This video aims to give you the basic knowledge about how to acquire material
for further experimentation.
But before we start, I think it's important to make clear why sea urchin eggs and early
development represent interesting systems for this purpose.
Unfertilised eggs are physiologically blocked in G1.
Fertilisation triggers the entry into the cell cycle with DNA duplication and the
first mitotic divisions, and mitotic division is rapid and synchronous.
Sea urchin eggs are therefore powerful systems for studying the cell cycle in
physiological conditions, as opposed to having to synchronise them with drugs,
which is the case for cultured cells.
The main objective of the first video is to show how to handle sea urchins and
obtain gametes. We will show you how to set up a classic fertilisation experiment
using one of our favourite models, the purple or edible sea urchin
Sphaerechinus granularis.
For this kind of experiment, we classically need some natural filtered sea water,
a piece of gauze,
a pair of scissors,
needles and a syringe,
we also need a few transfer pipettes, some 50ML plastic tubes,
two beakers,
a graduated cylinder,
some Petri dishes,
a few 1.5 ml Eppendorf tubes,
a solution of Acetylcholine at the concentration of 0.1 molar,
and a solution of Glycine at 10% concentration in natural filtered sea water.
We will need a set of micropipettes,
and the corresponding tips.
We will also need a low-speed centrifuge.
Now we need two sea urchins, one male and one female.
Here we are in the historic room, called the research aquarium, where the animals are
kept for various experiments.
Sea urchins are kept in tanks containing natural sea water.
For our experiment we will use the purple sea urchin Sphaerechinus granularis.
The sex of the animals can be defined beforehand by producing the release of
some gametes by a weak electric shock.
Males and females are kept in culture in different tanks.
After taking a male and female, sea urchins are brought to the laboratory where we can
proceed to the experiment. It is pretty easy to distinguish the dorsal region
from the ventral region.
The dorsal part has the anus in the central region and around are five gonopores from
which the gametes will be expelled.
The ventral part presents the mouth where the ends of the masticatory apparatus
called the lantern of Aristotle is visible. Around the mouth is the
peristomal membrane which the needle can cross.
We inject 1ml of 0.1 molar acetylcholine into the body cavity for the peristomal
membrane to induce spawning.
Then we invert the female on the beaker of natural filtered sea water and we have
just to wait for a little to see the release of the gametes.
If we observe orange cells, then we can be sure that we have a female.
We proceed in the same manner to obtain the gametes from the male.
We inject acetylcholine into the general cavity of the sea urchin,
and after injection we shake it gently and we wait for little. If we observe a
white or milky release, we can say that we have a male.
We invert the male on an empty Petri dish
and collect undiluted sperm that we called "dry sperm".
This dry sperm is collected using the micropipette
and transfered into a 1.5 ml Eppendorf tube.
This dry sperm is then put on ice.
It can be stored in the fridge before use for several days.
At the end of this step we obtain many gametes that we can now use for
different experiments.
Before proceeding with fertilisation, it is necessary to remove the faeces or spines
that have fallen with the eggs.
For this purpose, we will proceed to a rapid filtration through gauze.
A piece of gauze is cut and is placed on the top of a Falcon tube.
The eggs are then gently filtered through the gauze.
At the end of this filtration the gauze is removed.
Then the tubes are filled up to 50ml with natural filtered sea water,
and using a swinging rotor, the eggs are centrifuged at a low speed,
2,000 runs per minute.
After centrifugation, we can see the eggs pelleted at the bottom of the tube.
And after several washes, the eggs are diluted to a final concentration
of 5% of egg suspension.
We can now proceed to the fertilisation of the eggs.
Around 10 millilitres of the 5% egg suspension are transferred into a
Petri dish, and it will be used for the fertilisation experiment.
Dry sperm needs to be diluted before use. For this, 1ml of natural filtered sea water
is transferred into an Eppendorf tube.
50 microliters of the dry sperm initially kept at 4°C are transferred into
a tube containing sea water.
The diluted sperm suspension is then carefully homogenized using the micropipette.
The diluted sperm is added to the egg suspension at the final concentration of
2 microliters of diluted sperm with 1mL of 5% egg suspension. It is important not to
add too many sperm to avoid polyspermy
that will affect the correct dynamics of the embryonic cell cycle.
Once sperm have been added to the eggs,
we can start the chronometer, and add the experiment's start time.
Fertilised eggs can be easily monitored directly under a light microscope.
Fertilisation envelope elevation occurs within one minute after sperm-egg fusion
and acts as a mechanical block to polyspermy.
This even represents the first visible sign that the egg activation is mediated by the sperm.
Following a population of several eggs, it is remarkable to observe that the first
mitotic division occurs synchronously two hours following fertilisation.
This natural and synchronous division is essential for cell cycle studies.
At a higher focal length, this time lapse makes it possible to follow the first three divisions.
Two hours after fertilisation, we observe the two blastomere stage.
One hour later, we observe the four blastomere stage,
and finally, the eight blastomere stage is visible three hours following fertilisation.
You now know how to obtain and use the gametes for triggering fertilization of
the eggs and early embryonic development of sea urchins.
Before coming to the practical, please remember the three following points:
First, the animal is easy to handle, and it is simple to obtain a large number
of eggs from a single female.
Second, eggs are physiologically blocked in G1,
and fertilisation triggers entry into the cell cycle.
Finally, mitotic divisions of an egg population are synchronous.
This last point allows us to develop many different biochemical approaches to
analyse protein synthesis in relation with cell cycle regulation.