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biol 309 > Fertilization (2) > Flashcards

Fertilization (2) Flashcards

1
Q

What was found to be the chemoattractant in eggs?

A
  • resact - 14 amino acid peptide in egg jelly of the sea urchin Arbacia punctulata
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2
Q

How does the resact concentration gradient change the way the sperm moves?

A
  • shift from linear motility to curvilinear motility
  • at low: “curvi” linear motility
  • at high: “circi” linear motility
  • probability of contacting egg increases with “circi” linear motion
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3
Q

What causes the shift to circi-linear motility?

A
  • resact binds to a guanylyl cyclase receptor
  • guanylyl cyclase cleavse GTP to cGMP
  • cGMP binds to CNG-gated channel and leads to influx of calcium in the cell
  • calcium elevates the ATP production in sperm
  • this changes the motion
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4
Q

What shows specificity of this chemoattractant?

A
  • resact doesn’t attract Stronglyocentrotus sperm

- but they do secrete a functionally similar peptide “speract”

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5
Q

What is a broadcast spawner? What are the challenges with this?

A
  • an organism that releases gametes directly into the sea for external fertilization
  • a lot of sperm and eggs could be wasted or could have sperm fertilizing another species egg resulting in non-viable offspring
  • this is why there is a need for species specificity
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6
Q

Is there a need for sperm chemotaxis in mammals?

A
  • no risk of sperm fertilizing a different species egg

- but would help sperm since it needs to travel cms and chemotaxis would increase chances of fertilization

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7
Q

Where is the fertilization site?

A
  • in the fallopian tubes

- this means the sperms path is long and bilateral

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8
Q

What mechanism do vertebrate sperm use as an attractant? How was this studied?

A
  • thermotaxis: a temperature gradient
  • put sperm in a tube with a temperature gradient from 34 to 39 and count sperm in each chamber
  • sperm favoured higher temperature
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9
Q

What plays an important role in thermotaxis?

A
  • rhodopsin

- rhodopsin in photoreceptor cells in eyes respond to light to change conformation

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10
Q

How was the role of rhodopsin studied?

A
  • they produced a rhodopsin knockout mouse and used fluorescence to show it is not expressed in it
  • rhodopsin knockout mouse has defects in thermotaxis across temp gradient
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11
Q

Which chemicals might vertebrates use for chemotaxis?

A
  • progesterone is found in the oviduct and around the egg and receptors are found on the sperm
  • bourgeonal and lyral are not watr soluble and are aromatics that are usually detected by smell/olfactory receptors
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12
Q

What study has been determine if chemotaxis is in vertebrates?

A
  • screen testis cDNA to see if any or (olfactory receptors) genes expressed
  • found hOR 17-4 through correlative data
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13
Q

What is the role of g-protein coupled receptors?

A
  • g-protein coupled receptors may be activated by an agonist (odorant molecule) where beta and gamma and alpha are released to go onto downstream targets
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14
Q

How did they test if hOR 17-4 is a chemoattractant?

A
  • compare a capillary tip with HTF control and bourgeonal
  • in the control condition, sperm went in all directions
  • in the bourgeonal condition, sperm only went towards the capillary tip
  • there is still not definitive proof that this mechanism occurs in vivo
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15
Q

How do sperm among organisms compare?

A
  • similar sperm structures in most organizations

- drosophila: different longest sperm which functions to block the oviduct and prevent other males from mating

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16
Q

What makes C elegans a good model organism?

A
  • lives in soil all over

- easy to raise

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17
Q

What are the characteristics of C. elegans?

A
  • most are hermaphrodites (small amount are male)
  • no organelles in the pseudopod
  • filled almost exclusively with a dense fiber network composed of the Major Sperm Protein
  • the sperm appears to crawl
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18
Q

How does fertilization occur in c. elegans?

A
  • eggs move left to right as they are fertilized and become embryos
  • sperm are kept in spermatheca
  • sperm from male must enter the vulva and travel from the right to the left
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19
Q

What are the main parts of the egg?

A
  • jelly layer (protects egg by acting as shock absorber)
  • vitelline envelope (fibrous, glycoprotein-containing mat)
  • cell membrane
  • cortical granules, mitochondria, and nucleus
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20
Q

How do the sperm get through the jelly layer?

A
  • acrosome reaction
  • 1) fusion of acrosomal vesicle with sperm membrane
  • 2) extension of acrosomal process (actin form filaments that protrude)
  • acrosomes contain enzymes that eat through the jelly
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21
Q

How is species specificity determined through the acrsome reaction?

A
  • receptors on sperm recognize fucose sulfate polymer in jelly
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22
Q

What is the cellular process of acrosome reaction?

A
  • receptors on sperm recognize jelly
  • phospholipase activation
  • production of IP3
  • Ca++ release from internal stores
  • exocytosis
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23
Q

What is a possible hypothesis for how the sperm binds to the egg surface?

A
  • isolated a 35 kDa protein (bindin) from sperm acrosome
  • it is an insoluble protein that forms egg clumps in solution
  • hypothesis: this protein is involved in sperm-egg adhesion
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24
Q

What experimental design was used to test the bindin protein?

A
  • “agglutination” assay: eggs clump together when bindin added
  • bindin immunolabeling: correlative data, shows expression at acrosomal process and vitelline envelope of egg
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25
Q

How does immunolabeling work?

A
  • use an antibody that will bind to a target protein

- the antobody is invisible so must use a second antibody to make it visible

26
Q

What experiment shows the species specificity of bindin?

A
  • beads are coated with bindin receptor EBR1
  • same species sperm and ERB1 bind together well
  • different species sperm with ERB1 did not bind well together
  • either species sperm with blank beads do not bind well (negative control)
27
Q

What are the layers of a vertebrate egg?

A
  • cumulus layer: layer of cells surrounding eggs

- zona pellucida: extracellular matrix similar to vitelline envelope

28
Q

Does the acrosome reaction happen for vertebrates?

A
  • yes

- it occurs once the sperm hits the zona pellucida layer with ZP proteins to digest through the layer

29
Q

What are the zona pellucida proteins?

A
  • ZP1, ZP2, ZP3

- glycoproteins form matrix of the zona pellucida

30
Q

What have mouse knockout studies found about ZP proteins?

A
  • ZP2 and ZP3 are required for sperm binding and fertilization
31
Q

What is the process of ZP proteins?

A
  • galactosyltransferase receptor on sperm binds to N-acetylglucosamine residues on ZP3 (thought to induce acrosome reaction)
  • ZP2 cleavage after initial sperm binding prevents further binding of sperm
32
Q

How was the effect of ZP2 cleavage studied? Why is this important?

A
  • using fluorescence markers to visualize sperm nuclei, we can see that before ZP2 cleavage there are many sperm around the egg and after there are no longer sperm bound to the egg surface
  • this is important for preventing polyspermy
33
Q

What is polyspermy?

A
  • fertilization of egg by more than one sperm

- leads to polyploidy, a genetic state (not diploid) where embryos are not viable

34
Q

How can polyploidy occur?

A
  • majority due to polyspermic fertilizations
  • can also be from meiosis defects
    1. digyny: oocyte with defect in 1st or 2nd meiosis retains 2N chromosomal complement
    2. diandry: fertilization with diploid sperm
35
Q

What happens to triploid embryos?

A
  • die at various stages after conception

- some go to term (are born but die shortly after)

36
Q

What is a strategy used by sea urchins and mammals to prevent polyspermy?

A
  • “slow block”: 15-60 seconds after entry of first sperm they release cortical granule material from the egg
  • “fast block”: within 1 s, membrane depolarization occurs
37
Q

How does the “slow block” work?

A
  • cortical granules are released
  • they contain: cortical granule serine protease, mucopolysaccharides, peroxidase/transglutaminase, hyalin
  • this breaks tethers of vitelline envelope (serine), creates an osmotic gradient that swells up the egg, and transfer eggs to a rigid structure
38
Q

What actually triggers the slow block to occur?

A
  • ultimately an increase in concentration of Ca++
  • bindin on sperm binds to receptor
  • unknown processes happen
  • PLC is activated and cleaves PIP2 to DAG and IP3
  • IP3 binds to an endoplasmic reticulum receptor which allows Ca++ to flow in
  • this causes CG exocytosis
39
Q

For which animals does the “fast block” occur?

A
  • marine invertebrates, marine vertebrates
40
Q

How does the “fast block” occur in sea urchins?

A
  • membrane depolarization due to opening of sodium channels

- influx of Na+ ions into the eggs

41
Q

How does the “fast block” occur in amphibians?

A
  • due to chloride channels and efflux of Cl- ions
42
Q

Why does the “fast block” prevent polyspermy?

A
  • the depolarization is thought to alter the surface proteins on the egg and block other sperm from binding
43
Q

There are many steps that precede entry of sperm pronucleus into the egg, including?

A
  • genes that are required for proper fertilization
  • genes for sperm maturation, motility and fertilization
  • Dnahc f
  • many many genes related to fertility defects
44
Q

What are the factors related to infertility?

A
  • genetics, age, anatomical, gamteogenesis, fertilization, viruses, environment
45
Q

What are some treatments for infertility?

A
  • drugs
  • surgery
  • assisted reproduction technologies
46
Q

What is one type of assisted reproductive technology?

A
  • in vitro fertilization
  • first test tube baby born in 1978
  • more than 2 million born
  • remove eggs, and sperm are microinjected in (if underlying genetic problem this may not work)
  • not a problem if sperm are immotile because they are directly injected in
47
Q

What study was conducted to determine if having a final chromosomal content of 2N sufficient for normal embryogenesis?

A
  • pronuclear transplantation experiments to examine “equivalency” of mammalian male and female pronuclei
  • had zero surviving progeny for two female protonuclei and two male protonuclei
  • shows there is non-equivalence of mammalian pronuclei
48
Q

What is pronuclear transfer?

A
  • removal of one pronuclei in vitro using needle

- replaced with pronuclei from another embryo

49
Q

How are male and female pronuclei differentiated?

A
  • female pronuclei is smaller
50
Q

What control did they use in the pronuclear transfer experiment?

A
  • they removed a male protonucleus and added one back in
  • they had a small amount surviving
  • this shows that it’s not just the procedure that is killing the embryos
51
Q

What mechanism explains why only a sperm protonuclei and egg protonuclei produce viable offspring?

A
  • epigenetic modification of male and female genomic DNA

- imprinting through DNA methylation

52
Q

What is gene silencing?

A
  • in some cases it mediates activation
  • addition of methyl groups blocks the binding of factors that are necessary for transcription
  • methylated CpGs are recognized by methyl-binding domain proteins and can recruit histone-modification enzymes and alter the local chromatin environment
53
Q

How do sperm and egg DNA compare in terms of methylation?

A
  • egg DNA is highly methylated/imprinted with maternal ICRs (imprinting control regions)
  • males are not
54
Q

What are imprinted genes important for?

A
  • cell proliferation
  • embryonic and extraembryonic development
  • fitness and behaviour
55
Q

What study was conducted to determine if being imprinted is important for viability?

A
  • similar experiment to before using nuclear transplantation experiments
  • 2 female pronuclei not imprinted: not viable
  • 2 female pronuclei one imprinted: not viable
56
Q

Why does having one non-imprinted female pronuclei not make a viable offspring?

A
  • because the region of imprinting at sperm ICR is vital
  • they mutated the regions to see if they play an important role for viability
  • two important genes imprinted in male sperm: Igf2 and H19
57
Q

How do the mechanisms of gene expression for H19 and Ifg2 differ?

A
  • in females: the unmethylated ICR allows CTCF to bind, CTCF inhibits the Ifg2/H19 enhancer and neither genes are activated
  • in males: the methylated ICR prevents CTCF from binding, this allows the Ifg2/H19 enhancer to be active and both genes are expressed
58
Q

What experiment was conducted to show that Igf2/H19 is important for viability?

A
  • they did a nuclear transplantation experiment with one unimprinted female pronucleus and one correctly imprinted (by mutating so CTCF can’t bind)
  • this produced viable offspring
59
Q

What is viability?

A
  • the ability to reach adulthood and reproduce
60
Q

What is Silver-Russell Syndrome?

A
  • intra-uterine and postnatal growth restriction, facial dysmorphism, and dwarfism
  • in about half of the patients there is loss of DNA methylation at the ICR regulating IGF2 and H19

biol 309 (14 decks)

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