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Reproductive Biology 3 > L11: Fertilisation & Early Embryo Development > Flashcards

L11: Fertilisation & Early Embryo Development Flashcards

1
Q

What are the main parts of female reproductive organs responsible for in vivo sperm selection?

A
  • vagina, uterotubal junction
  • cervix
  • fallopian tubes
  • cumulus mass
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2
Q

How does vagina and uterotubal junction contribute to in vivo sperm seelction?

A

Anatomical barrier, acidic pH and leukocytes

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3
Q

How does cervix contribute to in vivo sperm selection?

A
  • cervical mucus penetration
  • blind-ended cervical crypts
  • responsiveness to secretory molecules from the reproductive tract
  • cervical mucus eliminates sperm with poor motility
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4
Q

How do fallopian tubes contribute to in vivo sperm seelction?

A
  • binding to epithelial cells
  • rheotaxis (swim against the current)
  • thermotaxis (reacting to increasing temperature)
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5
Q

How does cumulus mass contribute to in vivo sperm selection?

A
  • cumulus mass penetration
  • hyaluronic acid interaction
  • chemotaxis
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6
Q

What is the role of oviduct in fertilisation?

A
  • utero-tubal junction (UTJ): sperm selection and reduction of sperm numbers. (differing mechanisms between species)
  • isthmus: stores sperm, preserving viability
  • ampulla: holds oocytes, site of fertilisation
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7
Q

What is the specific protein in mice that is required for sperm to pass through UTJ? Is this a crucial protein in humans as well?

A
  • Mice require presence of a specific protein to be recognised and to pass through UTJ - ADAM3. it is a glycosylated membrane protein present on surface of sperm
  • in mammals: ADAM3 is not as critical, but it is known that in order to get through UTJ sperm need to be live, motile, normal morphology, uncapacitated, acrosome intact. Once the sperm is through UTJ, they will bind to the cilia of isthmus and will be able to rest, where they can stay for several days.
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8
Q

What are the 3 stages of fertilisation?

A
  1. sperm preparation
  2. sperm binding and fusion
  3. cortical reaction
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9
Q

What is the first stage of sperm preparation? What happens during it?

A

Sperm preparation - capacitation:
- ejaculated sperm need to acquire capacity to fertilise
- takes place in female reproductive tract
- triggered by alkaline environment
- physiological and molecular changes
- plasma membrane reorganisation: loss of cholesterol, phospholipids and glycoproteins on surface
- increase in ROS generation, Ca2+ influx, tyrosine phosphorylation
- sperm become less stable but have increased motility and ability to respond to chemoattractants
- visible sign: whiplash movements of tail (hyperactivation)

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10
Q

Where does capacitation take place?

A

female reproductive tract

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11
Q

What is capacitation triggered by?

A

triggered by alkaline environment

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12
Q

How is plasma membrane of sperm reorganised during capacitation?

A

loss of cholesterol, phospholipids and glycoproteins on surface

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13
Q

What are the visible signs of sperm capacitation?

A

whiplash movements of tail – hyperactivation

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14
Q

What are the stages of sperm capacitation?

A
  1. insemination (non-capacitated sperm)
  2. initial capacitation – as they start to go through slightly alkaline pH in uterus
  3. creation of a transient sperm reservoir in the isthmic region of the oviduct
  4. hyperactivation – generates propulsive forces necessary to: pull sperm away from oviduct epithelium and penetrate the dense cumulus complex
  5. sperm penetration of the cumulus mass
  6. zona penetration
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15
Q

Why is hyperactivation important?

A

generates propulsive forces necessary to:
- pull sperm away from oviduct epithelium and
- penetrate the dense cumulus complex

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16
Q

What is the 2nd stage of sperm preparation? What happens during it?

A

Acrosome reaction - fusion of sperm plasma membrane and outer acrosomal membrane (OAM) creates pores
- Acrosome: at the head of the sperm, filled with proteolytic enzymes – acrosin, trypsin, hyaluronidase (to get through sticky hyaluronan) and proteases
- induced by progesterone (from follicular fluid)
- releases enzymes
- exposes new set of surface antigens (e.g. Izumo1) to bind to oocyte (oolemma)
- only capacitated sperm can undergo acrosome reaction and only acrosome-reacted sperm can bind to the oolemma

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17
Q

What enzymes does acrosome contain?

A

Acrosome: at the head of the sperm, filled with proteolytic enzymes – acrosin, trypsin, hyaluronidase (to get through sticky hyaluronan) and proteases

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18
Q

What induces acrosome reaction?

A

progesterone (from follicular fluid)

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19
Q

What is exposed on the sperm after acrosome reaction?

A

exposes new set of surface antigens (e.g. Izumo1) to bind to oocyte (oolemma)

20
Q

What is the importance of zona pellucida?

A
  • mediates species-specific recognition (protein glycosylation with N- and O- glycans)
  • precents polyspermy
  • protects preimplantation embryo from reabsorption
21
Q

How does binding to the zona pellucida take place?

A

Difficult to identfiy ZP protein for sperm recognition, current view: ZP2 (N-terminal domain) as the primary ligand via unidentified sperm receptor.
Multimolecular event - requires the involvement of sperm surface receptors with complementary ZP carbohydrates

22
Q

What structure on ZP proteins confers to species specificity?

A

Carbohydrate residues on ZP proteins confer species specificity

23
Q

What are the responible proteins for sperm-oolemma binding?

A
  • Izumo1 protein on sperm and Juno receptor on oolemma;
  • CD9 and CD81 don’t actually bind but stabilize.
  • CD9 required for normal microvilli biogenesis and organisation. Mediates and stabilises the binding.
  • CD81 stabilises binding of JUNO and IZUMO1
24
Q

How does fusion of sperm and oocyte happen?

A
  • microvilli on oolemma engulf the sperm starting at the equatorial segment
  • anterior is engulfed by pinocytosis
  • spermatozoon stop moving
  • swelling (fertilisation cone) forms at the point of fusion
  • sperm head passes into the oocyte cytoplasm
  • two gametes have now fused

for diagramm check L11, slide 15

25
Q

What is cortical reaction? Why is it important?

A
  • Cortical reaction prevents other sperm from entering the egg,
  • cortical granules fuse with plasma membrane, contents released into perivitelline space.
  • Granules contain proteases, peroxidases, polysaccharides and zinc.
26
Q

What is perivitelline space?

A

between oolemma and zona pellucida

27
Q

What are the post-fertilisation blocks to reduce polyspermy?

A
  1. Zinc shield (few min) - start to harden zona pellucida, it can also dysregulate zinc signalling in the sperm
  2. Membrane block (40 min) – JUNO vesicles are shed from the membrane into perivitelline space
  3. zona block (4h) – zona pellucida starts to harden, basically cleaving one of the ZP proteins
28
Q

How does the membrane block happen?

A
  • JUNO receptor is gradually shed from plasma membrane and relocated within vesicles with PV space
  • reorganisation of the microvilli distribution
  • two fold decrease in CD9 protein
29
Q

How does zona pellucida block happen?

A

Ovastacin (from cortical granules) cleaves ZP2 (hardens zona):
- oligosaccharides removed from ZP3
- adjacent ZP molecules are cross-linked

  • Fetuin-B inhibits ovastacin and maintains ZP permeability until after gamete fusion
  • Premature ZP hardening can cause infertility in mice
  • Cortical granules are leaky, so Fetuin-B helps avoid premature ZP hardening
30
Q

What is polyploidy? What happens during it?

A
  • embryo lethal in mammals
  • vast majority of polyploid embryos die during cleavage stage or by 1st trimester
  • mismanagement of centrosomes - multiple spindles form, results in asynchronous cell divisions
  • some species (e.g. birds) can tolerate polyspermy
  • extremely rare case of semi-identical twins (sesquizygosity). Egg fertilised by two sperm which then divided to form two embryos
31
Q

How is oocyte activated to finish meiosis? What happens during oocyte activation?

A
  • sperm releases sperm-derived oocyte activating factor (phospholipase C zeta 1)
  • stimulates release of calcium from intracellular stores, re-fill and empty again
  • results in a series of rapid intracellular calcium spikes, continues for a few hours after fertilisation
  • stops then PN (pronucleus) form

calcium oscillation orchestrates a series of events:
- cortical reaction
- resumption of meiosis
- 2nd polar body extrusion
- translation of maternal mRNA
- Without this calcium influx, fertilisation would fail

32
Q

At which stage of meiosis is oocyte arrested during ovulation?

A

metaphase II

33
Q

What happens in female pronucleus upon the formation of the zygote?

A
  • resumption of meiosis
  • sister chromatids are segregated, and half are eliminated into 2nd polar body
  • maternal chromosomes enclosed in nuclear membrane and decondensed
34
Q

What happens in male pronucleus upon formation of the zygote?

A
  • sperm undergoes dramatic transformation
  • nuclear membrane forms around sperm DNA
  • sperm DNA decondensed and actively demethylated
  • histones replace protamines in sperm chromatin
35
Q

What are protamines?

A

nuclear proteins wrapped around the DNA. Sperm DNA tightly packed and inactive.

36
Q

What are the stages of pronuclear formation in mouse? Briefly explain what happens.

A
  • PN1- female pronucleus on the side to polar bodies, male pronucleus in an opposite pole of the cell
  • PN2 - pronuclei enlarging
  • PN3 - pronuclei enlarging, getting closer to the middle
  • PN4 - pronuclei enlarging, getting closer to the middle
  • PN5 - pronuclei not joining but overlapping
37
Q

How does first mitotic division and cleavage happen?

A
  • as male and female pronuclei migrate to the centre, DNA in both pronuclei is duplicated (separately) takes approx. 12h
  • pronuclear envelope breaks down and the chromosomes of both pronuclei align on first mitotic spindle
  • cleavage to form the 2-cell embryo
  • process takes 24h
38
Q

What is defined as preimplantation period?

A

Preimplantation is defined from 1-cell stage up to late blastocyst stage (>100 cell stage)

39
Q

What is compaction?

A

takes place during preimplantation period, cells start to merge together, cannot differ between individual cells

40
Q

What is the potency of the cells till morula stage?

A
  • Cells are highly totipotent till morula stage, can form any cell, even placenta
41
Q

What is the potency of ICM?

A
  • ICM is pluripotent – forms in morula and blastocyst – can form any cells, except for placenta, placenta needs to come from trophectoderm cells
42
Q

When does zygotic genome get activated?

A
  • at zygote maternal genome is fully responsible
  • in humans: zygotic genome gets activated at around 6-8 cell stage
  • before that it relies on maternal protein transcripts, that’s one of the reasons why the egg is so big
43
Q

How and when does cavitation happen?

A
  • stage of blastocyst formation
  • starts at 16-32 cell stage as small intracellular fluid-filled vacuoles appear in the outer trophectoderm
  • at 32 cell stage fluid initially accumulates in small cavities between inner cells
  • at 64 cell stage fuse to form one large blastocele cavity that compresses ICM to one side
44
Q

What is the mechanism of cavitation?

A

Fluid accumulation requires:
- Na+/K+ ATPase pumps
- tightly sealed epithelial trophectoderm
- aquaporins

TE undergoes epithelialisation: maturation of tight junctions (=) seal TE

  1. Na+/K+ pumps in TE cells establish an ion gradient
  2. drives water uptake through aquaporins in the TE
45
Q

What are the main primary lineages in blastocyst?

A
  • epiblast
  • primitive endoderm
  • trophectoderm (mural - next to cavity, polar - next to ICM)
46
Q

What are the destinies of the blastocyst lineages?

A
  • epiblast - fetus
  • trophectoderm - placenta
  • primitive endoderm - yolk sac

Reproductive Biology 3 (21 decks)

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