Fertilisation journey Flashcards
During what some key molecular changes occur?
The sperms journey through the reproductive tract.
Why do the sperms go through a journey through the reproductive tract?
In order to become fertilisation competent.
Where are sperms restored with successful spermatogenesis?
In the epididymis.
Until when are the sperms restored in the epididymis?
Ejaculation.
Die.
From what must the sperms release themselves?
The semen.
What must the sperms begin to do after they release themselves from the semen?
The challenging journey of navigating the female tract.
Why do sperms must begin the journey of navigating the female tract?
To find the egg.
How many eggs can be successful?
1.
How does the massive rubbing rate going?
From hundreds of millions in the vagina to just a few in the upper tract.
What does each part of the tract represent?
Its own challenges.
Why are the sperms specially designed?
To overcome their challenges in the co-evolution process.
How many excess phenomenon are observed in vitro?
3.
Why are the three excess phenomenon in vitro proposed?
To facilitate sperm journey to the egg.
What might exist along the tubule?
A temperature gradient.
What might the sperm do in response to a temperature gradient?
Swim up.
How is the process of the sperm swimming up to the temperature gradient called?
Thermotaxis.
From where are steroids released?
The cumulus-oocyte-complex.
What do the steroids stimulate?
Calcium influx.
Sperm motility.
What happens in the process of chemotaxis?
Sperm swim up the concentration gradient towards the egg.
From where are chemical released?
The tubules.
What do chemicals facilitate?
Sperm swim up the concentration gradient towards the egg.
What are the sperms observed to do?
Swim against a fluid flow.
What mechanism is the swimming of sperm against fluid flow?
Rheotactic = rheumatoid.
Where does the rheotactic mechanism help the sperm?
Swim against the fluid that maybe moved by the cilia of the tubule in the uterus direction.
What is a spermatozoon?
A stripped down.
A refined.
–> machine.
By what is motility driven?
The tail with mitochondria in the midpiece.
What are the mitochondria in the midpiece?
An ATP supply.
Of what does the head consist?
The nucleus and the acrosome.
From where is the acrosome derived?
The golgi apparatus.
What does the acrosome contain?
Enzymes.
Where do the enzymes contained in the acrosome help?
The sperm digest its way through the cumulus-oocyte-complex.
Why is it extremely difficult to replicate or advance many of the observations now about sperm transport in the female reproductive tract?
Due to ethical issues.
How the observations remain today of sperm transport in the female reproductive tract?
Important insights.
How many ejaculated human sperm out of 14,000,000 reach the oviduct?
1.
How many ejaculated human sperm reach the oocyte out of 14,000,000?
Not known.
What does the arrival of genetic engineering mean?
We can make observations in mice.
To what do the observations in mice help us?
Understand the process of spermatogenesis and fertilisation.
Why are there many limitations of making observations in mice?
Because mice produce litters.
We now appreciate that there are many fundamental molecular differences between human and mouse sperm.
What is the association between number and sperm?
The higher the number, the older the sperm.
What is the reason of the green fluorescence?
Due to the production of an acrosome-specific enzyme.
What is the acrosome-specific enzyme?
Genetically altered.
Why is the acrosome-specific enzyme genetically altered?
To have a GFP tag on it.
Why must the acrosome-specific enzyme have a GFP tag on it?
So it can be seen under a fluorescent microscope.
Where is a single sperm bound?
To the zona pellucida.
Where does the single sperm lie?
On its side.
Not bound by the hook.
What does powerful high resolution microscopy permit?
The observation of proteins at super resolution.
What does tubulin form?
The main structure of the tail.
What is not the tubulin?
Naturally concentrated down the middle.
Where is the glucose transporter found?
In the plasma membrane.
How is the glucose transporter distributed in the plasma membrane?
Evenly.
What does the sperm specific calcium channel CatSper have?
A very distinct spatial resolution along the plasma membrane.
How many stripes does the sperm calcium channel CatSper form down the tail?
4.
Why and how the 4 stripes down the tail of the sperm specific calcium channel CatSper occur?
It is unknown.
How do the sperm swim in normal media?
Normal.
How can azoospermia be diagnosed?
By using the software.
Where can sperm swim?
In a viscous medium.
How is the sperm tail movement characterised?
Elegant.
What does the sperm do as it moves forward?
It rotates.
By what is the sperm driven when it is rotating?
The tail.
What is the tail movement?
It is rolling around a central point.
To what does the sperm tail not move?
Site-to-site.
How is the semen composition characterised?
Complex.
What does semenogelin give to the semen?
Its gelatinous form.
o what is the semenogelin inhibitory ?
To sperm function.
How does the enzyme PSA act?
To degrade the semenogelin.
Help liberate the sperm.
To where are the sperm exposed during storage?
Fluids of lower pH.
To where are the sperm exposed during storage?
Fluids of lower pH.
Where does lower pH help?
Suppress motility.
What is bicarbonate?
A buffering component.
Where are the buffering components added to at ejaculation?
Raise the pH.
What is Zn?
An important ion.
Where does Zn have a role?
In DNA compaction.
PSA, SOD, NOS activity.
As what can fructose and glucose act?
Energy substrates.
Where might prostaglandin have effects on?
Sperm.
Female tract.
What does Zn inhibit?
Hyperactivation.
Capacitation.
When do ion concentrations radically change?
When the sperm are ejaculated in the seminal fluid and in the female tract.
Where is K+ significantly reduced and the Na+ and HCO3- concentrations are significantly increased?
In the female tract.
For what is the regulation of intracellular calcium ion concentration critical?
Regulating the sperm function.
By which factors is semen composed?
Sperm. Semenogelin. PSA. pH buffering components. Ions: citrate, Zn, Ca, Na, Cl, K, Mg. Fructose/glucose. Prostaglandins.
What do superoxide and NO control?
cAMP levels.
Changes in protein phosphorylation.
Nitration.
By what is ROS generation controlled?
Ca.
How is the regulation of intracellular calcium levels characterised?
Critical.
What are some important processes that are necessary in order to permit motility?
Elevated pH - sufficient alone to activate, in vitro.
Semenogelin to breakdown.
Generation of ROS.
For what is elevation of pH vital?
Sperm activation.
When does pH elevation occur?
At ejaculation as the sperm are mixed with the seminal fluids.
What does increasing pH stimulate?
Sperm metabolism.
Why does increasing pH stimulate sperm metabolism?
To supply the cell with the energy required for motility.
What do calcium ions directly activate?
The machinery in the tail.
What effect do calcium ions have when they activate the tail machinery?
A hyperpolarising effect.
Where do calcium ions have a hyperpolarising effect?
On membrane potential.
Why do calcium ions have a hyperpolarising effect on membrane potential?
Due to activation of the calcium-sensitive potassium channel.
What do sperm also have?
A channel.
How is the channel of sperm called?
A proton channel.
HV1.
Where does the proton channel of sperm occur?
In the tail.
What does the proton channel of sperm allow?
Hydrogen ions to leave.
Why does the proton channel of sperm allow hydrogen ions to leave?
To help raise the cytoplasmic pH.
How is pH elevated?
Combination of proteins, ionic buffer, bicarbonate > 5-25mM.
What does alkalinisation stimulate?
Sperm metabolism.
What does calcium influx activate directly?
The axoneme.
What does calcium influx hyperpolarise?
The membrane.
What does recombinant Sg inhibit when added to sperm, over time?
Motility.
How where the experiments done?
By using purified seminal vesicle extracts.
What do the graphs of testing show?
A drop in motility.
A speed of swimming over time.
What does Zinc stabilise?
Sg.
How does Zinc stabilise Sg?
Due to its inhibitory action on PSA.
Where is there a delay upon ejaculation?
In liquefaction.
Why is there a delay in liquefaction in ejaculation?
As the zinc ions must diffuse away.
What do the zinc ions allow when they diffuse away?
The PSA to become active.
How is the Sg protein characterised?
Very influential.
Where does Sg protein affect?
On motility.
Capacitation.
What properties does PSA protein have?
Antibacterial.
How are the antibacterial properties characterised?
Beneficial.
For what are the antibacterial properties of Sg protein beneficial?
For the sperm in the vagina.
For cervical regions.
Where does semenogelin (Sg ) protein originate?
In seminal vesicle.
What does Sg constitute?
The major protein type in semen.
What happens to the sperm motility in the absence of calcium?
It runs down over time.
For what are calcium ions essential?
For maintaining activity.
How can we become a scientist abut regulation in sperm?
Questions about calcium.
- Observe phenomenon: data collection/analysis.
- Interpretation.
- Hypothesis.
From which 2 sources can calcium come?
Intra.
Extracellular.
What are sperm proposed to have?
A calcium sore in the region of the RNE.
Where does RNE sit?
At the base of the head.
With what are sperm loaded?
A calcium sensitive dye.
Along where is the staining for CatSper occur?
The tail.
What is CatSper?
An ion channel.
what is CatSper responsible?
Controlling extracellular calcium influx.
Under what influence does CatSper control extracellular calcium influx?
The influence of progesterone and some prostaglandins.
What do agonists (progesterone and prostaglandins) activate?
The channel.
What do agonists (progesterone and prostaglandins) activate?
The channel.
Why do agonists activate CatSper channel?
To cause a large increase in intracellular ion concentration.
How many methods are there, to measure Ca in cells?
2.
Which are the 2 methods to measure Ca in cells for?
Studying calcium handling of cells.
Which are the 2 methods for studying calcium handling of cells?
- Use of Ca sensitive fluorescent dyes.
2. Ca sensitive electrophysiology (ephys).
What does fluorescence involve?
Measurement of population of cells/single cells.
For what is the method of fluorescence?
Measuring populations of cells.
How many wells does one plate have?
96.
What does each well of a 96 well plate contain?
Sperm loaded with a Ca-sensitive dye.
What does the addition of progesterone activate?
A calcium influx.
By what can a calcium influx be measured?
The detector.
The trace plotted on a graph with time on the x-axis.
Change in fluorescence relative to baseline on the y-axis.
What data does the single cell imaging give?
‘Messier’.
More insightful.
What does added progesterone give to each cell?
A unique response.
What can cells show in intracellular calcium before addition?
Oscillations = ταλαντώσεις.
What is the pattern of oscillations before and after addition of P4 for all cells?
Different.
What is consistent about the cells to progesterone addition?
They all give an immediate response to progesterone.
How does the trace look, when combining the data from all cells that show oscillations after the addition of P4?
More regular.
With clearly defined calcium oscillations.
What is the challenge in progesterone addition to cells and measuring of oscillations?
Working out why only a small proportion of cells do this.
The purpose of them.
What is now widely recognised?
That CatSper is sensitive to Progesterone.
What are some prostaglandins and oestrogen and the channel responsible for?
The characteristic immediate increase in fluorescence upon addition of these agonists in the plate reader experiments.
How is the mechanism of how progesterone and oestrogen activate CatSper characterised?
Still not clear.
What do prostaglandins also activate?
CatSper.
Through what do prostaglandins activate CatSper?
A different mechanism.
How does calcium entry occur?
Through plasma membrane ion channel = CatSper.
What do mouse KO experiments show?
It is needed for in vivo and in vitro fertilisation of zona intact eggs.
What does not occur with the lack of CatSper?
Fertilisation.
What does removal of zona restore?
Fertilisation.
What is one proposed functional deficit in CatSper-null mouse sperm?
The loss of hyperactivated motility.
With what use is the characteristic wide flagella beat pattern observed?
When using CASA.
What happens to the wide flagella beat pattern in CatSper-null sperm?
Is lost.
How are the experiments of sperm in humans characterised?
Impossible.
Where do we must rely on?
On finding men with mutations of CatSper but can still produce otherwise normal sperm.
How is this challenge of CatSper experiments in humans characterised?
Serious.
Why is CatSper in humans a serious challenge?
Because it requires men to consent to research and attending a clinic that is actively involved in research of CatSper function.
What happens in human sperm experiments?
Need to find a natural ‘knock out’.
Logistical and ethical issues.
What happens in the intracellular calcium response to progesterone in normal donor sperm and sperm from 2 failed IVF patients?
There is no response to progesterone in either case.
A flat line.
Where does the missing of 6 base pairs in epsilon gene of CatSper result?
In a reading frame shift.
What does a reading frame shift cause?
The loss of channel function.
What does progesterone not stimulate, into viscous media?
Motility.
What happens to unstimulated penetration when progesterone is added?
Is normal.
What can the sperm exhibit?
Hyperactivated motility.
How are the questions around why the sperm fail IVF characterised?
Unknown.
What is CatSper essential for?
Male fertility.
With what is a microdeletion in exon 18 of the CatSper auxiliary subunit epsilon gene associated?
The loss of CatSper function in sperm.
Where does the loss of CatSper function in sperm result?
In infertility.
Where does the fact of being homozygous for a 6-base pair in frame deletion in exon 18 of CatSper epsilon result?
In a loss of Methionine799 and Alanine800 in the putative extracellular domain of CatSper epsilon protein.
Where does the loss of Methionine799 and Alanine800 in CatSper result?
In production of ICatSper-null sperm.
What do ICatSper-null sperm not have?
Any obvious signs of sperm dysfunction.
Sperm concentration and sperm motility were normal.
To what does P4 fail?
To detach an increase in calcium concentration in cell populations.
By what was ICatSper confirmed to be absent?
By electrophysiology.
What is the consequence of loss of CatSper function?
Failed fertilisation.
What can ICatSper-null human sperm not do?
Fertilise at IVF.
What can P4 not induce?
An increase in calcium concentration.
Increase penetration into viscous media above basal levels.
Within what range was the number of cells exhibiting hyperactivation?
Within a normal range.
By what treatment can cells exhibiting hyperactivation be boosted?
With 4-aminopyridine (4-AP).
Why do molecular and cellular impairment might arise?
Due to CatSper dysfunction.
What do molecular and cellular impairment include?
Abnormal tyr/ser/thr-phosphorylation.
Deficiency of adaptive motility patterns.
Loss of zona/egg binding.
Failure of the acrosome reaction.
How are the observations in vitro characterised?
Informative.
How can the observations in vitro only serve?
As a basis for hypothesis for what happens in vivo.
What have several studies attempted to?
Literally shine a light on the mysteries of in vivo fertilisation.
How have several studies attempted to shine a light on the mysteries of in vivo fertilisation?
By using genetically engineered mouse sperm.
What is the genetically engineered mouse sperm?
An ex vivo imaging where the reproductive tracts from mated female mice are place in the microscope.
How many sperm are at the AIJ of the tubule 4 hours post coitus?
Very few.
What is AIJ region of the tubule?
The region nearest the ampulla.
Where does the sperm enter?
To the site of fertilisation.
How is the entry of the sperm to the entry of fertilisation?
Very difficult to get to.
What does the tract regulate?
Sperm entry.
How are the eggs fertilised?
Sequentially one by one from a period of 2-8 hours.
How many regions does isolated mouse oviduct have?
5.
Which are the 5 regions of the isolated mouse oviduct?
- Uterotubal junction (UTJ).
- Lower/
- Mid.
- Upper isthmus.
- Ampulla.
What does an oviduct flushed with TYH medium contain?
WGA-FITC.
What does an oviduct flushed with TYH medium show?
Many grooves created by mucosal folds.
What does a frame captured from Supplemental Movie S4 show?
A motile, GFP-negative speermatozoon in the lower isthmus.
What does a frame captured from Supplemental Movie S5 show?
A motile GFP-negative spermatozoon in the midisthmus.
Due to what was the long delay for human in vitro fertilisation success?
Due to the lack of understanding around the post ejaculation biochemical changes.
Where do post ejaculation biochemical changes must occur?
In a sperm.
Why must post ejaculation biochemical changes occur in a sperm?
For it to be able to fertilise.
How are sperm characterised at ejaculation?
Uncapacitated = Non-capacitated.
What does albumin remover, as sperm migrate along the tubule?
Cholesterol from the membrane.
What does cholesterol removal from the membrane by albumin permit?
Increased calcium and bicarbonate ion entry.
What does increased calcium and bicarbonate entry stimulate?
The intracellularly enzyme soluble adenylate cyclase.
What does intracellularly enzyme soluble adenylate cyclase use?
ATP.
Why does adenylate cyclase use ATP?
To produce cyclic adenosine monophosphate.
What does the second messenger activate?
Protein kinase A.
What does protein kinase A start?
Biochemical changes.
Where do biochemical changes from protein kinase A result?
In changes in motility pattern.
What are some changes in motility patterns that could occur due to biochemical changes?
Development of hyperactivation and wide spread protein tyrosine phosphorylation.
For what is capacitation also necessary?
For the sperm to acrosome react.
What happens in cAMP production in sperm after exposure to media containing elevated bicarbonate?
It changes rapidly.
Why is PKA important?
For capacitation.
How are PKA knock out mice characterised?
Infertile.
Sperm fail to fertilise zona-Intact eggs at IVF.
What do sperm have?
Defective hyperactivation.
What is there not in sperm , where the null mouse sperm are negative for phosphorylation proteins?
Change in protein phosphorylation.
How is calcium entry characterised in PKA knock out-mice?
Impaired.
What does the fact of the impaired calcium entry at PKA knock out mice suggest?
PKA acts to facilitate CatSper activity.
For wat is calcium essential?
Sperm function.
What does CatSper regulate?
Calcium entry.
What does progesterone regulate?
CatSper function.
What does knock out mice data show about CatSper?
That CatSper is necessary for in vivo and in vitro fertilisation.
What does removal of zona pellucida restore?
Fertilisation.
What does the fact that removal of zona pellucida restores fertilisation, mean?
That CatSper is necessary for regulating functions that are required of zona-intact eggs.
How is the reason for fertilisation failure of human CatSper null sperm characterised?
Not known.
What does CatSper represent in fertilisation?
A sperm-specific contraceptive target.
What is fertilisation for the sperm?
A massive challenge.
What target in not the egg?
Easy.
By what is the plasma membrane of the oocyte protected?
The zona pellucida.
What is zona pellucida?
A glycoprotein shell.
As what does zona pellucida serve?
A physical barrier which the sperm must penetrate.
What happens to zona pellucida after fertilisation?
It is modified.
Why is zona pellucida modified after fertilisation?
So that it cannot be penetrated by another sperm.
What is this mechanism of zona pellucida being modified after fertilisation for?
To prevent polyspermy.
Of how many proteins is the zona made?
4.
What does the sperm posse?
A binding partner.
Why does the sperm posse a binding partner?
So that it can attach to the zona.
When does the acrosome reaction begin in human sperm? Before or at zona?
It is unclear.
When do most mouse sperm begin the acrosome reaction?
Prior to contact with the zona.
What do zona proteins trigger?
A large calcium response and acrosome reaction.
To what are large calcium response and acrosome reaction necessary?
To help weaken the zona so the sperm can drill into the gaps in the filamentous structure.
By what is the weakening of zona and drilling of sperm into the filamentous structure facilitated?
By hyperactivated tail movement.
What does the acrosome do?
It swells.
What happens to the outer membrane when the acrosome swells?
Begins to be shed.
With what is the reacted sperm left when the acrosome swell and the outer membrane begins to be shed?
With the inner acrosome membrane exposed.
Where is the importance of the zona highlighted?
In women with mutations that causes the production of eggs with a very thin or no zona.
Where does the loss of zona result?
In many sperm binding to the membrane.
Why does the loss of zona results in many sperm binding to the membrane?
So polyspermy cannot be prevented.
Where does the sperm bind first?
The zona pellucida.
What does the sperm binding to the zona pellucida, the locally released progesterone and oestrogen trigger?
The acrosome reaction.
What does hyperactivated motility drive?
The sperm through the zona.
Where can the inner acrosome membrane bind to?
The out leaflet of the egg plasma membrane.
What can 2 membranes of acrosome and out leaflet of the egg plasma membrane do?
Fuse.
What does the sperm deliver into the cytoplasm, when the inner acrosome binds to the out leaflet of the egg plasma membrane?
Its cargo.
To what are the proteins necessary?
To facilitate sperm-egg binding.
What does the protein Juno on the egg membrane recognise?
Izumo on the sperm membrane.
When is Juno released?
After fusion occurs.
As what does Juno act when is released?
As a decoy for late arriving sperm.
What is the method of releasing Juno after fusion to act as a decoy for late arriving sperm for?
To prevent polyspermy.
Where do mechanisms involved in preventing polyspermy depend?
Upon egg activation.
What does egg activation involve?
Calcium waves being triggered in the egg upon sperm fusion.
What does sperm deliver to the nucleus?
An enzyme called phospholipase C zeta.
What do injection of sperm, sperm extract or recombinant PLC zeta cause?
Cyclical calcium oscillations in the egg.
What do cyclical calcium oscillations trigger?
Cortical granule release.
In what does cortical granule release result?
In the formation of a fertilisation envelope caused by the lifting of the vitelline envelope away from the egg plasma membrane.
What happens to most of the body after sperm enters the egg?
Is broken down.
Why most of the body is broken down after sperm enters the egg?
So only nucleus and centriole survive.
What does nucleus form when is decondensed?
Male pro-nucleus.
What is the eggs nucleus?
The female pro-nucleus.
For what is the sperm centriole required in most species?
For pronuclear fusion and later cleavage.
What does the sperm centriole use?
Egg-derived tubulin subunits.
Why does sperm centriole uses egg-derived tubulin subunits?
To form a microtubule aster that brings pro-nuclei together.
What does the contraction of the aster do?
It brings the pro-nuclei together.
When does the female pro-nucleus complete meiosis?
Before fusion.
What does the female pro-nucleus expel?
The second polar body.
Which phase do the male and female pro-nuclei undergo as they move towards each other?
S-phase.
How do the male and female pro-nuclei undergo mitosis?
By using the same spindle apparatus as in meiosis.
When does the first diploid nuclei form?
After the first cleavage division.
Where does sperm distribution occur?
In the uterus.
When does sperm distribution occur?
15 mins after coitus.
How often where vaginal plug examined?
Every 15 min after restricted mating.
