Lecture 5 - Fertilization

Question 1

what is capacitation, and where does it occur?

Answer 1

(occurs in mammals) - process by which sperm become able to penetrate zona pellucida

occurs in the fermale genital tract (can take a few hours)

Question 2

describe the process of capacitation

Answer 2

1) involves increase in metabolic activity leading to more rapid flagellar movements
2) PM undergoes changes in composition (lower cholesterol levels, possibly loss of proteins/carbs) to facilitate fusion
3) possible cytoskeletal changes, unmasking of egg binding sites. evidence that lipid rafts move to become clustered over anterior head (contain proteins that can bind to zona pellucida).
4) removal of cholesterol from mem increases perm. to Ca2+ and HCO3-; leads to increased cAMP production by adenylyl cyclase, increased tyrosine phosphorylation of sperm proteins, including those related to cell resp. and motility (activation of dynein?)

Question 3

what is chemoattraction/chemotaxis? how does it relate to fertilization?

Answer 3

chemoattraction - attraction of sperm to egg via chem. signals

chemotaxis - oriented movement in response to an external signal (like a gradient) - occurs in uterus and/or oviduct (and also in aquatic and marine enviros like w/ sea urchin egg/sperm)

molecules may be released by eggs themselves or the egg-containing structure (e.g. gonangium in Campanularia, related to jellyfish) - confers species specificity.

Question 4

what is an example of a purified chemoattractant in sea urchin?

Answer 4

resact (sperm activating protein) - from jelly layer of eggs of sea urchin species - 14 aa peptide bound by receptors on sperm PM. causes sperm to swim faster across gradient (lower to higher conc). increases sperm resp. rate in a guanylyl cyclase dependent manner

Question 5

what is the secondary messenger involved w/ sperm resp. after binding w/ resact? other results of binding resact?

Answer 5

cGMP synthesis via guanylyl cyclase

Ca2+ levels also increase activating motility (stimulates dynein ATPase activity).

peptide is species specific; others have slightly diff proteins (e.g. speract).

Question 6

what is allurin?

Answer 6

184 aa protein in xenopus; performs similar function to resact. allurin is related to cysteine-rich secretory proteins (CRISP) family in mammals, could play a role in fert. as well.

Question 7

what experiment first implicated receptors for egg-sperm signaling?

Answer 7

simple (“classical”) experiment w/ sperm, egg, and resact

1) sperm would be preincubated w/ resact
2) eggs would then after be introduced
3) sperm would not be able to find eggs - receptor sites were “blinded” by the fact that they already had resact bound on them. if they could still find eggs, it would be clear that receptor signaling was not in play

Question 8

describe chemotaxis in mammals

Answer 8

sperm respond to substance(s) released in follicular fluid during ovulation, includes progesterone secreted by cumulus cells (loose follicle cells surrounding the ovum)

only fraction of sperm are receptive - perhaps selects for those at peak ability to fert?

progesterone has been implicated in chemotaxis in mammals - more successful sperm are more sensitive to prog.

Question 9

what “signaling” occurs before chemotaxis in mammals?

Answer 9

thermotaxis - sperm can detect higher up in reproductive tract is warmer due to opsins (normally photosensors in vision in other contexts)

Question 10

how is the acrosome reaction initiated?

Answer 10

sperm reaches egg jelly (after resact binding), must penetrate it

comes into contact w/ jelly coat - one component (usually a glycoprotein) binds to receptor on sperm PM

leads to transient rise in Ca2+ in sperm, thus firing the acrosome reaction

Question 11

describe the anatomy of the acrosome.

Answer 11

acrosome consists of outer/inner membrane which has the acrosomal contents (hydrolytic enzymes + bindin)

subacrosomal space below inner acrosomal membrane contains globular actin - can rapidly polymerize into F-actin.

Question 12

describe the events that occur after the acrosome reaction has initiated.

Answer 12

1) exocytosis - outer acrosomal membrane + overlying portion of PM fuse and vesiculate, releasing acrosomal contents
2) enzymes lyse hole in jelly
3) acrosomal process (long extension) forms @ tip of sperm via rapid polym. of G-actin - sperm reaches vitelline envelope
4) adherence in sea urchin requires bindin - species specific (diff. aa compositions and size) - sticks to egg of own species

Question 13

what experiment did Glabe and Lennarz perform that indicated that bindins adhere to eggs in a species-specific manner?

Answer 13

purified bindin from 2 diff. species, matched w/ opposite species

glutination (sticking) only occurs when bindin matches w/ original species

Question 14

what occurs as a result of adherence of sperm to egg membrane?

Answer 14

egg activiation occurs due to bindin

also leads to formation of fertilization cone

a) egg forms structure which engulfs sperm
b) formation of fert. cone is dependent on actin polymerization

Question 15

fertilization in mammals - what does the sperm encounter first when reaching the oocyte?

Answer 15

layer of cumulus cells surrounding oocyte is first obstacle

these cells + ovum are released from ovary at ovulation

Question 16

describe sperm entry to get through granulosa cell layer

Answer 16

sperm has enzyme called PH20, degrades hyaluronic acid (principle ECM protein)

Question 17

when has the acrosome reaction occurred in mammals for successful sperm?

Answer 17

acrosomal rxn has already occurred in successful sperm by the time of reaching the cumulus cells (triggered by high levels of progesterone near ovum).

Question 18

what are zona pellucida proteins and what is their importance in fert.?

Answer 18

ZP1-4 are involved in sperm transport to egg cell membrane (though exact functions are under debate)

acrosome-reacted sperm interact w/ ZP2 + immediately begin making a channel into ZP. those w/ intact acrosome (no rxn yet) appear to bind ZP3, triggering acrosomal rxn. they then bind to ZP2 + tunnel inward.

Question 19

what happens after interactions w/ ZPs w/ mammalian sperm?

what is mammalian acrosomal rxn?

Answer 19

sperm continue to make burrowing movements to reach ZP

fusion + vesiculation of outer acrosomal memebrane w/ PM membrane = acrosomal rxn

Question 20

what occurs topologically w/ the sperm membrane as a result of the acrosomal rxn? does an acrosomal process occur?

Answer 20

vesiculation only occurs part of the way down (collar of membrane is left around head; now known as equatorial segment).

anterior sperm membrane now consists only of inner acrosomal membrane. region of sperm PM below eq. segment is called posteq. segment.

no, an acrosomal process does NOT form (don’t have massive actin polymerization)

Question 21

after acrosomal rxn occurs in mammals, what events occur?

Answer 21

sperm wiggles its way between ZP and egg PM (space in between called perivitelline space)

sperm PM binds w/ egg PM - but not at tip, like in sea urchins w/ acrosomal process. equatorial or postequatorial regions bind w/ egg PM - get modified during acrosomal rxn to become comepetent to bind (sperm not undergone acrosomal rxn cannot bind)

Question 22

what proteins are involved in sperm-egg mem. fusion? what is a result of mutations in these proteins?

Answer 22

Izumo (sperm) and Juno (egg) are thought to be involved in membrane fusion

mutations in either of these can block fertilization completely

Question 23

what happens after Juno/Izumo protein signaling?

Answer 23

entire sperm is taken in as a result of mem. fusion and actin polym. in the ovum

sperm mitochondria are generally destroyed soon after.

Question 24

what are the two main goals of activation?

Answer 24

1) block polyspermy
2) release from 2nd meiotic arrest

Question 25

fast block to polyspermy - what is it, in which animals does it occur?

Answer 25

established as soon as first sperm has fused w/ egg; due to mem. potential shift

egg membrane is polarized at -75 mV, due to relative conc. of Na+ and K+ in cytoplasm and outside the cell (e.g. sea water)

as soon as 1st sperm has fused, PM channels open so Na+ (sea urchins) can enter, increasing potential to +20 mV for about 1 min. this blocks further sperm fusion as it cannot occur w/ such a high mem. potential.

occurs in invertebrates and amphibians.

Question 26

is the fast block foolproof (as in, will it last forever)? is it necessary?

Answer 26

fast block is transient (to buy time for the slow block).

however, the positive “fertilization potential” is necessary for first sperm entry into egg.

Question 27

what are other mechanisms to block polyspermy

Answer 27

slow block - exocytosis of cortical granules; mechanistically prevents sperm from binding w/ egg membrane.

Question 28

what is the slow block in invertebrates? what is the consequence?

Answer 28

essentially physical; lifting away from the egg surface of the vitelline envelope together w/ sperm that are attached to it is a result of:

the release of proteases which cleave connections between VE and PM.

Question 29

what is formed after the cleavage of the vitelline envelope from the PM, and how?

Answer 29

fertilization envelope is formed

1) glycosaminoglycans (GAGs) are released from cortical granules - hydration of GAGs “poofs” the vitelline envelope up and moves it away from the PM - results in formation of hyaline layer (between VE and PM)
2) peroxidases harden vitelline envelope by crosslinking proteins therein, hardens fert. env.

Question 30

what happens to the sperm receptors on the vitelline envelope?

Answer 30

receptors appear to be modified so no sperm can adhere (any adhered will fall off).

Question 31

how does the slow block start in mammals?

Answer 31

cortical granules exocytose enzymes that remove carbohydrate groups from ZPs, blocking sperm binding and blocking elicitation of the acrosomal rxn (called the zona reaction).

proteins become chem. cross-linked so zona pellucida becomes impermeable to sperm.

Question 32

what is the “zinc-spark”? what does it have to do w/ blocking polyspermy?

Answer 32

prevents polyspermy; egg will release billions of zinc ions upon binding w/ first sperm (will inhibit at least two acrosomal enzymes in sperm).

Question 33

what role do the egg-sperm receptors play in blocking polyspermy?

Answer 33

Juno is released from the membrane, binding sperm receptors and preventing them from binding to egg.