(19) Fertilization

Question 1

(Fertilization)

Fertilization is the event that sees a single sperm nucleus unite with the egg nucleus within the activated oocyte cytoplasm

1. where does it occur?

Answer 1

1. ampulla region of the oviduct, adjacent the ampullary-isthmic junction

Question 2

(The oocyte at ovulation)

1. at ovulation, meiosis in the oocyte has been re-arrested at what stage?

exception?

2. oocyte surrounded by what?

space between vitelline membrane and ZP = ?

internal layer of the cumulus cells radiate out from the ZP, and are termed what?

Answer 2

1. metaphase II

canids - arrested at metaphase I (require extra post-ovulation time)

1. PM (vitelline membrane), zona pellucida (protective), granulosa cells (cumulus oophorus)

perivitelline space

corona radiata

(Coincident with the process of oocyte maturation following the LH surge, the cumulus undergoes expansion; changing from a tightly packed cell mass to one with a looser association in which the cumulus cells are held together by a viscous hyaluronic acid matrix)

Question 3

(Thee oocyte at ovulation - cont)

1. upon ovulation oocyte picked up by?

transported?

how?

2. Temporary fusion between what and what are required?
3. what prevents transport

Answer 3

1. fimbria of oviduct

through infundibulum (pper part) to ampulla

cilia beat - move fluid (for these oocyte needs to be in expanded cumulus matrix)

2. this complex and the apex of cilia on the fimbria
3. alterations in the COC adhesive properties (either increase of decrease)

Question 4

(Sperm Capacitation: Maturation of Sperm in the Female Reproductive Tract)

When the sperm left the testicle they were incapable of swimming and unable to bind to an egg and fertilize it. The potential for progressive motility was obtained on passage through the epididymis, but was not expressed until the sperm were diluted with contents of the accessory sex glands during the emission stage of ejaculation (activated motility).

1. Similarly, the potential for egg binding and fertilization is also developed when?

but in this case it is not expressed until when?

2. maturational changes sperm undergo in the female ttract = ?

reversible?

affect on lifespan?

Answer 4

1. during maturation in the epididymis
2. until further maturational changes occur to the sperm during their passage through the female reproductive tract

( It is the result of sequential sperm exposure to different molecules and fluids within the tract and interaction with the female epithelial surfaces.)

2. capacitation (capacity to bind egg)

no

drastically shortened

Question 5

Sperm Capacitation: Maturation of Sperm in the Female Reproductive Tract

cont

1. capacitation = modification of what?

what three modifications?

(it Results in….)

1. removal or modificaiton of what?
2. lowering of what?

function of what?

loss of cholestrol allows what?

3. hyperpolarization of what?

to do what?

5. phosphorylation of what?
6. Shearing forces generated when sperm swim through cervical mucus may be important in initiating these changes in ruminants and primates (removal of decapacitating factors on the sperm surface).
7. major changes occur where?

Answer 5

1. sperm PM and associated molecules

exposure of surface receptors in egg recognition, increase fusogenecitiy, alters Ca++ homeostasis

1. previously applied decapacitation factors
2. cholesterol/phospholipid ratio of sperm in PM

lipophilic moecules in female tract - starts after sperm are separated from seminal plasma

increased intracellular pH (increases sens for induction of acrosome rxn) - may also thin PM (^ sperm egg interaction)

3. region overlaying acrosome

increase Ca channel sens for acrosome rxn

4. lipid microdomains of PM that contain ZP binding protine on head
5. surface tyrosine residues
6. isthmus region of oviduct (around time of ovulation)

Question 6

Sperm Capacitation: Maturation of Sperm in the Female Reproductive Tract

cont

This is speculated to be mediated by changes in….

1. hormone milieu - increase in what?
2. contents of what?
3. increasing concentraction of what as you move closer to site of ovulation?

Answer 6

1. progesterone/prostaglandin from post-LH surge follicle
2. oviductal lumen (oligosaccharides and albumin) from ovulated follicular fluid
3. fluid bicarbonate concentration

Following capacitation the sperm, usually on interaction with the zona pellucida (ZP) of the oocyte, can undergo the acrosome reaction; after which they penetrate the ZP, and fuse with the plasma membrane of the oocyte.

Question 7

(Hyperactivated motility)

1. at the same time sperm become capacitated the sperm develop what?

is it reversible?

what for?

Answer 7

1. hyperactive motility (thrashing - side to side movement of head)

yes

detachment from oviduct, location and pentration of egg

Question 8

Does the female control the timing of sperm capacitation to coincide with ovulation?

(Two schools of thought)

1. what is first?
2. and the second?

Answer 8

1. ovulation sends signal to oviduct epithelium which triggers sperm (the female controls capacitaiton so that it is closely timed with arrival of oocyte)
2. during estrus there is constant release of sperm from storage site towards fert area - capacitance occur by changing conditions as they go - if no egg they die

(There does appear to be a slight increase in available sperm (~10%) immediately post-ovulation so there may be a minor role for female control of capacitation timing.)

Question 9

(Fertilizable Life of the Sperm and Oocyte)

1. sperm have longer fertile life than egg…. but after capacitation it becomes much shorter

Question 10

(Encounter of Sperm and Oocyte)

1. once cumulus-oocyte complex enters ampulla it adherees to what?

remains attached until when?

2. hyperactivty of sperm postulated by some to be for what purpose?

Answer 10

1. oviduct epithelium

after fertilization

2. so sperm can detect oocyte

(Hyperactivated sperm are better able to swim through the mucus found in the isthmus of the oviduct, turn and swim out of deep crypts in the oviductal wall, and have an effective search pattern within the confines of the oviduct.)

Question 11

Do the sperm randomly find the oocyte(s) by bumping into them within the confines of the oviduct, or are they specifically drawn towards them?

1. in what animals is there evidence of chemotaxis?

use what?

2. how do these substances work?
3. Does this happens in mammals?

at longer distances sperm show what?

Answer 11

1. non-mammalians (esp external fert)

small peptides/proteins/lipds (coral)/malic acid (ferns)

2. cause hyperpolarization –> lower Ca in sperm –> straight swimming
3. maybe

thermotaxis (which there is in oviduct)

At closer range to the oocyte they display some chemotactic responses. Follicular fluid (some enters oviduct at ovulation) is chemotactic for sperm in vitro and various substances in follicular fluid have been examined, including progesterone and atrial natriuretic peptide. Others suggest products of the oocyte/cumulus complex acting at short ranges. Most recently, a subset the same odorant receptors used by the olfactory system have been found on mature sperm and shown to cause hyperactivation and chemotaxis. Sperm also have surface progesterone receptors and the COC does secrete progesterone. The jury is still out.

Question 12

(Penetration of the Oocyte Vestments)

1. once sperm has located the oocyte it needs to penetrate - in order - to access the interior and fert?

(Penetration of the Cumulus oophorus)

2. only what can penetrate?

aided by what?

(Penetration of ZP)

1. sperm bind and undergo what?

Answer 12

1. cumulus oophorus (if present) –> ZP –> vitelline membrane
2. capacitated sperm (with hypermotility)

cell surface hyaluronidase (bound to sperm PM)

1. acrosome reaction

(The zona itself and the zona-binding proteins on sperm are fairly species specific, and generally sperm of one species will not bind to zona-intact eggs of another species)

Question 13

(The Acrosome Reaction)

1. fusion of what with what?

resulting in what?

(induction of acrosome rxn)

2. what two probable?

(Progesterone)

postulated that…

3. further mech of what?
4. or what?

(the ZP)

5. this is generally accepted model
6. made up of what glycoproteins?
7. How are these arranged?
8. only what bind to ZP?

Answer 13

1. outer acrosomal membrane with PM of sperm

exposure of the acrosomal content to the extracellular environment.

2. progesterone and ZP itself
3. sperm selection - induce AR in sub-optimal sperm
4. prime sperm for AR
5. ZP1, ZP2, ZP3
6. paris of ZP2 and ZP3 in chains - crosslinked by ZP1
7. acrosome intact sperm

Question 14

(The Acrosome Reaction)

(ZP binding cont)

(in mice)

1. sperm bind what?

inducing what?

2. cause rapid increase in cytolsoic increase in Ca rsulting in what three thigns?

Answer 14

1. ZP3 (primary binding)
2. AR
3. sperm PM fuses at mutliple points with outer acrosomal membrane –> membrane vesiculation

acrosomal content release –> exposing inner acrosomal membrane

inncer acrosomal membrane binds to Z2 (secondary binding)

(Secondary binding orients the sperm so the thrusting of the tail can drive it through the zona.)

Question 15

(The Acrosome Reaction)

(ZP binding cont)

The relative roles of the sperm thrusting its way through the ZP and the digestion of the ZP matrix by enzymes from the acrosome, both released and remaining bound to the inner acrosomal membrane, are the topic of some contention. The old paradigm was that the released enzymes were vital, and the thrashing of the sperm tail merely served to propel the sperm down the digested hole. Others argue that the physical thrusting is of primary importance, inducing a scything motion of the sperm head.
Relative importance may depend on species and order

1. what more important in marsupials?
2. in eutherians
3. On passing through the ZP the sperm enters what?

Answer 15

1. digestion
2. digestion + tail thrust
3. the perivitelline space, between the ZP on the outside and the oocyte plasma membrane on the inside.

Question 16

(Apart from penetration of the ZP, why does the Acrosome Reaction occur?)

1. only what are capable of binding to, and fusing with, the oocyte plasma membrane?
2. A second reason for the acrosome reaction is sperm selection - how does this work?

Answer 16

1. acrosome reacted sperm
2. Since prematurely acrosome reacted sperm cannot penetrate the ZP they are excluded from fertilization. (This removes these “aged” sperm from the fertilizing pool)

Question 17

(Fusion with the Vitelline Membrane (oocyte plasma membrane)

1. The head of sperm initially contacts the vitelline membrane how?
2. The sperm plasma membrane overlying the equatorial region does what?
3. The vitelline membrane and ooplasm (oocyte cytoplasm) adjacent this area well up to surround the sperm, and the engulfing vitelline membrane fuses to itself over the top of the sperm.

This results in what?

4. The thick fused sperm plasma membrane - vitelline membrane now does what?

Answer 17

1. “end on” and then reorients for lateral attachment.
2. fuses with the vitelline membrane.
3. sperm, contained within a membrane vesicle, becoming incorporated into the ooplasm.
4. breaks down, releasing the sperm nucleus into the egg cytoplasm, while the sperm plasma membrane becomes incorporated into the vitelline membrane.

(Most of the non-nuclear sperm elements degenerate within the ooplasm.)

Question 18

(Contributions of Sperm to next generation)

1-3. what three things?

Answer 18

1. paternal haploid genome
2. signal to initiate egg metabolism/activation
3. centrosome

Question 19

(Activation of the Egg)

1. Prior to fusion of the sperm with the vitelline membrane the oocyte is metabolically quiet. Incorporation of the sperm activates the egg:

does what?

2. activation of egg cause increases in levels of what?

(How does the Sperm increase Ca2+ in the Oocyte?)

many theories…

3. current evidence indicates that penetrating sperm induces what?

oscillogen = ?

Answer 19

1. escapes second meiotic arrest

undergoes the cortical rxn

2. intracellular Ca2+ levels

(In some species this calcium comes from stores within the ooplasm, in others it enters from the extracellular fluid.)

3. soluble sperm-derived factor “oscillin” or “oscillogen” which enhances the Ca2+-induced Ca2+-release from intracellular stores

sperm-specific phospholipase C: PLCζ. (Phospholipase C zeta)

Question 20

(Resumption of Oocyte Meiosis)

1. The second meiotic arrest of the oocyte is maintained by what?
2. Elevated Ca2+ in the oocyte does what?

Answer 20

1. a cytostatic factor (CSF)
2. destroys CSF and meiosis continues with the completion of division and expulsion of the second polar body.

Question 21

(I. Formation of Male and Female Pronuclei)

1. Upon entering the ooplasm, the sperm head swells and the head and tail separate. The sperm nuclear membrane degenerates, exposing the nuclear material to the ooplasm. The protamines, (that replaced histones during spermatogenesis to condense the nucleus) are removed, and histones from the oocyte are bound, resulting in decondensation of the paternal chromatin. A new nuclear envelope is formed and the resulting structure is termed the what?
2. Following completion of the second meiosis, the female chromatin is also decondensed and acquires a nuclear envelope, forming what?

Answer 21

1. male pronucleus
2. the female pronuclei

Question 22

(Pronuclear migration)

1. The sperm centriole now directs formation of what?
2. This growing structure does what?

Answer 22

1. the sperm aster, a growing microtubule spindle-like structure (protein for formation comes from the oocyte).
2. ushes the male pronucleus towards the center of the cell. When the aster has grown enough to reach the female pronucleus it draws it to the center, adjacent the male pronucleus.

Question 23

(K. Syngamy)

1. chromatin does what?

Answer 23

1. Chromatin in each pronucleus duplicates and condenses and the nuclear membranes break down.

(Note: syngamy as defined in invertebrates, the formation of a single diploid nucleus by fusion of male and female pronuclei prior to membrane breakdown, does not occur in mammals. Rather, the chromatin in each is duplicated and the chromosomes align on the equatorial plate; and a mitotic division commences at the metaphase stage. Quickly resulting in the first cleavage and a 2 cell embryo.)

Question 24

L. The Block to Polyspermy

1. Fertilization of the oocyte with more than one sperm can result in what?

(3 methods)

2. first one?
3. second way
4. ome species like the rabbit have a weak zona reaction and sperm can still penetrate to the perivitelline space (there may be tens to hundreds of sperm here in these animals. In these species the poorly understood what is predominant?

Answer 24

1. a polyploid embryo (3N, 4N vs normal 2N)
2. limiting number at fert site (AI can increase this –> polyspermy)

follwing egg activation by first sperm barriers are set up

3. cortical granules of ooplasm fusing with vitelline membrane –> discharge enzymes (proteases and oligosaccharaides) into periviteeline space –> zona reaction/hardening –> prevents further sperm penetration (from in out - so some sperm may be stuck halfway)
4. vitelline or membrane block (sperm unable to bind or fuse with vitelline membrane)

This may also function as a back-up system in species with a strong zona reaction.
Even with these in place the incidence of polyspermy is 1-2%, and increases dramatically if insemination occurs after ovulation (aged eggs are less able to establish these blocks).

Question 25

(triploidy)

1. Polyploidy is detected in 10-20% of spontaneous abortions in humans. The most common polyploidy is what?

can be

2. what?
3. or what?

Answer 25

1. triploidy (3N)
2. Failure of the block to polyspermy, so the single female pronucleus is fertilized by 2 male pronuclei (diandry).
3. Failure to extrude the second polar body (or other errors in meiosis 1 or 2)
   - 2 female pronuclei and 1 male (digyny).

(Most triploid embryos die early in embryogenesis but some survive longer and very rarely there are reports of live births. Survival rate may depend on mosaicism - what proportion of the cells are diploid.)

Question 26

(Parthogenesis)

N. Parthenogenesis
Parthenogenesis is the method of reproduction employed by some invertebrates and lower vertebrates, but is not a viable method in mammals. Oocytes may occasionally activate and develop parthenogenically, but most fail to continue beyond the blastocyst stage. In-vitro cultured mammalian oocytes can be encouraged to develop parthenogenically but fail at various stages due to abnpormal proliferation and differentiation of both embryonic and placental cell lineages. We saw in the sex determination section that the genome is maternally and paternally imprinted in mammals, and you need both for normal development - maternal and paternal genome sets are complementary.