Developing a Developmental Lab #4: Sea Urchin Fertilization

Welcome to my series documenting my trials and tribulations as I foray into academia.  I’ve recently been put in charge of a college-level course in developmental biology.  I do have a colleague handling the corresponding lecture, but ultimately the responsibility for the labs falls on me.  Labs are three hours long and are held once a week for 13 weeks total. 

Designing this course has been fun, but challenging.  The course hasn’t really been run before and most of the labs have to either be created from scratch or modified from protocols found within the Bio department or by searching the internet.  It's also hard to think of an appropriate lab that’s specific to developmental biology, but doesn't overlap with typical labs in a molecular biology, genetics, or anatomy course and fits within three hours.  I haven't been able to find many resources online for creating this type of course so I thought I'd post my labs on SteemSTEM after I make them.

In past weeks, the students have experimented on human placenta and living chick embryos.  This week we've moved onto a new organism:  The sea urchin.

Developing this Lab

Sea Urchins are one of the more unique organisms found in a developmental biology lab.  They’re primarily used as a model organism for egg fertilization experiments and not much else (https://askabiologist.asu.edu/explore/sea-urchins-do-research).   Fortunately, they’re very well suited for such experiments and it’s quite easy for students to learn to work with them.  A simple fertilization can be done in under an hour and variations to the fertilization experiment can be easily implemented.

This was one of the last labs my students performed before the end of the semester.  Previous labs had mostly stripped away the hand-holding aspects and I wanted to make the students do as much as the experimental design as they could handle.  Every student in lab was issued a digital lab manual of Developmental Biology: A Guide for Experimental Study by Mary S. Tyler (https://onlinelibrary.wiley.com/doi/abs/10.1002/ajmg.1320590229). This book contains a chapter on sea urchins that should have all the information needed to design a lab around them.  If this book is unavailable, Swarthmore has an online lab manual for sea urchins that can do in a pinch (https://www.swarthmore.edu/NatSci/sgilber1/DB_lab/DB_lab.html).

These lab manuals were used because they don’t clearly spell out a protocol.  Instead, students spent half of one lab reading through the manual to find certain variables like the components of sea water, normal fertilization temperature, and how to induce urchins to release gametes.  To make sure every student ended up with the same protocol, I created a power point that ultimately laid out the protocol as students successfully described each step.  They then spent the other half of the lab making necessary reagents (artificial sea water and 0.5 M KCl) and preparing glassware and other equipment.  By then end of this lab, students were expected to be fully prepared to begin working with sea urchins at the start of the second lab.

For my part, I had to make sure that at least 2 sea urchins (Lytechinus variegatus) were available for each group to work with.  These can be a bit tricky to find online, but companies like http://www.marinusscientific.com/ should have them available as long as they have a week or two notice.  These sea urchins arrived in a large insulated container with each sea urchin sequestered to a plastic bag filled with water.  We kept them at room temperature in the bags they shipped with.  Most survived several days in those bags will no ill effects.  We could tell that they were alive because they’d wiggle when we held them. 

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Several sea urchins used in this experiment

The full lab protocol is included below, but I’ll describe how it works with pictures here as well.  Sea urchins are spiny balls, but half two distinct sides.  One side as a mouth and the other has an anus.  All lab groups would grab a single sea urchin and inject 0.5 M KCl around the edges of the mouth with a syringe.

Injecting a sea urchin.

The syringe had to be sharp enough to pierce the soft tissue around the mouth of the sea urchin, but students had to be careful to not damage the organism too much or inject too much liquid.  The 0.5 M KCl would cause the sea urchins to release gametes out their back sides to the sea urchins then had to be placed ass down over a beaker.

A Sea urchin releasing eggs

Within minutes the sea urchin would start to release either sperm (white) or eggs (yellow).  Students would do this for multiple sea urchins until they had both a sperm and egg, then they’d mix the sperm and egg together in a petri dish to fertilize them.  We’d analyze the gametes under a microscope to see if fertilization occurred.  The presence of an envelope around an egg usually indicated successful fertilization.

A fertilized egg with an envelope

Students had to then come in the next day to calculate the percentage of eggs that had been fertilized.  Students were also instructed to design an additional experiment to push their protocol beyond a simple fertilization.  For example, they could adjust temperature and see if that affected fertilization efficiency.

Protocol

Materials

0.5 M KCl

Artificial Sea Water:

28.32 g NaCl

0.77 g KCl

5.41 g MgCl2·6H2O

7.13 g MgSO4·7H2O

1.18 g CaCl2

H2O to 1 Liter

Protocol for collecting sea urchin gametes

1. Induce spawning – inject 1 ml of 0.5 M KCL into the soft membrane around the mouth

2. Gametes should appear within minutes

Sperm are off-white

Eggs are tan-orange

3. Collect the sperm by inverting the male onto a small beaker à cover the sperm and place on ice

4. Collects the eggs by inverting the female onto a beaker filled with artificial sea water à allow eggs to settle. Pour out ASW and replace with fresh ASW.

Standard Fertilization protocol

1. Wash eggs with 10 mL artificial sea water (3 times)

2. Dilute 50 uL sperm in 5 mL ASL

3. Add 100 uL diluted sperm to 10 mL artificial sea water containing eggs in petri dish

4. Check for signs of fertilization under the dissecting scope

5. Cover petri dishes and wait 24 hours.  Calculate percentage of fertilized eggs

Turning this into an experiment

1. You will perform both a standard fertilization and an experimental fertilization

The experimental fertilization will change one variable to the standard protocol to see if it affects fertilization efficiency

2. Adjusting temperature, ion concentration, pH, light conditions, exposure to UV light, etc.

3. In your lab notebooks, you will include an extra paragraph in your conclusion explaining your results

Final Thoughts

This lab went very well. My only real complaint is that it was my first time working with these creatures and I wasn’t as effective an instructor due to my own inexperience.  Fortunately, my lab assistant had worked with them before and saved my bacon on a number of occasions.

I’m not sure if I’ll continue this series. I’ve highlighted my favorite labs and I may want to go back to covering research papers for a bit.  We’ll see what happens.  Thanks for reading!