Protocol - Paracentrotus lividus gametes preparation and egg fertilization
Paracentrotus lividus gametes isolation for fertilization and embryos development
Category:
In vivo experiments
Last revision: Dec 18, 2014
Contact
Name: Marta Di Carlo
Address: IBIM - CNR via Ugo La Malfa, 153, Palermo
Phone: 0916809538
Email: di-carlo@ibim.cnr.it
Figure legend:
Paracentrotus lividus fertilized egg.
After the contact of the first sperm the fertilization membrane is elevated to avoid polyspermy
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Paracentrotus lividus gametespreparation
|
|||
|
Open sea urchins with a scissor, remove the gonads with a teaspoon and put female gametes in a beker with 50ml of chilled seawater and male gametes in a beker without seawater
|
R.T.
|
||
|
Stir gently female gonads and place a drop of eggs suspension on a slide and examine with a 10x lens at microscope both morphology and the possibility to be already fertilized
|
R.T.
|
||
|
After eggs checking, the content of the beker is poured in a larger beker with 20 ml of seawater covered with a membrane for filtering eggs but no gonads.
|
R.T.
|
||
|
Transfer eggs in a centrifuge tube and centrifuge at 2000 rpm (for 2 min)
|
R.T.
|
2 min
|
|
|
Discard the supernatant, resuspend gently the pellet in seawater and centrifuge at 2000 rpm (for 2 minutes)
|
R.T.
|
2 min
|
|
|
Wash eggs in seawater twice
|
R.T.
|
||
|
Pour gently eggs in 30 ml (+/-4 ml )of seawater
|
R.T.
|
||
|
Eggs can be kept at 4°C or fertilized
|
R.T.
|
||
|
PARACENTROTUS LIVIDUS EGG FERTILIZATION
|
|||
|
Take with a pasteur pipette a small amount (5-10 microl) of spermatozoa directly from male gonad and put them in a beker with 50 ml of chilled seawater and stir it around a bit.
|
R.T.
|
||
|
Place a drop of sperm suspension on a slide without a coverslip, and examine it with a 20x lens to verify if the sperms are motile
|
R.T.
|
||
|
Add 1 ml (or one Pasteur pipette-full) of dilute sperm to the settled eggs, and stir them up.
|
R.T.
|
||
|
Wait
|
R.T.
|
2-3 minutes
|
|
|
Check a sample of the eggs under a microscope for the development of fertilization envelopes
|
R.T.
|
||
|
Centrifuge at 2000 rpm (for 2 min) for removing the excess of sperm
|
R.T.
|
2 min
|
|
|
Resuspend fertilized eggs in seawater (at 5000 embryos/ml) to permit embryos development
|
R.T.
|
||
|
PARACENTROTUS LIVIDUS EGG FERTILIZATION IN PABA
|
this procedure is used when, for experimental reasons, it is necessary to remove fertilization envelope
|
||
|
Place eggs collected from the female in 50 ml of chilled seawater containing PABA (2mM f.c.)
|
R.T.
|
||
|
Take with a pasteur a small amount (the 5-10 microl) of spermatozoa directly from male gonad and put them in a beker with 50 ml of chilled seawater containing PABA (2mM f.c.)
|
R.T.
|
||
|
Add 1 ml (or one Pasteur pipette-full) of dilute sperm to the settled eggs, and stir them up.
|
R.T.
|
||
|
Wait
|
R.T.
|
2-3 minutes
|
|
|
Repeat
|
R.T.
|
3-4 times
|
|
|
Wait
|
R.T.
|
2-3 minutes
|
|
|
Check a sample of the eggs under a microscope for the development of fertilization envelopes
|
R.T.
|
||
|
Centrifuge at 1600 rpm (for 2 min) and resuspend the pellet in seawater and transfer it in a beker containing 50 ml of chilled seawater
|
R.T.
|
2 min
|
|
|
Eliminate the fertilization membrane by getting through an appropriate syringe (50ml) in ice the fertilized egg suspension
|
R.T.
|
||
|
Check a sample of the eggs under a microscope to monitor removing of the fertilization envelope
|
R.T.
|
Other informations
Timetable of Paracentrotus lividus development at 16°C (+/- 2°C)
0 hr. Fertilization
~ 80 min. First cleavage.
~ 105 min. Second cleavage.
~ 130 min. Third cleavage.
2.5 hr.4 th cleavage
4hr. 6 th cleavage
7.5 hr. Hatched Blastula
9- 9.5 hr. Mesenchyme blastula
13.5 -14 hr. Gastrulation
19 hr. Prism stage
24 hr. Pluteus larva
Quality validation:
Yes
Validation info
The protocol has been published in a book and peer-reviewed journals.
| Citations |
|---|
| Di Carlo M, Romancino DP, Montana G, Ghersi G. Spatial distribution of two maternal messengers in Paracentrotus lividus during oogenesis and embryogenesis. Proceeding National Academy Science USA. 1994;91:5622-5626. |
| Giudice G. The sea urchin embryo. Springer-Verlag, Germany (1986). |
