are present within the cortex.
- Found among all mammalian eggs, many vertebrate eggs, and some invertebrate eggs.
- Golgi-derived structures containing proteolytic enzymes similar to acrosomal vesicles in sperm, and also contain materials involved in sperm entry or prevention of polyspermy.

Question 16

What is chemotaxis?

Answer 16

In many animals, sperm cells are attracted towards eggs of their species by chemotaxis (i.e., following a gradient of a chemical secreted by the egg).

Question 17

What is the acrosomal process?

Answer 17

The second part of the acrosomal reaction in sea urchins involves the extension of the acrosomal process. The acrosomal process is covered by bindin proteins. The acrosomal process contacts the vitelline envelope once the sperm has penetrated the egg jelly layer.

Question 18

What is bindin?

Answer 18

In sea urchins, the acrosomal process is covered by bindin. The bindin proteins attach to receptors on egg. The receptors on eggs of one species only recognize bindin proteins on sperm of the same species.

Question 19

What is capacitation?

Answer 19

Capacitation is an activation process that involves both enhancement of flagellar activity and the biochemical and structural changes in the plasma membrane of the sperm head that prepares sperm to undergo the acrosomal reaction so that it can penetrated and fertilize an egg.

Question 20

List 3 regions of a sperm cell (and explain what is found for each region.

Answer 20

1. Head:
   In the head region, one will find:
   – a haploid nucleus (or pronucleus)
   o The pronucleus is deposited into the egg (ovum) during fertilization and then merges with the pronucleus of the oocyte
   - a secretory vesicle called the acrosome, located over the anterior part of the pronucleus (derived from the Golgi, it is an organelle containing digestive enzymes that enable the pronucleus to enter the egg).
2. Midpiece:
   – many mitochondria to provide power (ATP) for sperm movement
   - centrioles that help form the flagellum as well as link the spermatozoon head and tail [in addition, it forms the major microtubule-organizing center of the zygote after fertilization (i.e., helps shape the zygote cytoskeleton)]
3. Tail:
   Contains axoneme (microtubular-based cytoskeletal structure that consists of nine peripheral microtubule doublets surrounding a central pair of microtubules – 9 + 2 microtubule structure)

Question 21

List the parts that make up the protective envelope of a sea urchin egg and mammalian egg (as well as briefly mention the function of this protective envelope).

Answer 21

Sea urchin:
1. Jelly layer
2. Vitelline envelope

Mammalian egg:
1. Cumulus layer (confirm whether this or corona radiata )
2. Zona pellucida

Function of protective envelope: The extracellular envelope serves to recognize the sperm and prevent fertilization by more than one sperm (polyspermy)

Question 22

Label parts of a sea urchin egg membrane.

Answer 22

1. Jelly layer
2. Vitelline envelope
3. Cell (plasma) membrane
4. Cortical granule
5. Mitochondria
6. Nucleus

Question 23

Label parts of a mammalian egg.

Answer 23

1. Cumulus layer
2. Corona radiata
3. Follicular (granulosa) cell
4. Zona pellucida
5. Cell (plasma) membrane
6. Nucleus
7. Cortical granule

Question 24

Explain the purpose of chemotaxis.

Answer 24

In many animals, sperm cells are attracted towards eggs of their species by chemotaxis (i.e., following a gradient of a chemical secreted by the egg).

Examples of chemoattractants are:
1. Resact is a small peptide expressed in Atlantic purple sea urchin, Arbacia punctulata (not only is it a chemoattractant, but resact is a sperm- activating peptide, in which it activates mitochondrial ATP synthesis – ultimately leads to an increase in motility of sperm cells)
2. Speract is also a small peptide expressed in Pacific purple sea urchin, Stronglyocentrotus purpuratus.

Question 25

List the summary of events leads to fusion of egg and sperm cell membranes of both the sea urchin.

Answer 25

1. First step:
   Sperm cell makes contact with jelly layer(the most outer one)
   Sperm cell completely intact at that point

Step 2: The acrosomal reaction is initiated when the sperm contacts the egg jelly layer. The proteolytic enzymes inside the acrosomal vesicle are released. These enzymes digest the jelly coat around the egg so that the sperm can make its way towards the vitelline envelope.

Steps 2 and 3: The second part of the acrosomal reaction involves the extension of the acrosomal process. Extension is mediated by the polymerization of the cytoskeletal protein actin into filaments that essentially push the process forward. The acrosomal process is covered by bindin proteins.

Steps 4: Once the sperm has penetrated the egg jelly layer, its acrosomal process contacts the vitelline envelope. The bindin proteins attach to receptors on egg. The receptors on eggs of one species only recognize bindin proteins on sperm of the same species.

Steps 4 and 5: The binding of the sperm cell to the egg cell completes the acrosomal reaction and allows the sperm’s cell membrane to fuse with that of the egg cell membrane.

Question 26

List the summary of events leads to fusion of egg and sperm cell membranes of a mouse (mammalian). - confirm the answer

Answer 26

First step:
Sperm capacitation (represented by the “activated” sperm in Step 1)

Second step:
Sperm penetrates cumulus layer instead of jelly layer
Acrosomal reaction is stimulated by contact with zona pellucida
No extension of acrosomal process (and no bindin proteins)

Step 2: The acrosomal reaction is initiated when the sperm contacts the zona pellucida. The proteolytic enzymes inside the acrosomal vesicle are released. These enzymes digest the cumulus layer around the egg.

Steps 2 and 3: No extension of acrosomal process (and no bindin proteins).

Steps 4 and 5: The binding of the sperm cell to the egg cell completes the acrosomal reaction and allows the sperm’s cell membrane to fuse with that of the egg cell membrane.

Question 27

Differences in the summary of events leading to fertilization between sea urchin and mouse.

Answer 27

1. Sperm capacitation (represented by the “activated” sperm in Step 1)
2. Sperm penetrates cumulus layer instead of jelly layer
3. Acrosomal reaction is stimulated by contact with zona pellucida
4. No extension of acrosomal process (and no bindin proteins)

Question 28

Describe the steps involved in acrosomal reaction involving sea urchin sperm cells.

Answer 28

1. The acrosomal reaction is triggered by a fucose sulfate rich polysaccharide in the egg jelly coat
2. This polysaccharide binds to a receptor molecule in the sperm plasma membrane that causes the sperm cell to be depolarized by influx of Na+
3. This induces voltage-gated Ca2+ channels on sperm plasma membrane to open … this causes:
   - Activation of proton (H+)/Na exchange pump (also on sperm plasma membrane) to increase intracellular pH
   - Exocytosis of acrosomal vesicles containing proteolytic enzymes, which causes break down the vitelline envelope so the sperm can reach egg plasma membrane (in addition, exocytosis of these vesicles expose bindin proteins)
4. The increase in intracellular pH (due to efflux of H+ ions) is responsible for:
   - Activation of dynein ATPase and a subsequent increase in sperm motility
   - Extension of acrosomal process

Question 29

Explain the purpose associated with sperm capacitation and what the actual process involves.

Answer 29

It is the first step involved the sperm cell being “activated” in the female reproductive tract – this activation is known as sperm capacitation.
- This process is part of the maturation of the spermatozoon in order to acquire the ability to bind to the egg and fuse with it (so prior to acrosomal reaction)
- Initiated when sperm cell travels through female reproductive tract for a particular period of time)
- Capacitation is an activation process that involves both enhancement of flagellar activity and the biochemical and structural changes in the plasma membrane of the sperm head that prepares sperm to undergo the acrosomal reaction so that it can penetrated and fertilize an egg
- Non-mammalian spermatozoa DO NOT require this capacitation step and are ready to fertilize an oocyte immediately after release from the male

Capacitation has two effects:
- Destabilization of the acrosomal sperm head membrane which allows it to penetrate the outer layer of the egg
- Chemical changes in the flagellar tail that allow a greater mobility in the sperm (otherwise known as hyperactivity)

Changes associated with capacitation are facilitated by removal of sterols (e.g. cholesterol) and non-covalently bound glycoproteins
The result is a more fluid membrane with an increased permeability to Ca2+

An influx of extracellular Ca2+ is linked to an increase in motility

Question 30

List the 3 glycoproteins associated with the zona pellucida (and the roles they play in the fusion of mammalian sperm and egg cells) - also be able to label these proteins, SED 1, AND Galt if given a drawing.

Answer 30

The zona pellucida is an extracellular matrix composed of three zona glycoproteins: ZP1, ZP2, and ZP3.

ZP3 is linked with ZP2 in a fibrous matrix and cross-links are made with ZP1

Binding to ZP3 allows the sperm to adhere to the zona pellucida and is a critical step in fertilization. The glycoprotein ZP3 (specifically the N- acetylglucosamine sugar on this protein) binds to the sperm in a species-specific manner. This triggers sperm head to undergo the acrosome reaction.