Uncovering the mystery of hybrid animalsIn animals that reproduce by external fertilization (like sea urchins), species-specificity is vital sothat the egg of one species recognizes and fuses with the sperm of the same species.Consequently, there are not one but several steps during fertilization that require species-specific recognition for fertilization to proceed. These steps include sperm attraction, initiation ofthe acrosomal reaction at the jelly coat, and sperm-egg binding. Interestingly, in mammals thatundergo internal fertilization, there are rare examples of interbreeding between species,including mules (horse x donkey) and zonkey (zebra x donkey).Consider the following scenarioYou are a developmental biologist on vacation in Florence, Italy. Talking to a local while having agelato, you hear of an animal reserve just outside of the city where a curious animal was born.The animal has a head of a donkey, and the stripes of a zebra on its body and legs! You instantlyguess it must be a zebroid, a hybrid between a zebra and another member of the horse family.Like any curious biologist, you drag your partner to the animal reserve to check out this curiouszebroid.When you arrive at the animal reserve, you immediately spot Ippo the zebroid running aroundher pen, and you go to talk to the caretakers at the reserve. The caretakers tell you that thezebras are kept in a fenced-in pen next to an area where an endangered species of donkey fromthe Amiata region are kept. One day Martin the zebra jumped the fence and mated with Giadathe donkey. Twelve months later, Giada gave birth to Ippo!Ippo has been such a sensation at the animal reserve that ticket sales to the park have soared.Hearing that you are a biologist, the owner strides over and asks you, how likely is a hybrid eventlike this to happen again? Could they produce other combinations of animals successfully to keepprofits up? Though you are concerned about the ethical implications, your curiosity about howanimals can mate to form hybrids is too much to resist. You can’t wait to return to yourlaboratory in Middletown and study this question.Back in the lab, you begin to read about the mechanism of fertilization. You learn that Deno is aprotein expressed on the acrosomal process of mammalian sperm (deno means ‘bind’ in Greek).In their discussion section, the authors of the paper speculate that Deno may recognizeglycoproteins in the zona pellucida, though no evidence is provided in the paper. The proteinsequence of Deno is 99% identical between horse, donkey and zebra, and 70% identical betweenhorse, human, mouse and rabbit.
Question 1: Test the hypothesis that Deno is a likely candidate protein for the recognition of sperm and egg.
a) How would you design an experiment to test this hypothesis?
b) What would you predict to find from your proposed experimental design?
Question 2: Once you prove that Deno is the recognition protein for sperm and egg, you need to prove its function
a) How would you approach testing that Deno is necessary for sperm-egg recognition? Include what you would predict to find and how would you know you had successful results.
b) How would you approach testing that Deno is sufficient for egg-sperm recognition? Include what you would predict to find and how would you know you had successful results.
a) To test the hypothesis that Deno is a likely candidate protein for the recognition of sperm and egg, one could design an experiment as follows:
1. Obtain samples of sperm and eggs from different species, such as horse, donkey, and zebra.
2. Isolate the protein Deno from each of the species.
3. Perform a binding assay using the isolated Deno protein and the respective species' eggs.
4. Measure the binding affinity and specificity of Deno to the eggs of each species.
5. Compare the binding affinity and specificity of Deno across the different species.
b) If Deno is a likely candidate protein for sperm and egg recognition, one would predict to find that the binding affinity of Deno is highest between the Deno protein from a species and its own eggs. Additionally, the binding affinity between Deno from closely related species (such as horse and donkey) may be higher compared to more distantly related species (such as horse and zebra).
Question 2:
a) To test whether Deno is necessary for sperm-egg recognition, one could use a knockout or gene silencing approach. This can be done by:
1. Select a suitable model organism with a similar Deno protein, such as a mouse.
2. Use gene-editing techniques to knockout or silence the Deno gene in the model organism.
3. Collect sperm and eggs from both the wild-type and Deno knockout/silenced individuals.
4. Perform a binding assay between the sperm and eggs from the wild-type and Deno knockout/silenced individuals.
5. Compare the binding affinity and success rate of fertilization between the two groups.
If Deno is necessary for sperm-egg recognition, one would predict to find a significant decrease in the binding affinity and success rate of fertilization in the Deno knockout/silenced group compared to the wild-type group.
b) To test if Deno is sufficient for egg-sperm recognition, one could use a transgenic approach. This can be done by:
1. Select a suitable model organism, such as a mouse, that does not naturally express Deno protein.
2. Introduce the Deno gene into the model organism, so it expresses Deno protein.
3. Collect sperm and eggs from the transgenic individuals expressing Deno and from non-transgenic individuals.
4. Perform a binding assay between the sperm and eggs from the transgenic and non-transgenic individuals.
5. Compare the binding affinity and success rate of fertilization between the two groups.
If Deno is sufficient for egg-sperm recognition, one would predict to find a significant increase in the binding affinity and success rate of fertilization in the transgenic group compared to the non-transgenic group.