BIOL473 - Unit 4 - Fertilization

when do sperm become fertile and progressively motile?

once they reach the caudal epididymis

what are the 4 main changes to sperm that occur in the epididymis?

1. cytoplasmic droplet migrates down tail (not all species)

2. membrane cholesterol and phospholipid changes

3. sialic acid containing mucopolysacchardies coated on sperm

4. acquisition of motility

What happens within the sperm to activate motility? Why?

increased intracellular cAMP → activates motor proteins

compare the sperm motility when exiting the male tract, adhering to the oviductal epithelium, and when close to oocyte

exiting male tract → swim vigorously in straight lines

oviductal epithelium → quiescent (waiting)

close to oocyte → hyperactivated

how does the cervix change the environment based on hormones?

high estrogen → watery, mucin strands in parallel make it easy to enter

high progesterone → highly viscous, strands disorganized making it hard to enter

How does the uterus help sperm transport? how do we know this?

uses contractions to move sperm → sperm cross the area too quickly (even if they are dead) to be doing it by themselves

why is the uterotubal junction a significant barrier to sperm?

sperm deposit directly into uterine lumen, and junction has very small opening

What happens to sperm once they reach the oviduct?

become immotile, adhere to epithelium, and act as a sperm ‘reservoir’

Define capacitation of sperm

the collective term for physiological and biochemical changes necessary for spermatozoa to acquire fertilization competency

what is the capacitation reaction necessary for?

being able to bind the zona, and the undergoing of the acrosome reaction

Compare the early vs late changes that occur during capacitation

early → changes in membrane properties and loss of surface proteins

late → changes in swimming pattern (hyperactivation) and acrosome reaction

What does the sperm plasma contribute to regulating capacitation? where is this from?

Fertilization Promoting Peptide (FPP) → from prostate

how does FPP work? What is its function?

binds to GPCR to activate a cAMP pathway → promotes early capacitation BUT prevents acrosome reaction

Can capacitation only happen in the uterus?

no! can occur anywhere because other signals are not specific to a site or species (can happen in the eye)

Explain the change in plasma membrane proteins between epididymal, ejaculated, and capacitated sperm

epididymal → proteins and CHOs on plasma membrane

ejaculated → FPP coats surface

capacitated → plasma coatings (including FPP) are removed to allow zona binding

Compare the epididymal sperm movement to the hyperactivated sperm movements

epididymal → regular wave-like flagellar beats

hyperactivated → wide amplitude “whip-lashing” beats (propels them forward in vigorous lurches)

What exactly triggers hyperactivation of sperm?

Sudden influx of calcium into sperm tails via specialized calcium channels

What are these specialized calcium channels? where are they located?

Catsper channels → extremely specific to the principal piece (PP), not midpiece or head

describe the structure of Catsper channels (subunits)

4 pore-forming subunits and (at least) 2 auxillary subunits

What are 2 suggestions for targets of male contraception?

1. blocking Catpser1 (reversible blockage)

2. calcineurin

What is calcineurin?

a calcium and calmodulin-dependent serine-threonine phosphatase that play a major role in calcium signalling

What do experiments knocking out vs inhibiting calcineurin demonstrate?

knock out → inflexible midpiece in sperm causes infertility

inhibition → same defects but fertility can be restored in a week after stopping treatment

Put the 4 structures a sperm encounters at the egg in order

1. Cumulus cells

2. Zona pellucida

3. Perivitelline space

4. Vitelline membrane

What are the major zona glycoproteins secreted by the growing oocyte?

ZP1, ZP2, ZP3, and ZP4

compare the functions of ZP2 and ZP3

ZP2 → binds inner acrosomal membrane to permit penetration of zona

ZP3 → binds sperm membrane and induces acrosome reaction (to expose inner acrosomal membrane)

Starting with the sperm binding to the zona, explain the key events that occur

1. sperm binds to ZP3

2. acrosome reaction

3. enzymes released

4. acrosomal membrane binds to ZP2

5. zona penetration

6. membrane fusion

What happens to the membranes during the acrosome reaction?

the sperm plasma membrane and outer acrosomal membrane breakdown

What 2 reasons make the acrosome reaction necessary?

1. release of acrosomal enzymes (hyaluronidase)

2. expose proteins required for membrane fusion (on inner acrosomal membrane to bind to ZP2)

How does the sperm enter the egg during membrane fusion?

the microvilli of the egg ‘engulf’ the sperm

how does membrane fusion trigger oocyte activation?

released the sperm factor into the egg

What is this sperm factor? how does it activate the cell?

phospholipase C zeta → hydrolyzes membrane phospholipids to generate IP3 that opens calcium channels on ER and causes an increase in cytosolic calcium

what are the 2 consequences (good) for increasing cytosolic calcium?

1. cortical granule release

2. resumption / completion of Meiosis 2

Describe the calcium spiking observed in eggs after sperm binding

multiple spikes occur over 2 hours, Ca2+ rushes across the whole cell in a line

what is polyspermy? is it normal in species?

multiple sperm fertilizing a single egg → normal in some species (birds) but in mammals is lethal

how do mammals prevent polyspermy?

the exocytosis of cortical granules into the perivitelline space!

In what two ways does the cortical granule reaction prevent polyspermy?

1. their enzymes degrade ZP2 (zona block)

2. they change the egg membrane (membrane block)

How is metaphase 2 arrest maintained in an egg?

cytostatic factor → stabilizes MPF and prevents activation of APC

How exactly does the spike in cytosolic calcium break the metaphase 2 arrest?

elevated calcium inactivates the cytosolic factor → no more inhibition of APC

how does APC help end metaphase 2 arrest?

degrades cyclin B to drop MPF activity, allows transition of metaphase 2 to anaphase 2

once sperm enters the cell, how is its DNA decondensed?

the sperm nuclear membrane breaks down and the protamines are replaced by histones

how do the male and female pronuclei form?

the two sets of haploid chromosomes become surrounding by new membranes

How do the two pronuclei replicate for the 1st mitosis?

pronuclei come together, initiating DNA synthesis before the membranes are broken down and mitosis occurs

What are the 2 contributions of sperm to the zygote?

1. centrosomes

2. half the chromosomes

(and also ‘sperm’ factor?)

What are the 5 contributions of the zygote?

1. plasma membrane

2. organelles, macromolecules

3. all the mitochondria

4. half the chromosomes

5. all the proteins, mRNA, to get through early embryo stages

(so essentially everything)

When does the embryo become a morula? Blastocyst?

morula → 32 cell stage

blastocyst → 200 cell stage?

when does compaction occur? when does trophectoderm / ICM form?

compaction → 64 cell stage

trophectoderm and inner cell mass → 500 cell stage?

where does fertilization occur in the uterine tube? when does it become a morula?

fertilized in ampulla, morula near the uteroduct