Fertilisation

Question 1

What are the events that occur during the process of fertilisation?

Answer 1

1. Capacitation & activation of sperm
2. Penetration and fusion
3. Activation of ovum and establishment of diploidy
4. Syngamy

Question 2

What are the overall types of changes that occur during sperm capacitation?

Answer 2

1. Change of motility pattern: Beating of flagellum changes from gentle wave-like motion to higher amplitude whiplash motion.
2. Change in surface membrane: Allowing sperm to respond to chemical factors released by the oocyte as well as fuse with it.

Question 3

What are the changes that occur in a sperm during capacitation?

Answer 3

1. Loss of membrane cholesterol: Binds to albumin in uterine secretions.
2. Loss of glycoproteins acquired in epididymis and seminal fluid: Changes in charge and organisation of the sperm PM, partly driven by loss of cholesterol and enzymes in uterine secretions.
3. Increased fusogenicity with oocyte: Loss of cholesterol causes increased PM fluidity, resulting in instability.
4. Biochemical changes: Removal of glycoproteins increase Ca²⁺ entry into the sperm, causing ↑ [cAMP]_i and subsequently ↑ PKA activity.
5. Increased motility: Caused by opening of Ca²⁺ channels.

Question 4

Where does spermatozoal capacitation take place?

Answer 4

In uterus and tubal-uterine junction

Question 5

What properties of the uterus allow for it to facilitate capacitation?

Answer 5

1. Secretion of proteolytic enzyme
2. Providing cholesterol-binding sinks
3. High ionic strength

Question 6

How can capacitation be reversed?

Answer 6

When capacitated sperm is added back to semen

Question 7

What are the changes that occur during sperm activation?

Answer 7

1. Change in spermatozoa tail motion
2. Acrosome reaction

Question 8

What changes to sperm tail motion occur during capacitation?

Answer 8

Wave-like movement → Whiplash movement (hyperactivity)

Question 9

What mediates changes in sperm tail motion during activation?

Answer 9

Ca²⁺ influx causing activation of PKC

Question 10

What is the acrosome reaction?

Answer 10

Fusion of outer acrosome membrane and sperm PM

Question 11

What are the changes that occur during the acrosome reaction?

Answer 11

1. Exocytosis of hyaluronidase: Facilitates penetration through cumulus cells around the oocyte.
2. Exocytosis of β-hexosaminidase: Breaks down ZP3 and prevents further binding of sperm.
3. Exposure of inner acrosomal membrane: Contains acrosin that facilitates penetration through zona pellucida.

Question 12

How many types of zona pellucida proteins (ZPs) are there?

Answer 12

4 (ZP1-4)

Question 13

What are the functions of the different ZP proteins?

Answer 13

• ZP1, 3, 4: Mediates capacitation of sperm
• ZP2: Mediates sperm penetration of zona pellucida

Question 14

What is a possible function of sperm immobilisation at the tubal-uterine junction?

Answer 14

• Once a sperm is activated, it has a very short lifespan.
• Sperm immobilisation in the isthmus, as this establishes a pool of sperm that can be released gradually over time, extending time window for fertilisation.

Question 15

How does the sperm penetrate the zona pellucida?

Answer 15

1. Proteolytic activity of acrosin
2. Physical movement of sperm

Question 16

What is the process of sperm binding to oocyte membrane?

Answer 16

1. Once inside the perivitelline space, microvilli on the surface of the oocyte envelop the head of the sperm, increasing SA for mediating fusion.
2. The sperm also binds to the PM (oolemma) of the oocyte via the middle (equatorial) and posterior half (post-acrosmoal) of its head. This binding is thought to be mediated by integrin-like molecules in oolemma and ADAM on the sperm PM.

Question 17

What is the proposed process of sperm/oocyte fusion?

Answer 17

1. Izumo1 on the sperm binds to Juno (Izumo1 receptor) on the oolemma
2. Accumulation of CD9 in the binding area of the sperm
3. Fusion may be a result of action of fusigenic proteins and is Ca²⁺-dependent

Question 18

What is the significance of the position of sperm fusion with the oocyte?

Answer 18

• It is usually on opposite pole to the oocyte pronucleus (in mice)
• Defines the plane of division for zygote (first cleavage division along pole defined by the site of fusion)

Question 19

What are the events that occur during activation of the ovum?

Answer 19

1. After fusion of sperm, there is a rise in [Ca²⁺]_i (mostly from intracellular stores) in a wave that sweeps across the oocyte lasting for 2-3 minutes, originating from the point of sperm entry.
2. After the initial wave, there is a more uniform rise in [Ca²⁺]_i across the whole oocyte every 3-15 minutes for the next 4-5 hours, each spike lasting 1-2 minutes.

Question 20

What is oocyte activation mediated by?

Answer 20

PLCζ from sperm

Question 21

What is the cortical reaction?

Answer 21

Waves of Ca²⁺ stimulates release of cortical granules from the oocyte. These contain many factors that mediate changes to prevent polyspermy.

Question 22

What are the changes that occur to prevent polyspermy?

Answer 22

1. Ovastacin cleaves ZP2
2. β-hexosaminidase B cleaves ZP3
3. Tyrosine residues between adjacent ZPs are cross-linked, preventing the zona pellucida from being further penetrated by sperm
4. Juno receptors on the oolemma are exocytosed to inhibit sperm fusion

Question 23

What are the events that take place during establishment of diploidy?

Answer 23

1. Ca²⁺ transient causes phosphorylation of a number of different proteins in the zygote that contribute towards re-entry into meiosis (from stalled metaphase II).
2. The zygote is triploid at this point, with diploid from oocyte and the addition of the haploid chromosomes from sperm.
3. In order to re-establish diploidy, one set of maternal chromosomes in the zygote is extruded into the 2^nd polar body.

Question 24

What is syngamy?

Answer 24

Process of coming together of the maternal and paternal chromosomes

Question 25

What is the sequence of events that occur to achieve syngamy?

Answer 25

1. Once fusion has occurred, the sperm nuclear membrane breaks down and sperm chromatin begins to swell. 2. Strands of chromatin are released into the zygotic cytoplasm. 3. Protamines in sperm chromatin are replaced by histones. 4. Pronuclei form around both maternal and paternal chromosomes (paternal slightly bigger). 5. DNA is duplicated within the pronuclei to form chromosomes containing 2 sister chromatids. 6. Pronuclei break down and maternal/paternal chromosomes line up in equator (mediated by metaphase spindle) ready for first mitotic division.

Question 26

What are the contributions of the mother to the zygote?

Answer 26

1. PM 2. Haploid DNA 3. Ribosomes 4. Mitochondria 5. Golgi apparatus 6. Cytoplasm 7. Centrosome

Question 27

What are the contributions of the father to the zygote?

Answer 27

1. Haploid DNA 2. Centriole 3. Non- coding RNA 4. Proteins and enzymes mediating oocyte activation and meiosis (e.g. PLCζ)

Question 28

What are the fates of components of the sperm not inherited by zygote?

Answer 28

Targeted for destruction by proteasome

Question 29

What is parthenognesis?

Answer 29

Development of a zygote that only contains maternal DNA

Question 30

Why does parthenogenesis lead to development failure in humans?

Answer 30

No centriole from sperm

Question 31

What is androgenesis?

Answer 31

Development of zygote that contains only paternal DNA

Question 32

What is parthenogenesis caused by?

Answer 32

Loss of male pronucleus and failure for second polar body to be expelled.

Question 33

What is androgenesis caused by?

Answer 33

Loss of female pronucleus and duplication of male nucleus.

Question 34

What are the clinical consequences of partheno-/androgenesis?

Answer 34

- Androgenesis: Hydatidiform mole - Parthenogenesis: Ovarian teratoma