Fertilisation Flashcards
How many sperm are produced in an ejaculate?
20 - 300 million
What are the different mechanisms to make sure the egg only gets the best sperm?
Acidic pH and leukocytes in the vagina
Cervical mucus traps poor motile sperm
Rheotaxis were the sperm are swimming against the current
Chemotaxis trying to destroy the sperm and makes it warmer
What is the role of the oviduct in fertilisation - the different parts of the oviduct?
Utero-tubul junction - sperm selection and reduction
Isthmus - stores sperm
Ampulla - hold oocyte and site of fertilisation
How is sperm selected in the uterotubul junction in mice?
The protein ADAM3 must be recognised in order for sperm to pass through. This is found on the surface of the sperm and we think it binds to the junction.
The sperm needs to be healthy e.g. motile, have acrosome, uncapcicated,
What happens to sperm in the isthmus region?
They can rest by binding to the cillia
What are the 3 stages of fertilisation?
Sperm preparation
Sperm binding and fusion
Corticol reaction
Sperm preparation - what is capacitation?
Ejaculated sperm need to go through this to fertilise. Takes place in the female reproductive tracts and is activated by its alkaline environment.
Brings about physiological and molecular changes including loss of cholesterol, phospholipids and glycoproteins. Increases ROS generation, calcium influx, tyrosine phosphorylation
What happens to sperm after capacitation?
They become less stable but have increased motility and can respond to chemoattractants (they are hyperactive).
What triggers capacitation?
Endocrine signal at ovulation induces change in sperm
Why do sperm need to be hyperactive?
Helps pull the sperm away from oviduct epithelium (isthma)
Penetrate dense cumulus complex
Sperm preparation - what happens during the acrosome reaction?
Progesterone induces sperm plasma membrane and outer acrosomal mebrane fuse to create pores which releases enzymes and exposes new set of surface antigens which can bind to the oocyte
What enzymes are in the acrosome?
Proteolytic enzymes, acrosin, trypsin, hyaluronidase and proteases which are needed until this point to get through the uterus
Can uncapacitated sperm undergo acrosome reaction?
No
What does the zona pellucida do?
Mediated species-specific recognition, prevents polyspermy and protect preimplantatin embryo from reabsorption.
Made of Zp1, ZP2, ZP3, and ZP4 in humans
How does the sperm get through the zona pellucida?
ZP2 is the primary ligand on the egg but we dont know about the sperm. It is however more likely to be a multimolecular event.
Once bound it digests its way through using the acrosome enzymes
Once the sperm gets through the zona pellucida what happens (sperm binding to oolemma)?
In the perivitelline space the izumo1 (ligand exposed after acrosome reaction) on the sperm binds to the Juno receptors on the oolemma.
CD9 and CD81 stabilise this complex
How does the sperm and oocyte fuse?
Microvilli on the oolema engulf the sperm, the anterior in engulfed by pinocytosis and the spermatozoon stops moving. The fertilisation cone forms at point of fusion and the sperm head passes into the oocyte cytoplasm.
How does the oocyte stop polyspermy? (cortical reaction)
Corticol granules fuse with plasma membrane and contents are released into the perivitelline space. These contain proteases, peroxidase, polysaccharides and zinc.
What are the blocks set up by the corticol reaction?
Zinc sheild - short transient sheild hardens the zona pellucida and disregulates the zinc signalling in the sperm.
Membrane block - juno vesicles are shed into the perivitelline space and any other sperm bind to it.
Zona block - zona pellucida hardens by cleaving one ZP
These all take different amounts of time
How does the membrane block work?
Juno receptor is shed and relocated. The microvilli are reorganised and there is a decreased in CD9 production
How does the zona pellucida block work?
In the cortical granules there is a protein - ovastacin - which cleaves ZP2 hardening the zona.
Oligiosaccharides removed from ZP3 and adjacent ZP moleculars are cross-linked.
What inhibits ovastacin until gamete fusion?
Fetuin-B to keep the ZP permeable because premature ZP hardening can cause infertility (when fertilisation happens the ovastacin released in huge so over rides this mechanism).
What is polyploidy?
Multiple copies of the genome which is embryonic lethal.
This occurs from mismanagement of centrosomes (multiple spindles form resulting in asynchronous cell division).
Extremely rare in semi-indentical twins (egg fertilised by 2 sperm and split).
Can some animals cope with polyploidy?
Yes mainly birds
How does oocyte activate?
Sperm gives the egg a oocyte activating factor which stimulates release of calcium stores resulting in rapid intracellular spiked of calcium (oscillations).
What do the calcium spikes do?
Release cortical reaction
causes 2nd polar boyd
Translation of maternal mRNA
Resumption of meiosis in the egg
Without this fertilisation would fail
What does the zygote have?
It has 2 pronuclei - male and female
What happens in the female pronucleus?
Meiosis resumes and sister chromatids are separated making 2nd polar body.
Maternal chromosomes enclosed in nuclear membrane and decondensed
All the pronuclear changes are caused by enzymes in the egg
What happens in the male pronucleus?
Nuclear membrane forms around sperm DNA
Sperm DNA decondenses and actively demethylates
Histones replace protamines in sperm chromatin
The pronuclei are small at either side of the zygote before growing and moving towards one another. Whilst this happens what is going on inside both pronuclei?
DNA in both nuclei duplicate, the pronuclear envelope break when they reach one another and the chromosomes of both pronuclei align on first mitotic spindle and cleave to form a 2 cell embryo
Do the cells go through many stages before the blastocyst stage?
Yes
Does the zygotic genome need to be activated?
Yes and until then there is maternal transcripts keeping the cell alive. These are on until the zygotes genome turns them off
What is the cavitation stage?
The embryo is the same size after the pronuclei has joined, during this stage the egg grows. Fluid filled vacuoles form inbetween the inner cells and eventually becomes one large cavity called the blastocoele cabity that compresses the Intra Cellular Mass to one side
What is the mechanism of cavitation?
Fluid accumulation requires sodium and potassium pumps, tightly sealed epithelial and aquaporins.
TE undergoes epithelialisation maturation of tight junctions.
1) Sodium/potassium pumps in TE cells establish ion gradient
2) Drives water uptake through aquaporins in the TE
What is the final stage of blastocyst development?
The epiblast, primitive endoderm and the trophectoderm differentiate within the zygote (the trophectoderm is now called the polar trophectoderm and the mural trophectoderm).
How does the embryo get out of the blastocyst?
A little split appears in the blastocyst and it breaks free
What does the epiblast become?
Fetus
What does the primitive endoderm become?
Yolk sac
What does the trophectoderm become?
Placenta
